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| United States Patent |
5,766,854
|
|
Wells
, et al.
|
June 16, 1998
|
Method for identifying active domains and amino acid residues in
polypeptides and hormone variants
Abstract
The invention provides methods for the systematic analysis of the structure
and function of polypeptides by identifying active domains which influence
the activity of the polypeptide with a target substance. Such active
domains are determined by substituting selected amino acid segments of the
polypeptide with an analogous polypeptide segment from an analog to the
polypeptide. The analog has a different activity with the target substance
as compared to the parent polypeptide. The activities of the
segment-substituted polypeptides are compared to the same activity for the
parent polypeptide for the target. A comparison of such activities
provides an indication of the location of the active domain in the parent
polypeptide. The invention also provides methods for identifying the
active amino acid residues within the active domain of the parent
polypeptide. The method comprises substituting a scanning amino acid for
one of the amino acid residues within the active domain of the parent
polypeptide and assaying the residue-substituted polypeptide so formed
with a target substance. The invention further provides polypeptide
variants comprising segment-substituted and residue-substituted growth
hormones, prolactins and placental lactogens.
| Inventors:
|
Wells; James A. (Burlingame, CA);
Cunningham; Brian C. (Piedmont, CA)
|
| Assignee:
|
Genentech, Inc. (San Francisco, CA)
|
| Appl. No.:
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483039 |
| Filed:
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June 6, 1995 |
| Intern'l Class: |
C12Q 001/68; G01N 033/566; C07K 016/00; C12N 015/00 |
| Field of Search: |
435/7.1,4,6,7.6,7.71,69.1,71.1
436/501
530/350,387.1,388.1,399,806,808
935/9-15,76-79,82,88
|
References Cited
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| |
| WO 88/07084 | Sep., 1988 | WO.
| |
| WO 88/07578 | Oct., 1988 | WO.
| |
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|
Primary Examiner: Achutamurthy; Ponnathapura
Attorney, Agent or Firm: Skjerven, Morrill, MacPherson, Franklin & Friel, LLP, Terlizzi; Laura, Haliday; Emily M.
Parent Case Text
This application is a continuation of application Ser. No. 08/190,723,
filed Feb. 2, 1994, which is a continuation of application Ser. No.
07/960,227, filed Oct. 13, 1992, now abandoned, which is a continuation of
07/875,204, filed Apr. 27, 1992, now abandoned, which is a continuation of
07/428,066, filed Oct. 26, 1989, now abandoned, which is a
continuation-in-part of 07/264,611, filed Oct. 26, 1988, now abandoned.
Claims
What is claimed is:
1. A method for identifying at least a first unknown active domain in a
region of known amino acid sequence of a parent polypeptide, which parent
polypeptide has been cloned and has a pre-identified biological activity,
said active domain capable of interacting with a first target when said
parent polypeptide is in its native-folded form, which interaction is
responsible for said biological activity, said method comprising:
(a) comparing amino acid sequence or polypeptide structure in the region of
known amino acid sequence of the parent polypeptide with amino acid
sequence or polypeptide structure in a region of known amino acid sequence
of an analog polypeptide to said parent polypeptide, said analog having at
least 15% sequence homology with said parent polypeptide or .alpha.-carbon
coordinates within about 2 to about 3.5 .ANG. of parent polypeptide
.alpha.-carbon coordinates for about 60% or more of the analog sequence,
wherein any interaction of said analog with said first target is different
from the interaction of said parent polypeptide with said first target;
(b) substituting DNA encoding a first analogous polypeptide segment from
said analog into DNA encoding substantially the full-length parent
polypeptide and expressing a first segment-substituted polypeptide;
(c) contacting said first segment-substituted polypeptide with said first
target to determine the interaction, if any, between said first target and
said segment-substituted polypeptide;
(d) repeating steps (b) and (c) using a second analogous polypeptide
segment from an analog to said parent polypeptide to form at least a
second segment-substituted polypeptide containing said second analogous
polypeptide segment, which is different from said first analogous
polypeptide segment; and
(e) comparing the difference, if any, between the activity relative to said
first target of said parent polypeptide and said first and second
segment-substituted polypeptides as an indication of the location of said
first unknown active domain in said parent polypeptide.
2. The method of claim 1 wherein said first unknown active domain comprises
at least two discontinuous amino acid segments in the primary amino acid
sequence of said parent polypeptide.
3. The method of claim 1 wherein at least a first selected polypeptide
segment of said parent polypeptide replaced by said first analogous
polypeptide segment of said analog contains at least one amino acid
residue located on the surface of the native-folded form of said parent
polypeptide.
4. The method of claim 3 further comprising repeating steps (b) and (c) to
form a plurality of segment-substituted polypeptides that, collectively,
contain substitutions of substantially all of the amino acid residues on
said surface of said parent polypeptide.
5. The method of claim 1 further comprising repeating steps (b) and (c) to
form a plurality of segment-substituted polypeptides that, collectively,
contain substitutions of analogous polypeptide segments covering about
15-100% of the amino acid sequence of said parent polypeptide.
6. The method of claim 1 further comprising repeating steps (b) and (c) to
form a plurality of segment-substituted polypeptides that, collectively,
contain substitutions of analogous polypeptide segments covering about
60-100% of the amino acid sequence of said parent polypeptide.
7. The method of claim 1 further comprising identifying a second unknown
active domain of said parent polypeptide, said second active domain
interacting with a second target, said method comprising repeating steps
(b) through (e) with said second target.
8. The method of claim 1 further comprising identifying at least a first
active amino acid residue within said first active domain, said method
comprising:
(f) substituting a scanning amino acid for a different first amino acid
residue within said first active domain to form a first
residue-substituted polypeptide;
(g) contacting said first residue-substituted polypeptide with said first
target to determine the interaction, if any, between said first target and
said first residue-substituted polypeptide;
(h) repeating steps (f) and (g) to substitute a scanning amino acid for at
least a second amino acid residue within said first active domain to form
at least a second residue-substituted polypeptide; and
(i) comparing the difference, if any, between the activity relative to said
first target of said parent polypeptide and each of said first and second
residue-substituted polypeptides as an indication of the location of said
first active amino acid residue in said first active domain.
9. The method of claim 8 further comprising repeating steps (b) through (i)
with a second target to identity a second active domain and at least one
active amino acid residue within said second active domain.
10. The method of claim 9 further comprising the step of substituting an
active amino acid residue in said first active domain with a different
amino acid to produce a polypeptide variant having a modified interaction
with said first target but which retains substantially all of the
interaction of said parent polypeptide with said second target.
11. The method of claim 10 further comprising the step of substituting an
active amino acid residue in said second active domain with a different
amino acid to produce a polypeptide variant having a modified interaction
with said second target but which retains the interaction of said parent
polypeptide with said first target.
12. The method of claim 9 wherein said first and said second active domains
have at least one common active amino acid residue, said method further
comprising substituting at least said one common active amino acid residue
with a different amino acid to produce a polypeptide variant having
modified interactions with each of said first and said second targets.
13. The method of claim 9 wherein said first and said second active domains
have at least one common active amino acid residue, said method further
comprising substituting at least one amino acid residue in said first
active domain, other than said at least one common active amino acid
residue, with a different amino acid to produce a polypeptide variant
having a modified interaction with said first target.
14. A method for identifying at least one active amino acid residue in a
parent polypeptide, which parent polypeptide has been cloned and has a
pre-identified biological activity resulting from an interaction with a
first target, comprising:
(a) substituting DNA encoding a scanning amino acid for DNA encoding a
first amino acid residue at residue number N within DNA encoding
substantially the full-length parent polypeptide and expressing an
N-substituted polypeptide;
(b) substituting DNA encoding a scanning amino acid for each of the amino
acid residues at residue numbers N+1 and N-1 to said first residue within
DNA encoding substantially the full-length parent polypeptide and
expressing N+1- and N-1-substituted polypeptides, respectively;
(c) contacting each of said substituted polypeptides expressed in steps (a)
and (b) with said first target to determine the interaction, if any,
between said first target and said substituted polypeptides; and
(d) comparing the difference, if any, between the activity relative to said
first target of said parent polypeptide and said substituted polypeptides
as an indication of the location of said active amino acid residue.
15. The method of claim 14 wherein residue N is a suspected active amino
acid residue and steps (b) through (d) are repeated until at least four
consecutive residues are identified wherein substitution of a scanning
amino acid at each of said four conservative residues has less than a
two-fold effect on activity with said first target as compared to said
parent polypeptide.
16. The method of claim 14 wherein said parent polypeptide is selected from
the group consisting of human growth hormone, human placental lactogen,
and human prolactin.
17. The method of claim 14 wherein at least one of said scanning amino
acids is an isosteric amino acid.
18. The method of claim 14 wherein the same scanning amino acid is employed
in steps (a) and (b), and said scanning amino acid is a neutral amino
acid.
19. The method of claim 18 wherein said neutral amino acid is selected from
the group consisting of alanine, serine, glycine, and cysteine.
20. The method of claim 19 wherein said scanning amino acid is alanine.
21. The method of claim 14 wherein said parent polypeptide is a hormone and
said activity is measured in an in vitro assay using a soluble hormone
receptor.
22. The method of claim 21 wherein said hormone is human growth hormone and
said soluble hormone receptor is shGHr.
23. The method of claim 21 wherein said hormone is human growth hormone and
said soluble hormone receptor is shPRLr.
24. The method of claim 14 wherein said interaction between said first
target and said parent polypeptide involves either binding or catalytic
interaction of said parent polypeptide with said first target.
25. The method of claim 24 wherein the activity between said first target
and any of said substituted polypeptides is increased greater than
two-fold as compared to said parent polypeptide.
26. The method of claim 24 wherein the activity between said first target
and any of said substituted polypeptides is decreased greater than
two-fold as compared to said parent polypeptide.
27. The method of claim 1 wherein said parent polypeptide is selected from
the group consisting of growth hormone, prolactin, placental lactogen,
.alpha.-interferon, .gamma.-interferon, TGF-.alpha..sub.1, EGF, IGF-1,
GM-CSF, TNF, tissue plasminogen activator, and CD-4 receptor.
28. The method of claim 27 wherein said parent polypeptide is selected from
the group consisting of human growth hormone, human placental lactogen,
and human prolactin.
29. The method of claim 1 wherein said activity is measured in an in vitro
assay.
30. The method of claim 29 wherein said parent polypeptide is a hormone and
said activity is measured in an in vitro assay using a soluble hormone
receptor.
31. The method of claim 30 wherein said hormone is human growth hormone and
said soluble hormone receptor is shGHr.
32. The method of claim 30 wherein said hormone is human growth hormone and
said soluble hormone receptor is shPRLr.
33. The method of claim 1 wherein said interaction between said first
target and said parent polypeptide involves either binding or catalytic
interaction of said parent polypeptide with said first target.
34. The method of claim 33 wherein the activity between said first target
and any of said substituted polypeptides is increased greater than
two-fold as compared to said parent polypeptide.
35. The method of claim 33 wherein the activity between said first target
and any of said substituted polypeptides is decreased greater than
two-fold as compared to said parent polypeptide.
36. The method of claim 1, wherein said analog has at least 15% amino acid
sequence homology with said parent polypeptide.
37. The method of claim 1, wherein said analog is naturally occurring.
38. The method of claim 1, wherein said analog is a tertiary analog.
39. The method of claim 1 wherein said parent polypeptide is human growth
hormone and said analog is selected from the group consisting of human
placental lactogen, porcine growth hormone, and human prolactin.
40. The method of claim 1 wherein the biological activity of the parent
polypeptide is of clinical utility.
41. The method of claim 1 wherein said parent polypeptide is selected from
the group consisting of a hormone, enzyme, antigen, receptor, enzyme
substrate, binding protein, and enzyme inhibitor.
42. The method of claim 1 wherein said first target is selected from the
group consisting of a hormone, enzyme, antibody, antigen, receptor, enzyme
substrate, binding protein, and enzyme inhibitor.
43. The method of claim 1 wherein said analog does not substantially
interact with said first target.
44. The method of claim 8 wherein at least one of said scanning amino acids
is an isosteric amino acid.
45. The method of claim 8 wherein the same scanning amino acid is employed
in steps (f) and (h), and said scanning amino acid is a neutral amino
acid.
46. The method of claim 45 wherein said neutral amino acid is selected from
the group consisting of alanine, serine, glycine, and cysteine.
47. The method of claim 46 wherein said scanning amino acid is alanine.
48. The method of claim 1 wherein said parent polypeptide is naturally
occurring.
49. The method of claim 14 wherein said parent polypeptide is naturally
occurring.
Description
FIELD OF THE INVENTION
The invention is directed to methods for identifying the active domains and
amino acid residues in polypeptides. It is also directed to hormone
variants.
BACKGROUND OF THE INVENTION
Polypeptides, i.e., peptides and proteins, comprise a wide variety of
biological molecules each having a specific amino acid sequence, structure
and function. Most polypeptides interact with specific substances to carry
out the function of the polypeptide. Thus, enzymes, such as subtilisin,
amylase, tissue plasminogen activator, etc., interact with and hydrolyze
specific substrates at particular cleavage sites whereas proteinaceous
hormones such as human growth hormone, insulin and the like interact with
specific receptors to regulate growth and metabolism. In other cases, the
interaction is between the polypeptide and a substance which is not the
primary target of the polypeptide such as an immunogenic receptor. Many
polypeptides are pluripotential in that they contain discrete regions
which interact with different ligands or receptors, each of which produces
a discrete biological effect. For example, human growth hormone (hGH) is
diabetogenic and lypogenic in adults and induces long bone growth in
children.
Efforts have been made to modify the primary functional properties of
naturally occurring polypeptides by modifying amino acid sequence. One
approach has been to substitute one or more amino acids in the amino acid
sequence of a polypeptide with a different amino acid. Thus, protein
engineering by in vitro mutagenesis and expression of cloned genes
reportedly has been applied to improve thermal or oxidative stability of
various proteins. Villafranca, J. E., et al. (1983) Science 222, 782-788,
Perry, L. J., et al. (1984) Science 226, 555-557; Estell, D. A., et al.
(1985) J. Biol. Chem. 260, 6518-6521; Rosenberg, S., et al. (1984) Nature
(London) 312, 77-80; Courtney, M., et. al. (1985) Nature (London) 313,
149-157. In addition, such methods have reportedly been used to generate
enzymes with altered substrate specificities. Estell, D. A., et al. (1986)
Science 223, 655-663; Craik, C. S., et al. (1985) Science, 291-297;
Fersht, A. R., et al. (1985) Nature (London) 314, 235-238; Winther, J. R.,
et al. (1985) Carlsberg Res. Commun. 50 273-284; Wells, J. A., et al.
(1987) Proc. Natl. Acad. Sci. 84, 1219-1223. The determination of which
amino acid residue should be modified has been based primarily on the
crystal structure of the polypeptide, the effect of chemical modifications
on the function of the polypeptide and/or the interaction of the
polypeptide with various substances to ascertain the mode of action of the
polypeptide. In some cases, an amino acid substitution has been deduced
based on the differences in specific amino acid residues of related
polypeptides, e.g. difference in the amino acid sequence in substrate
binding regions of subtilisins having different substrate specificities.
Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 5767. In other
cases, the amino acid sequence of a known active region of a molecule has
reportedly been modified to change that sequence to that of a known active
region of a second molecule. Wharton, R. P., et al. (1985) Nature 316,
601-605, and Wharton, R. P., et al. (1984) Cell 38, 361-369 (substitution
of recognition helix of phage repressor with recognition helix of
different repressor); Jones, P. T., et al. (1986) Nature 321, 522-525
(substitution of variable region of a mouse antibody for corresponding
region of human myeloma protein). While this approach may provide some
predictability with regard to the properties modified by such
substitutions, it is not a methodical procedure which would confirm that
all regions and residues determinative of a particular property are
identified. At best, empirical estimates of the energetics for the
strengths of the molecular contacts of substituted residues may be
ascertained. In this manner, the strengths of hydrogen bonds (Fersht, A.
R., et al. (1985) Nature 314, 235; Bryan, P., et al. (1986) Proc. Natl.
Acad. Sci. USA 83, 3743; Wells, J. A., et al. (1986) Philos. Trans. R.
Soc. London A. 317, 415), electrostatic interactions (Wells, J. A., et al.
(1987) Proc. Natl. Acad. Sci. USA 84, 1219; Cronin, C. N., et al. (1987)
J.Am. Chem. Soc. 109, 2222), and hydrophobic and steric effects (Estell,
D. A., et al. (1986) Science 233, 659; Chen, J. T., et al. (1987)
Biochemistry 26, 4093) have been estimated for specific modified residues.
These and other reports (Laskowski, M., et al. (1987) Cold Spring Harbor
Symp. Quant. Biol. 52, 545; Wells, J. A., et al. (1987) Proc. Natl. Acad.
Sci. USA 84, 5167; Jones, P. T., et al. (1986) Nature 321, 522; Wharton,
R. P., et al. (1985) Nature 316, 601) have concluded that mutagenesis of
known contact residues causes large effects on binding whereas mutagenesis
of non-contact residues has a relatively minor effect.
A second reported approach to understand the relationship between amino
acid sequence and primary function employs in vivo homologous
recombination between related genes to produce hybrid DNA sequences
encoding hybrid polypeptides. Such hybrid polypeptides have reportedly
been obtained by the homologous recombination of trp B and trp A from
E.coli and Salmonella typhimurium (Schneider, W. P., et al. (1981) Proc.
Natl. Acad. Sci., USA 78, 2169-2173); alpha 1 and alpha 2 leukocyte
interferons (Weber, H. and Weissmann, C. (1983) Nuc. Acids Res. 11, 5661);
the outer membrane pore proteins ompC and phoE from E.coli K-12
(Thommassen, J., et al. (1985) EMBO 4, 1583-1587); and thermophilic
alpha-amylases from Bacillus stearothermophilus and Bacillus lichiniformis
(Gray, G. L., et al. (1986) J. Bacterial. 166, 635-643). Although such
methods may be capable of providing useful information relating to amino
acid sequence and function as well as useful hybrid polypeptides, as
reported in the case of the hybrid alpha amylases, it is difficult to
utilize such methods to systematically study a given polypeptide to
determine the precise regions and amino acid residues in the polypeptide
that are active with one of the target substances for that particular
molecule. This is because the site of crossover recombination, which
defines the DNA and amino acid sequence of the hybrid, is determined
primarily by the DNA sequence of the genes of interest and the
recombination mechanism of the host cell. Such methods do not provide for
the predetermined and methodical sequential replacement of relatively
small segments of DNA encoding one polypeptide with a corresponding
segment from a second gene except in those fortuitous circumstances when
crossover occurs near the 5' or 3' end of the gene.
The interaction of proteinaceous hormones with their receptors has
reportedly been studied by several techniques. One technique uses hormone
peptide fragments to map the location of the receptor binding sites on the
hormone. The other technique uses competition between neutralizing
monoclonal antibodies and peptide fragments to locate the receptor binding
site by epitope mapping. Exemplary of these techniques is the work
reported on human growth hormone (hGH).
Human growth hormone (hGH) participates in much of the regulation of normal
human growth and development. This 22,000 dalton pituitary hormone
exhibits a multitude of biological effects including linear growth
(somatogenesis), lactation, activation of macrophages, insulin-like
effects and diabetagenic effects among others. See Chawla, R. K. (1983)
Ann. Rev. Med. 34, 519; Edwards, C. K., et al. (1988) Science 239, 769;
Thorner, M. O., et al. (1988) J. Clin. Invest. 81, 745. Growth hormone
deficiency in children leads to dwarfism which has been successfully
treated for more than a decade by exogenous administration of hGH. There
is also interest in the antigenicity of hGH in order to distinguish among
genetic and post-translationally modified forms of hGH (Lewis, U. J.
(1984) Ann. Rev. Physiol. 46, 33) to characterize any immunological
response to hGH when it is administered clinically, and to quantify
circulating levels of the hormone.
hGH is a member of a family of homologous hormones that include placental
lactogens, prolactins, and other genetic and species variants of growth
hormone. Nichol, C. S., et al. (1986) Endocrine Reviews 7, 169. hGH is
unusual among these in-that it exhibits broad species specificity and
binds monomerically to either the cloned somatogenic (Leung, D. W., et al.
(1987) Nature 330, 537) or prolactin receptor (Boutin, J. M., et al.
(1988) Cell 53, 69). The cloned gene for hGH has been expressed in a
secreted form in Eschericha coli (Chang, C. N., et al. (1987) Gene 55,
189) and its DNA and amino acid sequence has been reported (Goeddel, et
al. (1979) Nature 281, 544; Gray, et al. (1985) Gene 39, 247). The
three-dimensional structure of hGH is not available. However, the
three-dimensional folding pattern for porcine growth hormone (pGH) has
been reported at moderate resolution and refinement (Abdel-Meguid, S. S.,
et al. (1987) Proc. Natl. Acad. Sci. USA 84, 6434).
Peptide fragments from hGH have been used in attempts to map the location
of the receptor binding site in hGH. Li, C. H. (1982) Mol. Cell. Biochem.
46, 31; Mills, J. B., et al. (1980) Endocrinology 107, 391. In another
report, a fragment consisting of residues 96-133 was isolated after
proteolysis of bovine growth hormone. This fragment was reported to bind
to a growth hormone receptor. Yamasakin, et al. (1970) Biochemistry 9,
1107. However, when a larger peptide containing residues 1-133 was
produced by recombinant methodology, no detectable binding activity was
observed. Krivi, G. G., et al., International Symposium on Growth Hormone;
Basic and Clinical Aspects, Abstract I-18, Final Program, sponsored by
Serono Symposia, USA, Jun. 14-18, 1987. These results are clearly
irreconcilable and demonstrate the potential unreliability of using
peptide fragments to map receptor binding sites on a proteinaceous
hormone, especially for those where the binding site is composed of two or
more discontinuous and/or conformationally dependent epitopes.
The use of neutralizing monoclonal antibodies to locate the receptor
binding sites by epitope mapping has similar limitations. For example, a
monoclonal antibody was reportedly used in a receptor binding assay to
compete with the hGH receptor for a peptide consisting of residues 98-128
of hGH. Even though the peptide 98-128 of the hGH hormone only binds to
the neutralizing monoclonal antibody, it was proposed that this region
contains the receptor binding site based on these competitive studies.
Retegin, L. A., et al. (1982) Endocrinology 111, 668. Similar approaches
have been used in attempts to identify antigenic sites on the hGH hormone.
Epitope mapping of twenty-seven different monoclonal antibodies to hGH by
competitive binding reportedly resolved only four different antigenic
sites on the hormone. Surowy, T. K., et al. (1984) Mol. Immunol. 21, 345;
Aston, R., et al. (1985) Pharmac. Ther. 27, 403. This strategy, however,
did not locate the epitopes on the amino acid sequence of the hormone.
Another approach to defining antigenic sites has been to test the binding
of antibodies to short linear peptides over the protein of interest.
Geysen, H. M., et al. (1984) Proc. Natl. Acad. Sci. USA 81, 3998; Geysen,
H. M. (1985) Immunol. Today 6, 364. However, this approach suffers from
the same limitations of using linear peptide fragments to locate receptor
binding sites. To be useful, the linear sequence must be capable of
adopting the conformation found in the antigen for the antibody to
recognize it. Furthermore, based upon the known size of antibody epitopes
from X-ray crystallography (Sheriff, S., et al. (1987) Proc. Natl. Acad.
Sci USA 84, 8075; Amit, A. G., et al. (1986) Science 233, 747) it has been
estimated that virtually all antibody combining sites must be, in part,
discontinuous (Barlow, D. J., et al. (1986) Nature 322, 747) and as a
result linear fragments may not adequately mimic such structure.
Peptide fragments from hGH have also been studied by non-covalently
combining such fragments. Thus, several investigators have reported the
analysis of the combination of relatively large fragments of human growth
hormone comprising either the natural amino acid sequence or chemically
modified peptides thereof. Burstein, S., et al. (1979) J. of Endo. Met.
48, 964 (amino terminal fragment hGH-(1-134) combined with
carboxyl-terminal fragment hGH-(141-191)); Li, C. H., et al. (1982) Mol.
Cell. Biochem. 46 31; Mills, J. B., et al. (1980) Endocrinology 107, 391
(subtilisin-cleaved two-chain form of hGH).
Similarly, the chemically modified fragment hGH-(1-134) and a chemically
modified carboxy-terminal fragment from human chorionic somatomammotropin
(also called placental lactogen), (hCS-(141-191)), have been
non-covalently combined, as have the chemically modified fragments
hCS-(1-133) and hGH-(141-191). U.S. Pat. No. 4,189,426. These
investigators reported incorrectly that the determinants for binding to
the hepatic growth hormone receptor are in the first 134 amino-terminal
residues of growth hormone (Burstein, et al. (1978) Proc. Natl. Acad. Sci.
USA 75, 5391-5394). Clearly, such techniques are subject to erroneous
results. Moreover, by utilizing two large fragments this technique is only
potentially able to localize the function to one or the other of the two
fragments used in such combinations without identification of the specific
residues or regions actively involved in a particular interaction. A
review of some of the above techniques and experiments on hGH has been
published. Nichol, C. S., et al. (1986) Endocrine Rev. 7, 169-203.
An alternative approach has been reported wherein a 7 residue peptide
fragment from the "deletion peptide" of hGH (hGH-32-46) was modified to
contain amino acid residues from analogous segments of growth hormone from
other mammalian species. The effect, if any, of such substitutions,
however, was reported. See U.S. Pat. No. 4,699,897. Nonetheless, the
shortcomings of the use of short peptide fragments are apparent since the
linear sequence of such fragments must be capable of adopting the
conformation found in the intact growth hormone so that it may be
recognized in an in vitro or in vivo assay.
A number of naturally occurring mutants of hGH have been identified. These
include hGH-V (Seeberg, P. H. (1982) DNA 1, 239; U.S. Pat. Nos. 4,446,235,
4,670,393 and 4,665,180) and 20K hGH containing a deletion of residues
32-46 of hGH (Kostyo, J. L., et al. (1987) Biochemica et Biophysica Acta
925, 314; Lewis, U. J., et al. (1978) J. Biol. Chem. 253, 2679).
One investigator has reported the substitution of cysteine at position 165
in hGH with alanine to disrupt the disulfide bond which normally exists
between Cys-53 and Cys-165. Tokunaga, T., et al. (1985) Eur. J. Biochem.
153, 445. This single substitution produced a mutant that apparently
retained the tertiary structure of hGH and was recognized by receptors for
hGH.
Another investigator has reported the in vitro synthesis of hGH on a solid
resin support. The first report by this investigator disclosed an
incorrect 188 amino acid sequence for hGH. Li, C. H., et al. (1966) J. Am.
Chem. Soc. 88, 2050; and U.S. Pat. No. 3,853,832. A second report
disclosed a 190 amino acid sequence. U.S. Pat. No. 3,853,833. This latter
sequence is also incorrect. In particular, hGH has an additional glutamine
after position 68, a glutamic acid rather than glutamine at position 73,
an aspartic acid rather than asparagine at position 106 and an asparagine
rather than aspartic acid at position 108.
In addition to the foregoizng, hybrid interferons have been reported which
have altered binding to a particular monoclonal antibody. Camble, r. et.
al. "Properties of Interferon-.alpha.2 Analogues Produced from Synthetic
Genes in Peptides: Structure and Function," Proceedings of the Ninth
American Peptide Symposium. (1985) eds. Deber et. al., Pierce Chemical
Co., Chicago, Ill., pp.375-384. As disclosed therein, amino acid residues
101-114 from .alpha.-1 interferon or residues 98-114 from
.gamma.-interferon were substituted into .alpha.-2 interferon. .alpha.-2
interferon binds NK-2 monoclonal antibody whereas .alpha.-1 interferon
does not. This particular region in .alpha.-2 interferon apparently was
chosen because 7 of the 27 amino acid differences between .alpha.-1 and
.alpha.-2 interferon were located in this region. The hybrids so obtained
reportedly had substantially reduced activity with NK-2 monoclonal
antibody. When tested for antiviral activity, such hybrids demonstrated
antiviral activity on par with the activity of wild type .alpha.-2
interferon. Substitutions of smaller sections within these regions were
also reported. Sequential substitution of clusters of 3 to 7 alanine
residues was also proposed. However, only one analogue ›Ala-30,32,33!
IFN-.alpha.2 is disclosed.
Alanine substitution within a small peptide fragment of hen egg-white
lysozyme and the effect of such substitutions on the stimulation of 2A11
or 3A9 cells have also been reported. Allen, P. M., et. al. (1987) Nature
327,713-715.
Others have reported that binding properties can be engineered by
replacement of entire units of secondary structure units including antigen
binding loops (Jones, P. T., et al. (1986) Nature 321, 522-525) or DNA
recognition helices (Wharton, R. P., et al. (1985) Nature 316,601-605).
The references discussed above are provided solely for their disclosure
prior to the filing date of the present application, and nothing herein is
to be construed as an admission that the inventors are not entitled to
antedate such disclosure by virtue of prior invention or priority based on
earlier filed applications.
Given the state of the art as exemplified by the above references, it is
apparent that a need exists for a useful method for the systematic
analysis of polypeptides so as to ascertain the relationship between
structure and function. Accordingly, it is an object herein to provide
such methods to identify the active domains within the polypeptide which
contribute to the functional activity of the polypeptide.
It is a further object herein to provide methods for determining the active
amino acid residues which determine functional activity.
A further object of the invention is to provide methods for systematically
identifying the biologically active domains in a polypeptide.
Further, it is an object herein to provide hormone variants having
desirable biological, biochemical and immunogenic properties which are
different as compared to the same properties of the hormone from which
such variants are derived.
Still further it is an object herein to provide hormone variants having
diminished activity with one biological function and substantial or
increased activity with a second target substance.
Still further it is an object herein to provide hGH variants having
modified binding and/or biological activity with the somatogenic receptor
for hGH and increased potency.
Still further it is an object herein to provide hGH variants which retain
one or more desirable biological properties but which also have decreased
diabetogenic activity.
Further, it is an object herein to provide hPRL and hPL variants having an
increased binding activity with the somatogenic receptor of hGH.
Further, it is an object herein to provide DNA sequences, vectors and
expression hosts containing such vectors for the cloning and expression of
polypeptide variants including hGH variants.
SUMMARY OF THE INVENTION
In one aspect, the invention provides methods for the systematic analysis
of the structure and function of polypeptides by identifying unknown
active domains which influence the activity of the polypeptide with a
first target substance. Such unknown active-domains in one aspect of the
invention may comprise at least two discontinuous amino acid segments in
the primary amino acid sequence of the polypeptide. Active domains are
determined by substituting selected amino acid segments of the polypeptide
(referred to as the parent polypeptide) with an analogous amino acid
segment from an analog to the polypeptide. The analog has a different
activity with the target substance as compared to the parent polypeptide.
The segment-substituted polypeptides so formed are assayed to determine
the activity of each of the segment-substituted polypeptides with the
target substance. Such activities are compared to the same activity for
the parent polypeptide. Since the structurally analogous amino acid
segments are obtained from an analog that has a different interaction with
the target substance, a comparison of such activities provides an
indication of the location of the active domain in the parent polypeptide.
The method further comprises identifying the active amino acid residues
within the active domain of the parent polypeptide. The method comprises
substituting a scanning amino acid for one of the amino acid residues
within the active domain of the parent polypeptide and assaying the
residue-substituted polypeptide so formed with a target substance. The
activity of each of the residue-substituted polypeptides is compared to
the same activity of the parent polypeptide. These steps are repeated for
different amino acids in the active domain until the active amino acid
residues are identified.
In another aspect, the invention provides methods to identify different
active domains and active amino acid residues for different target
substances. Such methods comprise repeating the foregoing methods with a
second target.
In accordance with the foregoing method, polypeptide variants are
identified which have a different activity with one or more target
substance as compared to a parent polypeptide. Such variants are produced
based on the identification of the active domains or the identification of
the active amino acid residues in the active domain which determine the
activity of the parent polypeptide with a target substance.
The invention further comprises growth hormone, prolactin, and placental
lactogen variants comprising at least three portions. The first portion
corresponds to at least a part of the amino acid sequence of a parent
hormone, the third portion corresponds to the amino acid sequence of at
least part of the same parent hormone, and the second portion corresponds
to an amino acid sequence of an analog to the parent hormone. The second
portion is analogous to those amino acid residues of the parent hormone
not contained between the first and third portions of the polypeptide
variant.
The invention also includes specific human growth hormone, human prolactin
and human placental lactogen variants comprising segment-substituted and
residue-substituted variants of hGH.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the strategy used to identify active domains.
FIG. 2 shows the conserved and variable amino acid residues amongst the
amino acid sequences of hGH, hPL, pGH and hPRL.
FIG. 3 shows the putative low resolution structure of hGH and helical wheel
projections viewed from the N-terminal start residue for each helix.
Hydrophobic, neutral and charged residues are indicated by .largecircle.,
.box-solid. and .circle-solid. symbols, respectively.
FIG. 4 is a bar graph showing the relative reduction in binding of various
segment-substituted hGH variants to the soluble hGH receptor.
FIG. 5 depicts the analogous amino acids in the active domains A, C and F
which interact with the somatogenic hGH receptor.
FIG. 6 depicts the relative binding positions of the somatogenic receptor
and eight monoclonal antibodies to hGH. The top, middle, and bottom panels
in FIG. 6A show the binding positions of monoclonal antibodies 1, 8, and
7, respectively. In FIG. 6B, the top and bottom panels show the binding
positions of monoclonal antibodies 2 and 6, respectively, while the
binding position of the somatogenic receptor is shown in the middle panel.
The top, middle, and bottom panels in FIG. 6C show the binding position of
monoclonal antibodies 3, 4, and 5, repectively.
FIG. 7 is a bar graph showing the relative increase or decrease in binding
to the soluble hGH somatogenic receptor for various alanine-substituted
hGH variants. The stippled bar at T175 indicates that serine rather than
alanine is substituted. The broken bar at R178 indicates that asparagine
rather than alanine is substituted.
FIG. 8 depicts the DNA and amino acid sequence of the hGH gene used in the
examples.
FIG. 9 depicts the construction of vector pB0475 which contains a synthetic
hGH gene.
FIG. 10 is the DNA sequence of pB0475 showing the amino acid sequence for
hGH.
FIG. 11 depicts the construction of vector pJ1446.
FIG. 12 is the DNA sequence for pJ1446 showing the amino acid sequence for
the soluble portion of the somatogenic receptor from liver.
FIGS. 13 through 20 depict the epitope binding sites on hGH for each of
eight different monoclonal antibodies.
FIG. 21 shows the active amino acids involved in binding to the somatogenic
receptor in hGH and helical wheel projections for helices 1 and 4.
FIG. 22 shows the rat weight gain versus time for hGH and hGH, variants
administered at 50 micrograms/kg/day. The top panel shows weight gain in
rats treated with wild type hGH (WT), the E174A variant of hGH, and the
segment-substituted hGH variant hPRL (88-89). The bottom panel shows
weight gain in rats treated with wild type hGH as compared to hPRL.
FIG. 23 is a semilog plot of Kd ratio versus potency for hGH variants as
compared to wild-type hGH.
FIG. 24. Competitive binding curves of ›.sup.125 I!hGH and cold hGH to the
hGH binding protein isolated from either human serum (O) or from E. coli
KS330 cultures expressing the plasmid phGHr(1-238) (.circle-solid.). Bars
represent standard deviations from the mean. Inset shows Scatchard plots
that were derived from the competitive binding curves. The concentrations
of the binding protein from human serum and E. coli were 0.1 and 0.08 nM,
respectively.
FIG. 25. Structural model of hGH based on a folding diagram for pGH
determined from a 2.8 .ANG. resolution X-ray structure. Panel A shows a
functional contour map of the hGH receptor epitope and Panel B shows that
determined here for the hPRL receptor epitope. The size of the closed
circles corresponds to the magnitude of the disruptive effect for alanine
substitution at these residues. The small circles represent >2-fold
disruption whenever the larger circles represent >10-fold disruption. The
.tangle-solidup. in the hGH receptor epitope (Panel A) represents the
position of E174A that causes greater than a four-fold increase in binding
affinity.
FIG. 26. Plasmid diagram of pBO760 used for intracellular expression of
hPRL in E. coli.
FIG. 27. Location of residues in hGH that strongly modulate its binding to
the hGH binding protein. Alanine substitutions (serine or asparagine in
the case of T175 or R178, respectively) are indicated that cause more than
a 10-fold reduction (.largecircle.), a 4- to 10-fold reduction
(.box-solid.), or more than a 4-fold increase (.tangle-solidup.) in
binding affinity. Helical wheel projections in regions of .alpha.-helix
reveal their amphipathic quality and the fact that in helix 4 the most
important determinants are on its hydrophilic face (shaded).
FIG. 28. Circular dichroic spectra in the far UV (Panel A) or near UV
(Panel B) of hGH (-), wild-type hPRL (--) and hPRL variant D (----) (see
Table XXIII).
FIG. 29. Sequence comparison of hGH and hPRL in regions defined by homolog
and alanine scanning mutagenesis to be important for binding. Identical
residues are shaded and the numbering is based on the hGH sequence.
Residues are circled that when mutated cause more than a 4-fold change in
binding affinity. Asterisks above residues indicate sites at which
mutations cause a 2- to 4-fold reduction in binding affinity.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the method of the invention provides for the systematic
analysis of a parent polypeptide, such as human growth hormone or human
prolactin, to determine one or more active domains in the polypeptide that
are involved in the interaction of the parent polypeptide with a target
substance. To employ the method of the invention, one or more analogs to
the polypeptide of interest must exist which exhibit a different activity
with the target substance of interest.
Accordingly; as used herein, "parent polypeptide" refers to any polypeptide
for which an "analog" exists that has a different activity with a target
substance as compared to the same activity for the parent polypeptide.
Examples of such polypeptides, analogs and target substances are shown in
Table I.
TABLE I
______________________________________
Parent Target or Assay
Polypeptide
Analog Containing Target
______________________________________
Human growth
Human placental
Receptors for somatogenic,
hormone lactogen, human
lactogenic, diabetagenic,
prolactin and
lipolytic, nitrogen,
porcine growth
retention, macrophage
hormone activation and insulin-like
effects of hGH; rat tibia
assay, rat weight gain
assay, insulin resistance
assay in OB/OB mice or dog,
receptors on human liver,
adipose, lymphocytes,
thymocytes and ovary tissue
hPRL pGH Binding to human prolactin
receptor
Rabbit GH Human GH Binding to rabbit GH
receptor receptor
.alpha.-interferon
Related human
Binding to .alpha..sub.1 interferon
interferons and
receptor
animal interferons
human tissue
human TGF-.beta..sub.2
Human hemopoietic cell
growth factor
or inhibins growth modulation
(TGF-.beta..sub.1)
Epidermal growth
TGF-.alpha. Carotinocyte proliferation
factor (EGF)
Mouse Tumor
Human Tumor Mouse TNF receptor
Necrosis Necrosis activity
Factor (mTNF)
Factor (hTNF)
human mouse granulocyte
Growth and differentiation
granulocyte
macrophage colony
of human bone marrow stem
macrophage
stimulating cells
colony
stimulating
factor (hGMCSF)
factor (mGMCSF)
human CD-4
mouse CD-4 gp-120 from HIV virus
receptor receptor
Subtilisin
Subtilisin succinyl-ala--ala--pro--glu--
Bacillus Bacillus P-Nitroanilyd
Amylilquifaciens
licheniformis
human Related human
Activation of human
.gamma.-interferon
interferons and
interferon receptor
animal interferons,
e.g., from mouse
Insulin like
Insulin IGF-1 receptor growth
growth factor growth modulation receptor
(IGF-1)
Tissue Trypsin, urokinase
Plasminogen (cleavage)
Plasminogen fibrin (binding)
Activator (tPA)
______________________________________
The parent polypeptides, analogs and target substances in Table I, of
course, are exemplary only. Parent polypeptides also include proteinaceous
material comprising one or more subunits, e.g. succinyl coenzyme A
synthetase, mitochondrial ATPase, aminoacyl tRNA synthetase, glutaine
synthetase, glyceraldehyde-3-phosphate dehydrogenase and aspartate
transcarbamolase (see, Huang, et al. (1982), Ann. Rev. Biochem, 51,
935-971). In such multi-subunit parent polypeptides, active domains may
span the two or more subunits of the parent polypeptide. Accordingly, the
methods as described in more detail hereinafter can be used to probe each
of the subunits of a particular polypeptide to ascertain the active domain
and active amino acid residues for a particular target which may be
partially contained on one subunit and partially on one or more other
subunits.
The parental polypeptide and analog typically belong to a family of
polypeptides which have related functions. Moreover, such parental
polypeptides and analogs ordinarily will have some amino acid sequence
identity, i.e., conserved residues. Such sequence homology may be as high
as 90% but may range as low as about 15% to 20%.
In addition to primary sequence homology, an analog to a parent polypeptide
may be defined by the three-dimensional framework of the polypeptide and
analog. Thus, an analog may be divergent from a parent polypeptide in
amino acid sequence but structurally homologous to the parent polypeptide
based on a comparison of all, or part, of the tertiary structure of the
molecules. Chothia, C., et al. (1986) Embo. J. 5, 823.
In general; tertiary analogs can be identified if the three-dimensional
structure of 4 possible analog is known together with that of the parent
polypeptide. By performing a root means squared differences (RMS) analysis
of the .alpha.-carbon coordinates, (e.g., Sutcliffe, M. J., et al. (1987)
Protein Engineering 1, 377-384), the superposition of regions having
tertiary analog y, if any, are identified. If the .alpha.-carbon
coordinates overlap or are within about 2.ANG. to about 3.5.ANG. RMS for
preferably about 60% or more of the sequence of the test analog relative
to the .alpha.-carbon coordinates for the parent polypeptide, the test
analog is a tertiary analog to the parent polypeptide. This, of course,
would exclude any insertions or deletions which may exist between the two
sequences.
Although the above parent polypeptide and analogs disclose naturally
occurring molecules, it is to be understood that parent polypeptides and
analogs may comprise variants of such sequences including naturally
occurring variants and variations in such sequences introduced by in vitro
recombinant methods. Variants used as parent polypeptides or analogs thus
may comprise variants containing the substitution, insertion and/or
deletion of one or more amino acid residues in the parent polypeptide or
analog. Such variants may be used in practicing the methods of the
invention to identify active domains and/or amino acids or to prepare the
polypeptide variants of the invention. Thus, the naturally occurring
variants of hGH or the recombinantly produced variant containing the
substitution of Cys-165 with Ala may be used as parent polypeptide or an
analog so long as they have some activity with a target. Such naturally
occurring and recombinantly produced variants may contain different amino
acid residues which are equivalent to specific residues in another parent
polypeptide. Such different amino acids are equivalent if such residues
are structurally analogous by way of primary sequence or tertiary
structure or if they are functionally equivalent.
Further, it should be apparent that many of the parent polypeptides and
analogs can exchange roles. Thus, non-human growth hormones and their
related family of analogs each can be used as a parent polypeptide and
homolog to probe for active domains. Further, targets such as the CD-4
receptor for the HIV virus, may be used as a parent polypeptide with
analog CD-4 receptors to identify active domains and amino acids
responsible for binding HIV and to make CD-4 variants.
As used herein, a "target" is a substance which interacts with a parent
polypeptide. Targets include receptors for proteinaceous hormones,
substrates for enzymes, hormones for proteinaceous receptors, generally
any ligand for a proteinaceous binding protein and immune systems which
may be exposed to the polypeptides. Examples of hormone receptors include
the somatogenic and lactogenic receptors for hGH and the receptor for
hPRL. Other targets include antibodies, inhibitors of proteases, hormones
that bind to proteinaceous receptors and fibrin which binds to tissue
plasminogen activators (t-PA).
Generally, targets interact with parent polypeptides by contacting an
"active domain" on the parent polypeptide. Such active domains are
typically on the surface of the polypeptide or are brought to the surface
of the polypeptide by way of conformational change in tertiary structure.
The surface of a polypeptide is defined in terms of the native folded form
of the polypeptide which exists under relevant physiological conditions,
i.e. in vivo or under similar conditions when expressed in vitro. The
amino acid segments and amino acid residues may be ascertained in several
ways. If the three dimensional crystal structure is known to sufficient
resolution, the amino acid residues comprising the surface of the
polypeptide are those which are "surface accessible". Such surface
accessible residues include those which contact a theoretical water
molecule "rolled" over the surface of the three dimensional structure.
The active domain on the surface of the polypeptide may comprise a single
discrete segment of the primary amino acid sequence of the polypeptide. In
many instances, however, the active domain of a native folded form of a
polypeptide comprises two or more discontinuous amino acid segments in the
primary amino acid sequence of the parent polypeptide. For example, the
active domain for human growth hormone with the somatogenic receptor is
shown in FIG. 5. As indicated, domain A, C and F of the active domain are
each located on discontinuous amino acid segments of the hGH molecule.
These amino acid segments are identified in FIG. 4 by the letters A, C and
F. Discontinuous amino acid segments which form an active domain are
separated by a number of amino acid residues which are not significantly
involved in the interaction between the active domain and the target.
Typically, the separation between discontinuous amino acid segments is
usually at least about five amino acids.
The methods of the invention are directed to the detection of unknown
active domains in the amino acid sequence of a parent polypeptide. Except
for those few cases where a three dimensional crystal structure of a
polypeptide with its target are available, e.g. the crystal structure of
enzymes with inhibitors or transition state analogs, most active domains
for a vast array of polypeptides remain unknown.
As used herein an "analogous polypeptide segment" or "analogous segment"
refers to an amino acid sequence in an analog which is substituted for the
corresponding sequence in a parent polypeptide to form a "segment
substituted polypeptide". Analogous segments typically have a sequence
which results in the substitution, insertion or deletion of one or more
different amino acid residues in the parent polypeptide while maintaining
the relative amino acid sequence of the other residues in the selected
segment substituted in the parent. In general, analogous segments are
identified by aligning the overall amino acid sequence of the parent
polypeptide and analog to maximize sequence identity between them.
Analogous segments based on this sequence alignment are chosen for
substitution into the corresponding sequence of the parent polypeptide.
Similarly, analogous segments from analogs showing tertiary homology can
be deduced from those regions showing structural homology. Such analogous
segments are substituted for the corresponding sequences in the parent. In
addition, other regions in such tertiary homologs, e.g., regions flanking
the structurally analogous region, may be used as analogous segments.
The analogous segment should be selected, if possible, to avoid the
introduction of destabilizing amino acid residues into the
segment-substituted polypeptide. Such substitutions include those which
introduce bulkier side chains, and hydrophilic side chains in hydrophobic
core regions.
Typically, the amino acid sequence of the parent polypeptide and analog are
known and in some cases three-dimensional crystal structures may be
available. An alignment of the amino acid sequence of the parent
polypeptide with that of one or more analogs readily reveals conserved
amino acid residues in the sequences which should not be altered, at least
in the preliminary analysis. Sequence alignment will also reveal regions
of sequence variation which may include the substitution, insertion or
deletion of one or more amino acid residues. Those regions containing such
variations determine which segments in the parent may be substituted with
an analogous segment. The substitution of an analogous segment from an
analog may therefore result not only in the substitution of amino acid
residues but also in the insertion and/or deletion of amino acid residues.
If three-dimensional structural information is available, it is possible to
identify regions in the parent polypeptide which should not be subjected
to substitution with an analogous segment. Thus, for example, the
identification of a tightly packed region in a hydrophobic face of an
amphiphilic helix in the parent polypeptide should not be substituted with
an analogous segment. Residues identified as such should be retained in
the polypeptide variant and only surface residues substituted with
analogous residues.
Generally, analogous segments are 3 to 30 amino acid residues in length,
preferably about 3 to 15 and most preferably about 10 to 15 amino acid
residues in length. In some instances, the preferred length of the
analogous segment may be attenuated because of the insertion and/or
deletion of one or more amino acid residues in the analogous segment as
compared to the homolog or parent polypeptide. If a three-dimensional
structure is unavailable for the parent polypeptide, it is generally
necessary to form segment-substituted polypeptides with analogous segments
covering most, if not all, of the parent polypeptide. Segment-substitution
of the entire amino acid sequence, however, is not always necessary. For
example, fortuitous segment-substitutions covering only a portion of the
total amino acid sequence may provide sufficient information to identify
the active domain for a particular target. Thus, for example, the
segment-substitution of about 15% of the amino acid sequence of the parent
polypeptide may provide sufficient indication of the active domain. In
most instances, however, substantially more than about 15% of the amino
acid sequence will need to be segment-substituted to ascertain the active
domain. Generally, about 50%, preferably about 60%, more preferably about
75% and most preferably 100% of the amino acid sequence will be
segment-substituted if no structural information is available.
As used herein, "analogous amino acid residue" or "analogous residue"
refers to an amino acid residue in an analogous segment which is different
from the corresponding amino acid residue in the corresponding segment of
a parent polypeptide. Thus, if the substitution of an analogous segment
results in the substitution of one amino acid, that amino acid residue is
an analogous residue.
Once the parent polypeptide and one or more analogs are identified, the
analogous segments from one or more of the analogs are substituted for
selected segments in the parent polypeptide to produce a plurality of
segment-substituted polypeptides. Such substitution is conveniently
performed using recombinant DNA technology. In general, the DNA sequence
encoding the parent polypeptide is cloned and manipulated so that it may
be expressed in a convenient host. DNA encoding parent polypeptides can be
obtained from a genomic library, from cDNA derived from mRNA from cells
expressing the parent polypeptide or by synthetically constructing the DNA
sequence (Maniatis, T., et al. (1982) in Molecular Cloning, Cold Springs
Harbor Laboratory, New York).
The parent DNA is then inserted into an appropriate plasmid or vector which
is used to transform a host cell. Prokaryotes are preferred for cloning
and expressing DNA sequences to produce parent polypeptides, segment
substituted polypeptides, residue-substituted polypeptides and polypeptide
variants. For example, E. coli K12 strain 294 (ATCC No. 31446) may be used
as well as E. coli B, E. coli X1776 (ATCC No. 31537), and E. coli c600 and
c600hfl, E. coli W3110 (F-, .gamma.-, prototrophic, ATCC No. 27325),
bacilli such as Bacillus subtilis, and other enterobacteriaceae such as
Salmonella typhimurium or Serratia marcesans, and various pseudomonas
species. The preferred prokaryote is E. coli W3110 (ATCC 27325). When
expressed in prokaryotes the polypeptides typically contain an N-terminal
methionine or a formyl methionine, and are not glycosylated. These
examples are, of course, intended to be illustrative rather than limiting.
In addition to prokaryotes, eukaryotic organisms, such as yeast cultures,
or cells derived from multicellular organism may be used. In principle,
any such cell culture is workable. However, interest has been greatest in
vertebrate cells; and propagation of vertebrate cells in culture (tissue
culture) has become a repeatable procedure (Tissue Culture, Academic
Press, Kruse and Patterson, editors (1973)). Examples of such useful host
cell lines are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell
lines, W138, BHK, COS-7 and MDCK cell lines.
In general, plasmid vectors containing replication and control sequences
which are derived from species compatible with the host cell are used in
connection with these hosts. The vector ordinarily carries a replication
site, as well as sequences which encode proteins that are capable of
providing phenotypic selection in transformed cells. For example, E. coli
may be transformed using pBR322, a plasmid derived from an E. coli species
(Mandel, M. et al. (1970) J. Mol. Biol. 53, 154). Plasmid pBR322 contains
genes for ampicillin and tetracycline resistance and thus provides easy
means for selection. A preferred vector is pB0475. See Example 1. This
vector contains origins of replication for phage and E. coli which allow
it to be shuttled between such hosts thereby facilitating mutagenesis and
expression.
"Expression vector" refers to a DNA construct containing a DNA sequence
which is operably linked to a suitable control sequence capable of
effecting the expression of said DNA in a suitable host. Such control
sequences include a promoter to effect transcription, an optional operator
sequence to control such transcription, a sequence encoding suitable mRNA
ribosome binding sites, and sequences which control termination of
transcription and translation. The vector may be a plasmid, a phage
particle, or simply a potential genomic insert. Once transformed into a
suitable host, the vector may replicate and function independently of the
host genome, or may, in some instances, integrate into the genome itself.
In the present specification, "plasmid" and "vector" are sometimes used
interchangeably as the plasmid is the most commonly used form of vector at
present. However, the invention is intended to include such other forms of
expression vectors which serve equivalent functions and which are, or
become, known in the art.
"Operably linked" when describing the relationship between two DNA or
polypeptide regions simply means that they are functionally related to
each other. For example, a presequence is operably linked to a peptide if
it functions as a signal sequence, participating in the secretion of the
mature form of the protein most probably involving cleavage of the signal
sequence. A promoter is operably linked to a coding sequence if it
controls the transcription of the sequence; a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to permit
translation.
Once the parent polypeptide is cloned, site specific mutagenesis (Carter,
P., et al. (1986) Nucl. Acids Res. 13, 4331; Zoller, M. J., et al. (1982)
Nucl. Acids Res. 10, 6487), cassette mutagenesis (Wells, J. A., et al.
(1985) Gene 34, 315), restriction selection mutagenesis (Wells, J. A., et
al. (1986) Philos. Trans. R. Soc. London SerA 317, 415) or other known
techniques may be performed on the cloned parent DNA to produce
"segment-substituted DNA sequences" which encode for the changes in amino
acid sequence defined by the analogous segment being substituted. When
operably linked to an appropriate expression vector, segment-substituted
polypeptides are obtained. In some cases, recovery of the parent
polypeptide or segment-modified polypeptide may be facilitated by
expressing and secreting such molecules from the expression host by use of
an appropriate signal sequence operably linked to the DNA sequence
encoding the parent polypeptide or segment-modified polypeptide. Such
methods are well-known to those skilled in the art. Of course, other
methods may be employed to produce such polypeptides and
segment-substituted polypeptides such as the in vitro chemical synthesis
of the desired polypeptide (Barany, G., et al. (1979) in The Peptides
(eds. E. Gross and J. Meienhofer) Acad. Press, N.Y., Vol. 2, pp. 3-254).
Once the different segment-substituted polypeptides are produced, they are
contacted with a target for the parent polypeptide and the interaction, if
any, of the target and each of the segment-substituted polypeptides is
determined. These activities are compared to the activity of the parent
polypeptide with the same target. If the analog has a substantially
altered activity with the target as compared to the parent polypeptide,
those segment-substituted polypeptides which have altered activity with
the target presumptively contain analogous segments which define the
active domain in the parent polypeptide.
If the analog has an activity with the target which is greater than that of
the parent polypeptide, one or more of the segment-substituted
polypeptides may demonstrate an increased activity with that target
substance. Such a result would, in effect, identify an active domain in
the analog and an appropriate region in the parent polypeptide which can
be modified to enhance its activity with that target substance. Such an
event is most likely when the region in the analog responsible for the
target interaction is contained primarily within one continuous amino acid
segment. If the "active domains" of the analog comprise discontinuous
regions in the amino acid sequence of the analog, enhanced activity in the
segment-substituted polypeptide is less likely since the demonstration of
such enhanced activity may require the simultaneous introduction of all
active domains from the analog into the segment-substituted polypeptide.
Accordingly, it is preferred that the analog have an activity with the
target which is less than that for the parent polypeptide. In this manner,
a loss in activity is observed in the segment-substituted polypeptide.
However, once the active domains in a parent polypeptide are determined,
that polypeptide may be used as an analog to sequentially or
simultaneously substitute such active domains into a second parent
polypeptide which lacks activity with the target for the first parent
polypeptide.
Active domains in polypeptides are identified by comparing the activity of
the segment-substituted polypeptide with a target with the activity of the
parent polypeptide. Any number of analytical measurements may be used but
a convenient one for non-catalytic binding of target is the dissociation
constant Kd of the complex formed between the segment-substituted
polypeptide and target as compared to the Kd for the parent. An increase
or decrease in Kd of about 1.5 and preferably about 2.0 per analogous
residue-substituted by the segment-substitution indicates that the segment
substituted is an active domain in the interaction of the parent
polypeptide with the target.
In the case of catalytic interaction with a target, a suitable parameter to
measure activity relative to the parent enzyme is to compare the ratios of
kcat/Km. An increase or decrease in kcat/Km relative to the parent enzyme
of about 1.5 and preferably 2.0 per analogous residue-substituted
indicates that an active domain has been substituted.
As used herein, a "scanning amino acid" is an amino acid used to identify
active amino acids within a parent polypeptide. A "residue-substituted
polypeptide" is a polypeptide variant containing at least a single
substitution of an amino acid in the parent polypeptide with a scanning
amino acid. A "residue-substituted DNA sequence" encodes a residue
substituted polypeptide. Such DNA and polypeptide sequences may be
prepared as described for the preparation of segment-substituted DNA and
polypeptides.
The "active amino acid residue" identified by the amino acid scan is
typically one that contacts the target directly. However, active amino
acids may also indirectly contact the target through salt bridges formed
with other residues or small molecules such as H.sub.2 O or ionic species
such as Na.sup.+, Ca.sup.+2, Mg.sup.+2 or Zn.sup.+2.
In some cases, the scanning amino acid is substituted for an amino acid
identified in an active domain of the parent polypeptide. Typically, a
plurality of residue-substituted polypeptides are prepared, each
containing the single substitution of the scanning amino acid at a
different amino acid residue within the active domain. The activities of
such residue-substituted polypeptides with a particular target substance
are compared to the activity of the parent polypeptide to determine which
of the amino acid residues in the active domain are involved in the
interaction with the target substance. The scanning amino acid used in
such an analysis may be any different amino acid from that substituted,
i.e., any of the 19 other naturally occurring amino acids.
TABLE II
______________________________________
Isosteric
Polypeptide Scanning
Amino Acid Amino Acid
______________________________________
Ala Ser, Gly
Glu Gln, Asp
Gln Asn, Glu
Asp Asn, Glu
Asn Aln, Asp
Leu Met, Ile
Gly Pro, Ala
Lys Met, Arg
Ser Thr, Ala
Val Ile, Thr
Arg Lys, Met, Asn
Thr Ser, Val
Pro Gly
Ile Met, Leu, Val
Met Ile, Leu
Phe Tyr
Tyr Phe
Cys Ser, Ala
Trp Phe
His Asn, Gln
______________________________________
This table uses the following symbols for each amino acid:
______________________________________
Amino Acid
or residue 3-letter
1-letter
thereof symbol symbol
______________________________________
Alanine Ala A
Glutamate Glu E
Glutamine Gln Q
Aspartate Asp D
Asparagine Asn N
Leucine Leu L
Glycine Gly G
Lysine Lys K
Serine Ser S
Valine Val V
Arginine Arg R
Threonine Thr T
Proline Pro P
Isoleucine Ile I
Methionine Met M
Phenylalanine Phe F
Tyrosine Tyr Y
Cysteine Cys C
Tryptophan Trp W
Histidine His H
______________________________________
Most preferably, the scanning amino acid is the same for each residue
substituted polypeptide so that the effect, if any, on the activity of the
residue-substituted polypeptides can be systematically attributed to the
change from the naturally occurring amino acid residue to a uniform
scanning amino acid residue.
In some cases, the substitution of a scanning amino acid at one or more
residues results in a residue-substituted polypeptide which is not
expressed at levels which allow for the isolation of quantities sufficient
to carry out analysis of its activity with a target. In such cases, a
different scanning amino acid, preferably an isosteric amino acid, can be
used.
The most preferred scanning amino acids are relatively small, neutral amino
acids. Such amino acids include alanine, glycine, serine and cysteine.
Alanine is the preferred scanning amino acid among this group because it
eliminates the side-chain beyond the bet.alpha.-carbon and is less likely
to alter the main-chain conformation of the residue-substituted
polypeptide. Alanine is also preferred because it is the most common amino
acid. Further, it is frequently found in both buried and exposed positions
(Creighton, T. E., in The Proteins (eds. W. H. Freeman & Co., New York);
Chothia, C. (1976) J. Mol. Biol. 150, 1). If alanine substitution does not
yield adequate amounts of residue-substituted polypeptide, an isosteric
amino acid can be used. Alternatively, the following amino acids in
decreasing order of preference may be used: Ser, Asn and Leu.
The use of scanning amino acids is not limited to the identification of
active amino acids in an active domain ascertained by the analysis of
segment-substituted polypeptides. If, for example, one or more amino acids
in a parent polypeptide are known or suspected to be involved in the
interaction with a target, scanning amino acid analysis may be used to
probe that residue and the amino acid residues surrounding it. Moreover,
if the parent polypeptide is a small peptide, e.g., about 3 to 50 amino
acid residues, scanning amino acid analysis may be carried out over the
entire molecule.
Once the active amino acid residues are identified, isosteric amino acids
may be substituted. Such isosteric substitutions need not occur in all
instances and may be performed before any active amino acid is identified.
Such isosteric amino acid substitution is performed to minimize the
potential disruptive effects on conformation that some substitutions can
cause. Isosteric amino acids are shown in Table II.
Active amino acid residues can be identified by determining the activity of
the residue-substituted polypeptide with a target as compared to the
parent. In general, a two-fold increase or decrease in Kd indicates that
the residue substituted is active in the interaction with the target.
Similarly, in the case of catalytic interaction with a target, a two-fold
increase or decrease in kcat/Km relative to the parent enzyme indicates
that an active residue has been substituted.
When a suspected or known active amino acid residue is subjected to
scanning amino acid analysis the amino acid residues immediately adjacent
thereto should be scanned. Three residue-substituted polypeptides are
made. One contains a scanning amino acid, preferably alanine, at position
N which is the suspected or known active amino acid. The two others
contain the scanning amino acid at position N+1 and N-1. If each
substituted polypeptide causes a greater than about two-fold effect on Kd
or kcat/Km for a target, the scanning amino acid is substituted at
position N+2 and N-2. This is repeated until at least one and preferably
four residues are identified in each direction which have less than about
a two-fold effect on Kd or kcat/Km or either of the ends of the parent
polypeptide are reached. In this manner, one or more amino acids along a
continuous amino acid sequence which are involved in the interaction with
a particular target can be identified.
The methods of the invention may be used to detect the active domain for
more than one target of a particular parent polypeptide. Further, active
amino acid residues within the different active domains may be also
identified by the methods herein. Once two or more active domains and
active amino acid residues are identified for the different targets of a
particular polypeptide, various modifications to the parent polypeptide
may be made to modify the interaction between the parent polypeptide and
one or more of the targets. For example, two active domains on the surface
of hGH have been identified for the somatogenic and prolactin receptor. In
this particular case, the active domains overlap. Accordingly, there are a
number of common active amino acid residues which interact with the
somatogenic and prolactin receptors. Various modifications to hGH may be
made based on this information as described in more detail hereinafter.
In some instances, the active domain for different targets will not
overlap. In such situations, the active amino acids in the parent
polypeptide for one receptor can be substituted with different amino acids
to reduce or enhance the interaction of that active domain with its
target, thus shifting the physiological effect of such a variant.
As used herein, the term "modified interaction" refers to a polypeptide
variant wherein one or more active domains have been modified to change
the interaction of the variant with a target as compared to the parent
polypeptide. A modified interaction is defined as at least a two-fold
increase or decrease in the interaction of the polypeptide variant as
compared to the interaction between the parent polypeptide and a
particular target.
The interaction between a target and a parent polypeptide, polypeptide
variant, segment-substituted polypeptide and/or residue-substituted
polypeptide can be measured by any convenient in vitro or in vivo assay.
Thus, in vitro assays may be used to determine any detectable interaction
between a target and polypeptide, e.g. between enzyme and substrate,
between hormone and hormone receptor, between antibody and antigen, etc.
Such detection may include the measurement of colorimetric changes,
changes in radioactivity, changes in solubility, changes in molecular
weight as measured by gel electrophoresis and/or gel exclusion methods,
etc. In vivo assays include, but are not limited to, assays to detect
physiological effects, e.g. weight gain, change in electrolyte balance,
change in blood clotting time, changes in clot dissolution and the
induction of antigenic response. Generally, any in vivo assay may be used
so long as a variable parameter exists so as to detect a change in the
interaction between the target and the polypeptide of interest.
Exemplary of the present invention is a preferred embodiment wherein the
active domains and active amino acids of human growth hormone which
determine its activity with its somatogenic receptor are identified. In
carrying out this embodiment of the invention, human growth hormone
variants, including segment-substituted and residue-substituted hGH
variants, have been made or identified which have different binding
interactions with the somatogenic receptor for growth hormone as compared
to naturally occurring human growth hormone. At least one of these human
growth hormone variants has a higher affinity for the somatogenic receptor
and enhanced potency for somatogenesis in rats. Others have a decreased
activity with the somatogenic receptor. Such hGH variants are useful as
hGH agonists or antagonists and may have a higher potency for stimulating
other receptors for human growth hormone since such variants will be freed
from substantial interaction with the somatogenic receptor. Further, such
variants are useful in immunoassays for hGH as an hGH standard or tracer.
In one instance, a variant has been identified which has a significant
decrease in reactivity with human and mouse serum containing anti-hGH
polyclonal antibodies. Another has the same binding affinity for the
somatogenic receptor as hGH but increased potency to stimulate growth.
The method for determining the active domains for human growth hormone
which interact with its somatogenic receptor from liver is shown
schematically in FIG. 1. In this approach, segments of hGH were
systematically replaced with analogous sequences from analogs of hGH that
are known to have greatly reduced affinities for the cloned hGH liver
receptor and for monoclonal antibodies raised against hGH. Such analogs
for hGH include human placental lactogen (hPL), porcine growth hormone
(pGH) and human prolactin (hPRL). These analogs have binding affinities
for the cloned hGH receptor that are reduced by about 100 to 10,000-fold
for the somatogenic hGH receptor (hGHr) (Harrington, A. C., et al. (1986)
J. Clin. Invest. 77, 1817; Baumann, G., et al. (1986) J. Clin. Endocrinol.
Metab. 62, 137. Such analogs are used because homologous proteins are
known to have similar three-dimensional structures even though they may
have a large sequence divergence (Chothia, C., et al. (1986) EMBO J. 5,
823). In so doing, the likelihood is increased that analogous sequence
substitutions will be readily accommodated without grossly disrupting the
native folding of the molecule. The amino acid sequence for human growth
hormone and the analogs hPL, pGH and hPRL are shown in FIG. 2. These
latter three analogs share a sequence identity with hGH at the level of
85%, 68% and 23%, respectively.
Referring to FIG. 1, the overall strategy is shown for identifying one or
more active domains in human growth hormone which interact with the
somatogenic receptor for human growth hormone (a "target" for hGH). As
indicated, hGH has a positive binding activity with the target receptor,
in this case, the somatogenic receptor. The hPRL, hPL and pGH analogs,
however, have a greatly reduced activity with that target as indicated by
the minus sign. Six segment-substituted growth hormones, identified by
letters A through F, are formed by substituting a selected amino acid
segment of hGH with an analogous amino acid segment from the hPRL analog.
Each of these selected segments are different and are chosen to probe
either the entire amino acid sequence of the hGH molecule or those regions
which are expected to contain the active domains. After the
segment-substituted human growth hormones are prepared each is assayed
against the hGH somatogenic receptor to determine its activity. The
results of such an assay as compared to hGH are indicated by + or - under
the segment-modified human growth hormones in FIG. 1. As can be seen in
FIG. 1, segment-substituted human growth hormones C and F in this
schematic do not bind the somatogenic receptor. Based on these results,
those regions human growth hormone corresponding to the analogous segments
from the analog in the growth hormone variants C and F are identified as
active domains involved in the binding of hGH to its somatogenic receptor.
As indicated, it is not necessary to probe the entire amino acid sequence
of human growth hormone or other parental polypeptides if structural
information or other data are available. Thus, low-resolution or
high-resolution structural information from crystallographic studies can
provide important information so as to avoid destabilizing substitutions
of selected amino acid segments from a homolog. For example, the X-ray
coordinates for human growth hormone are not available. However, helix
wheel projections from the pGH folding model, based on the low resolution
X-ray crystal structure of pGH, reveal that three of the four helices
(helix 1, 3 and 4) are amphipathic with strong hydrophobic moments. See
FIG. 3. Eisenberg, D., et al. (1984) J. Mol. Biol. 179, 125. Since the
hydrophobic core in polypeptides is very tightly packed (Ponder, J. W., et
al. (1987) J. Mol. Biol. 193, 775), changes in such buried amino acid
residues are generally destabilizing (Alber, T., et al. (1987) Biol. Chem.
26, 3754; Reidhaar-Olson, J. F. (1988) Science 241, 53).
In addition, regions of high amino acid sequence conservation amongst
members of the polypeptide family, for example the human growth hormone
family, in general, need not be probed, at least initially. This is
because the disruption of such conserved sequences is likely to disrupt
the folding of the molecule. Further, other data may suggest that certain
regions of the parent polypeptide are not involved in the interaction with
a particular target substance. For example, deletion of the N-terminal 13
amino acids of hGH by mutagenesis (Ashkenazi, A., et al. (1987)
Endocrinology 1, 414) and a natural variant of hGH which deletes residues
32 to 46 (the 20, Kd variant; Lewis, U. J., et al. (1980) Biochem.
Biophys. Res. Commun. 92, 5111) have been reported not to affect
dramatically the binding to the somatogenic receptor. In addition, the
production of a two-chain derivative of hGH by limited proteolysis, which
deletes some or all of the residues between 134 and 149, does not markedly
affect binding to the somatogenic receptor. Li, C. H. (1982) Mol. Cell.
Biochem. 46, 31; Mills, J. B., et al. (1980) Endocrinology 107, 391.
Based on this information, six segments of the amino acid sequence of hGH
were selected for substitution with the corresponding analogous amino acid
segments from a number of analogs to hGH. These selected segments are
identified as A through F in FIG. 2. These segments are separated either
by disulfide bonds, by borders of secondary structure (see FIG. 4), by
areas of high sequence conservation in the growth hormone family or by
regions previously identified as not being involved in binding to the
somatogenic receptor. Seventeen segment-substituted hGH variants were
prepared which collectively substituted 85 out of the 191 residues in hGH.
The regions identified as A through F in FIG. 2 and the
segment-substituted hGH variants prepared within each region are
summarized in Table III.
TABLE III
__________________________________________________________________________
Segment-
Actual K.sub.d
Region
Substituted
Substitution
Mutagenesis
(variant)
probed
hGH Variant
Introduced
method
K.sub.d (nM)
K.sub.d (wt)
__________________________________________________________________________
hGH None 0.34
1.0
A 11-33
hPL (12-25)
N12H, F25L
r.s..sup.1 /
1.4 4.1
pGH (11-33)
D11A, M14V, H18Q
cassette.sup.2 /
1.2 3.4
R19H, F25A, Q29K,
E33R
hPRL (12-33)
N12R, M14V, L15V,
cassette
3.6 11
R16L, R19Y, F25S,
D26E, Q29S, E30Q,
E33K
hPRL (12-19)
N12R, M14V, L15V,
r.s. 5.8 17
R16L, R19Y
hPRL (22-33)
Q22N, F25S, D26E,
r.s. 0.29
0.85
Q29S, E30Q, E33k
B 46-52
hPL (46-52)
Q46H, N47D, P48S,
r.s. 2.5 7.2
Q49E, L52F
pGH (48-52)
P48A, T50A, S51A,
r.s. 0.94
2.8
L52F
C 54-74
hPL (56-64)
E56D, R64M
cassette
10 30
pGH (57-73)
S57T, T60A, S62T,
cassette
5.8 17
N63G, R64K, E65D,
T67A, K70R, N72D,
L73V
hPRL (54-74)
F54H, S55T, E56S,
cassette
23 69
I58L, P59A, S62E,
N63D, R64K, E66Q,
T67A, K70M, S71N,
N72Q, L73K, E74D
D 88-104
hPRL (88-95)
E88G, Q91Y, F92H,
r.s. 0.47
1.4
R94T, S95E
hPRL (97-104)
F97R, A98G, N99M,
r.s. 0.47
1.6
S100Q, L101D,
V102A, Y103P,
G104E
E 108-136
hPL (109-112)
N109D, V110D,
cassette
0.61
1.8
D112H
hPRL (111-129)
Y111V, L113I,
cassette
0.52
1.5
K115E, D116Q,
E118K, E119R,
G120L, Q122E,
T123G, G126L,
R127I, E129S
hPRL (126-136)
R127D, L128V,
cassette
0.58
1.7
E129H, D130P,
G131E, S132T,
P133K, R134E,
T135N
F 164-190
pGH (164-190)
Y164S, R167K,
hybrid.sup.3 /
>34 >100
M170L, D171H,
V173A, F176Y,
I179V, V180M,
Q181K, S184R,
I184F, G187S,
G190A
pGH (167-181)
R167K, D171H,
r.s. 9.2 27
I179V, Q181K
__________________________________________________________________________
.sup.1 /Restriction selection Wells, J. A., et al. (1986) Philos. Trans.
R. Soc. London SerA 317, 415.
.sup.2 /Cassette mutagenesis Wells, J. A., et al. (1985) Gene 34, 315.
.sup.3 /Recombination mutagenesis Gray, G. L., et al. (1986) J.
Bacteriol. 166, 635.
The segment-substituted hGH variants are generally identified by the
analogous segments substituted into the human growth hormone sequence.
However, in some instances, not all of the analogous residues in the
substituted analogous segment were maintained in a particular
construction. Thus, in Table III hPL (12-25) identifies a
segment-substituted hGH variant wherein amino acids 12 through 25 of human
placental lactogen (hPL) are substituted for amino acid residues 12
through 25 in the parent hGH. The effect of substituting this analogous
segment can be determined by comparing the amino acid sequence of hGH and
hPL in this region in FIG. 2. Four amino acid substitutions are generated
in an hPL (12-25) variant where no other changes are made. These residues
are 12, 16, 20 and 25 for hPL (12-25).
The actual amino acid substitutions in the hPL (12-25) variant and the
other segment-substituted variants are shown in Table III. Each
substitution is represented by a letter followed by a number which is
followed by a letter. The first letter and number correspond to the amino
acid at that residue number in the unmodified hGH. The last letter
corresponds to the amino acid which is substituted at that position. Thus,
N12, H indicates that the asparagine at position 12 in hGH is substituted
by histidine in the hPL (12-25) variant.
As can be seen, some of the actual substitutions introduced do not
correspond to the totality of substitutions indicated by the corresponding
segments in FIG. 2. Thus, hPL (12-25) would contain the four substitutions
N12H, R16Q, L20A and F25L if the entire hPL (12-25) segment were
substituted. The actual variant made, however, maintained R16 and L20 and
therefore incorporated only two of the four substitutions, i.e., N12H and
F25L, as shown in Table III. Other segment substituted variants which
maintained one or more resudues of the parent hGH include those covering
regions A and E and the segment substituted variants hPL (46-52) and pGH
(167-181).
Each of the segment-substituted human growth hormone variants were assayed
in an in vitro system comprising displacement of ›.sup.125 I!hGH from the
extracellular portion of the cloned soluble hGH receptor to quantify the
relative affinities of the segment-substituted variants to the
extracellular domain of the somatogenic receptor. Leung, D. W., et al.
(1987) Nature 330, 537. This truncated form of the somatogenic receptor
exhibits the same selectivity for hGH as the membrane form of the receptor
(Spencer, S. A., et al. (1988) J. Biol. Chem. 263, 7862) albeit with about
a slight reduction in binding affinity (K.sub.d= 0.3 nM).
As will be described in more detail in the examples, selected segments A, C
and F, comprising residues 11-19, 54-74 and 164-191, respectively, are
active domains in the hGH molecule interactive with the somatogenic
receptor. This is based on the observed decrease in Kd of ten-fold or
greater for most of the segment-substituted hGH variants containing
analogous segments for hGH analogs over these regions. See FIG. 4. Of
course, this does not mean that each of the amino acid residues within
these active domains comprise the binding residues for the somatogenic
receptor. Rather, such domains define the amino acid sequence within which
such active residues can be found.
The active domains A, C and F were further localized. For example, the
variant hPRL (12-33) was dissected into the amino and carboxy terminal
variants, hPRL (12-19) and hPRL (22-33). The results from this experiment
further localized this active domain of hGH to residues 12 through 19.
Similarly, the amino terminal portion of region F (pGH (167-181)) exhibits
a large reduction in binding affinity. One of the most dramatic effects
was the 30-fold reduction in binding caused by hPL (56-64) which
introduced only two mutations, E56D and R64M. Although regions A, C and F
are widely separated in the primary sequence of hGH, the tertiary folding
of the hormone brings them within close proximity. See FIG. 5. These
active domains form a patch that contains the amino terminus of helix 1
(active domain A), the loop from Cys-53 to the start of helix 2 (active
domain C) and the central portion of helix 4 (active domain F).
In addition, eight Mabs against hGH were assayed against
segment-substituted hGH variants to map the epitopes of hGH. Further, the
Mab's were used in a competitive assay with hGH and hGH variants to
evaluate the ability of each of the Mabs to block the binding of the hGH
receptor to hGH.
The collective results obtained from these experiments provide several
lines of evidence that the substitution of analogous segments into hGH do
not grossly disrupt the native folding of the molecule and that the
observed activity is due to a direct effect on the interaction between the
somatogenic receptor and the segment-substituted hGH variants. Firstly,
the segment-substituted variants are highly selective in disrupting
binding to the somatogenic receptor or the Mabs. Secondly, the somatogenic
receptor and Mabs recognize conformation as well as sequence. The receptor
and at least four of the Mabs recognize discontinuous epitopes that are
sensitive to the protein tertiary structure. Thirdly, circular dichroic
spectra of all of the purified variants are virtually identical to
wild-type hGH. Fourthly, all of the variants, with the exception of pGH
(164-190), were expressed in essentially wild-type amounts. Resistance to
proteolysis in vivo has been used as a screen for conformational
integrity. Hecht, M. H., et al. (1984) Proc. Natl. Acad. Sci. USA 81,
5685; Shortle, D., et al. (1985) Genetics 110, 539.
The alteration in binding activity for segment-substituted hGH variants
does not necessarily indicate that the substituted residues in such
variants make direct contact with the somatogenic receptor. A disruptive
mutation may not only remove a favorable interaction but may introduce an
unfavorable one. For example, the N12R mutation in the hPRL (12-19)
segment-substituted hGH variant not only changes the hydrogen bonding
amide function of Asn12, the Arg substitution also introduces a bulkier
side chain that is positively charged. Furthermore, a number of the
binding contacts may be conserved between the analogs so that not all
contacts, or even regions, may be probed by generating segment-substituted
hGH variants. Further, the substitution of analogous segments generates
the substitution of multiple amino acid residues in the hGH molecule.
In order to identify the specific active amino acids within the active
domains A, C and F in FIG. 2, a fine structure analysis of these active
domains was performed. In this analysis, residues in these three active
domains were replaced sequentially with alanine. A total of 63 single
Alanine mutants were made and each of their binding constants were
determined for the soluble hGH receptor (shGHr) by Scatchard analysis.
Leung, D. W., et al. (1988) J.
Biol. Chem. 263, 7862.
Based on this analysis, the amino acid residues listed in Table IV comprise
residues within the hGH molecule which are actively involved in the
interaction with the somatogenic receptor. This is based on the more than
four-fold effect on the relative dissociation constant caused by the
substitution of alanine for these residues as compared to wt hGH. See FIG.
7. Preferred amino acid substitutions for these residues to form hGH
variants are shown.
TABLE IV
______________________________________
Preferred amino
hGH Residue acid substitution
______________________________________
F10 GEMARQSYWLIV
F54 GEMARQSYWLIV
E56 GMFARQSDNKLH
I58 GEMFARQSVT
R64 GEMFAQSH,KDN
Q68 GEMFARSHKDN
D171 GEMFARQSHKN
K172 GEMFARQSHDN
E174 GMFARQSHDNKL
T175 GEMFARQSVI
F176 GEMARQSYWLIV
R178 GEMFAQSHKDN
C182 GEMFARQS
V185 GEMFARQSITLYW
______________________________________
Other amino acid residues which are less active with the somatogenic
receptor are listed in Table V.
These residues demonstrate generally less than two-fold increase in
relative Kd when substituted with alanine.
TABLE V
______________________________________
I4 N12 S55 E66 Q181
P5 M14 S57 K70 R183
L6 L15 P59 S71 G187
S7 R16 S62 K168
R8 R19 N63 I179
______________________________________
Amino acid residues in hGH showing a relative decrease in Kd when
substituted with alanine (and consequently greater affinity for the
somatogenic receptor) are listed in Table VI.
TABLE VI
______________________________________
P2 E65 S184
T3 Q69 E186
L10 L73 S188
H18 R167 F191
R64 E174
______________________________________
One residue substituted hGH variant, E174A, surprisingly resulted in a
significant decrease (almost five-fold) in the dissociation constant with
the somatogenic receptor. This variant, in addition to showing an
increased binding affinity for the somatogenic receptor also exhibited an
increased somatogenic potency relative to hGH in a rat weight gain assay.
This and other specific residue substitutes that enhance somatogenic
binding by >1.4 fold are presented in Table VII.
TABLE VII
______________________________________
hGH variants having enhanced somatogenic binding
Substituted
hGH residues amino acid
______________________________________
H18 A
R64 K
E65 A
L73 A
E174 A,N,Q,S,G
E186 A
S188 A
F191 A
______________________________________
Other variants containing alanine substitutions not shown in FIG. 7 are
listed in Table VIII.
TABLE VIII
______________________________________
Variant K.sub.d (mM)
K.sub.d (var/K.sub.d (wt)
______________________________________
H21A NE --
K172A/F176A 201 543
N47A 0.84 2.3
P48A NE --
Q49A 0.36 1.0
T50A 0.38 1.0
S51A
Q46A NE --
V173A NE --
______________________________________
Note:
Ne--not expressed in shake flasks at levels which could be easily isolate
(i.e., <.about.5% of wildtype expression levels).
Once identified, the active amino acid residues for the somatogenic
receptor in hGH are analyzed by substituting different amino acids for
such residues other than the scanning amino acid used for the preliminary
analysis. The residue substituted variants in Table IX have been made.
TABLE IX
______________________________________
Variant K.sub.d (nM)
K.sub.d (var)/K.sub.d (wt)
______________________________________
R77V 0.44 1.3
L80D 0.78 2.3
F176Y 3.2 8.6
E174G 0.15 0.43
E174D NE --
E174H 0.43 1.2
E174K 1.14 3.1
E174L 2.36 6.4
E174N 0.26 0.7
E174Q 0.21 0.6
E174S 0.11 0.3
E174V 0.28 0.8
E174R NE --
R64K 0.21 0.6
E65K NE --
E65H NE --
K172R NE --
I58L NE --
F25S NE --
D26E NE --
Q29S NE --
E30Q NE --
R178K NE --
R178T NE --
R178Q NE --
I179M NE --
D169N 3.6 10.5
______________________________________
Ne--not expressed in shake flasks at levels which could be easily isolate
(i.e., <.about.5% of wildtype expression levels).
In addition to the hGH variants that have been made, Table X identifies
specific amino acid residues in hGH and replacement amino acids which are
expected to produce variants having altered biological functions.
TABLE X
______________________________________
wT hGH amino
acid residue Replacement amino acid
______________________________________
S43 GEMFARQHDKN
F44 GEMARQSYWLIV
H18 GEMFARQSKDNY
E65 GEMFARQSHDNKL
L73 GEMFARQSIVY
E186 GMFARQSHDNKL
S188 GEMFARQHDNKY
F191 GEMARQSYWLIV
F97 GEMARQSYWLIV
A98 GEMFRQSDNHK
N99 GEMFARQSDKY
S100 GEMFARQHDNKY
L101 GEMFARQSIVY
V102 GEMFARQSITLYW
Y103 GEMFARQSWLIV
G104 EMFARQSP
R19 GEMFAQSHKND
Q22 GEMFARSKKDN
D26 GEMFARQSHKN
Q29 GEMFARSKKDN
E30 GMFARQSHDNKL
E33 GMFARQSHDNKL
______________________________________
In another embodiment, the binding epitope of hGH for the prolactin
receptor was determined. hGH can bind to either the growth hormone or
prolactin (pRL) receptor. As will be shown herein, these receptors compete
with one another for binding to hGH suggesting that their binding sites
overlap. Scanning mutagenesis data show that the epitope of hGH for the
hPRL receptor consists of determinants in the middle of helix 1
(comprising residues Phe25 and Asp26), a loop region (including Ile58 and
Arg64) and the center portion of helix 4 (containing residues K168, K172,
E174, and F176). These residues form a patch when mapped upon a structural
model of hGH. This binding patch overlaps but is not identical to that
determined for the hGH receptor as disclosed herein and by B. C.
Cunningham and J. A. Wells (1989) Science 244, 1081-1085. By mutating the
non-overlap regions of these receptor binding sites on hGH, the preference
of hGH was shifted toward the hGH receptor by >2000-fold or toward the
hPRL receptor by >20-fold without loss in binding affinity for the
preferred receptor. Similarly, by mutating the overlap regions it is
possible to reduce binding to both receptors simultaneously by >500-fold.
Such receptor-selective variants of hGH should be useful molecular probes
to link specific receptor binding events to the various biological
activities of hGH such as linear growth or lactation.
In a further embodiment, the receptor-binding determinants from human
growth hormone (hGH) were placed into the normally nonbinding homolog,
human prolactin (hPRL). The alanine scanning mutagenesis disclosed herein
and Cunningham, B. C. & Wells, J. A. (1989) Science 244, 1081-1085
identified important residues in hGH for modulating binding to the hGH
receptor cloned from human liver. Additional mutations derived from hPRL
were introduced into hGH to determine which hPRL substitutions within the
hGH receptor binding site were most disruptive to binding. Thereafter, the
cDNA for hPRL was cloned and expressed in Escherichia coli. It was then
mutated to sequentially introduce those substitutions from hGH that were
predicted to be most critical for receptor binding. After seven iterative
rounds of site-specific mutagenesis, a variant of hPRL containing eight
mutations whose association constant was strengthened over 10,000-fold for
the hGH receptor was identified. This hPRL variant binds only six-fold
weaker than wild-type hGH while sharing only 26% overall sequence identity
with hGH. These results show the structural similarity between hGH and
hPRL, and confirm the identity of the hGH receptor epitope. More
generally, these studies demonstrate the feasibility to borrow receptor
binding properties from distantly related and functionally divergent
hormones that may prove useful for the design of hybrid hormones with new
properties as agonist or antagonist.
The following is presented by way of example and is not to be construed as
a limitation to the scope of the invention.
EXAMPLE 1
hGH Mutagenesis and Expression Vector
To facilitate efficient mutagenesis, a synthetic hGH gene was made that had
18 unique restriction sites evenly distributed without altering the hGH
coding sequence. The synthetic hGH DNA sequence was assembled by ligation
of seven synthetic DNA cassettes each roughly 60 base pairs (bp) long and
sharing a 10 bp overlap with neighboring cassettes to produce the 405 bp
DNA fragment shown from NsiI to BglII. The ligated fragment was purified
and excised from a polyacrylamide gel and cloned into a similarly cut
recipient vector, pB0475, which contains the alkaline phosphatase promoter
and StII signal sequence (Chang, C. N., et al. (1987) Gene 55, 189), the
origin of replication for the phage f1 and pBR322 from bp 1205 through
4361 containing the plasmid origin of replication and the .beta. lactamase
gene. The sequence was confirmed by dideoxy sequence analysis (Sanger, F.,
et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463).
pB0475 was constructed as shown in FIG. 9. fI origin DNA from filamentous
phage contained on a DraI, RsaI fragment 475bp in length was cloned into
the unique PvuII site of pBR322 to make plasmid p652. Most of the
tetracycline resistance gene was then deleted by restricting p652 with
NheI and NarI, filling the cohesive ends in with DNA polymerase and dNTPs
and ligating the large 3850bp fragment back upon itself to create the
plasmid p.DELTA.652. p.DELTA.652 was restricted with EcoRI, EcoRV and the
3690bp fragment was ligated to a 1300bp EcoRI, EcoRV fragment from phGH4R
(Chang, C. N., et al. (1987) Gene 55, 189) containing the alkaline
phosphatase promoter, STII signal sequence and natural hGH gene. This
construction is designated as pBo473. Synthetically derived DNA was cloned
into pB0473 in a three-way construction. The vector pB0473 was restricted
with NsiI, BglII and ligated to a 240pb NsiI, HindIII fragment and a
1170bp HindII, BglII fragment both derived from synthetic DNA. The
resulting construction pB0475 contains DNA coding for the natural
polypeptide sequence of hGH but possesses many new unique restriction
sites to facilitate mutagenesis and further manipulation of the hGH gene.
The entire DNA sequence of pB0475 together with the hGH amino acid
sequence is shown in FIG. 10. The unique restriction sites in the hGH
sequence in pB0475 allowed insertion of mutagenic cassettes (Wells, J. A.,
et al. (1985) Gene 34, 315) containing DNA sequences encoding analogous
segments from the analogs pGH, hPL and hPRL. Alternatively, the hGH
sequence was modified by site specific mutagenesis in the single-stranded
pB0475 vector followed by restriction-selection against one of the unique
restriction sites (Wells, J. A., et al. (1986) Philos. Trans. R. Soc.
London SerA 317, 415).
The 17 segment-substituted hGH variants in Table III were prepared. Each
was secreted into the periplasmic space of E. coli at levels comparable to
wild-type hGH and at levels that far exceeded the hGH-pGH hybrid described
infra. The hGH and hGH variants were expressed in E. coli W3110, tonA
(ATCC 27325) grown in low phosphate minimal media (Chang, C. N., et al.
(1987) Gene 55, 189).
The hGH and hGH variants were purified as follows. To 200g of cell paste
four volumes (800 ml) of 10 mM TRIS HCl Tris(hydroxymethyl)aminomethane
hydrochloride pH 8.0 was added. The mixture was placed on an orbital
shaker at room temperature until the pellets were thawed. The mixture was
homogenized and stirred for an hour in a cold room. The mixture was
centrifuged at 7000 g for 15 min. The supernatant was decanted and
ammonium sulfate was added to 45% saturation (277 g/1) and stirred at room
temperature for one hour. After centrifugation for 30 minutes at 11,000 g,
the pellet was resuspended in 40ml 10 mM Tris HCl
(hydroxymethyl)aminomethane hydrochloride pH 8.0. This was dialyzed
against 2 liters of 10 mM Tris HCl (hydroxymethyl)aminomethane
hydrochloride pH 8.0 overnight. The sample was centrifuged or filtered
over a 0.45 micron membrane. The sample was then loaded on a column
containing 100 ml of DEAE cellulose (Fast Flow, Pharmacia, Inc.). A
gradient of from zero to 300 mM NaCl in 10 mM Tris HCl
(hydroxymethyl)aminomethane hydrochloride 8.0 in 8 to 10 column volumes
was passed through the column. Fractions containing hGH were identified by
PAGE, pooled, dialyzed against 10 mM TRIS HCl
Tris(hydroxymethyl)aminomethane hydrochloride pH 8.0 overnight. Samples
were concentrated to approximately mg/ml by Centri-Prep10 ultrafiltration.
EXAMPLE 2
Homologous Recombinants of hGH and pGH
A random hybrid library containing various N-terminal lengths of hGH linked
to the remaining C-terminal portion of porcine growth hormone (pGH) was
constructed by the method of random recombination of tandomly linked
genes. Gray, G. L., et al. (1986) J. Bacteriol. 166, 635.
The EcoRI site of pB0475 was removed by restricting the plasmid with EcoRI,
filling in the cohesive ends by addition of DNA polymerase and dNTPs, and
ligating the plasmid back together. A new EcoRI site was then introduced
just following the 3' end of the hGH gene. This was accomplished by
subcloning the 345bp BglII, EcoRV fragment of hGH-4R which contains such
an EcoRI site, into a similarly restricted vector from the EcoRI.sup.-
pB0475 construction. The pGH gene (Seeburg, P. H., et al. (1983) DNA 2,
37) was then introduced just downstream and adjacent to the 3' end of the
hGH gene in this construction. This was accomplished by doping an EcoRI,
HindIII (filled in) fragment containing pGH cDNA into the large fragment
of a EcoRI, EcoRV digest of the construction described above. The
resulting plasmid, pB0509, contains an intact hGH gene with a unique EcoRI
site at its 3' end followed by an intact pGH gene reading in the same
direction. Due to the homology between the hGH and pGH genes, a percentage
of the pB0509 plasmid underwent in vivo recombination, to make hybrid
hGH/pGH genes when transformed into E. coli rec.sup.+ MM294 (ATCC 31446).
These recombinants were enriched by restricting pool DNA with EcoRI to
linearize plasmids which had not undergone recombination resulting in the
loss of that EcoRI site. After two rounds of restriction selection and
transformation into E. coli rec.sup.+ MM294 nearly all the clones
represented hybrid hGH/pGH recombinants. Sequence analysis of 22 clones
demonstrate that the hGH/pGH hybrids contained amino terminal hGH sequence
followed by pGH sequence starting at amino acid residues +19, +29, +48,
+94, +105, +123 and +164.
Seven hGH-pGH hybrids having cross-over points evenly distributed over the
hGH gene were obtained. However, only the extreme carboxy terminal hybrid
(hGH (1-163)-pGH (164-191)) was secreted from E. coli at levels high
enough to be purified and analyzed. This hGH-pGH hybrid introduces three
substitutions (M170L, V173, A and V180M) that are located on the
hydrophobic face of helix 4. Accordingly, most of the sequence
modifications in the helical regions A, D, E and F in FIG. 2 were designed
to avoid mutations of residues on the hydrophobic face of the helices. For
example, the above hybrid hGH-pGH variant was modified to-retain M170,
V173, F176 and V180 because these residues are inside or boarding the
hydrophobic face of helix 4.
EXAMPLE 3
Expression and Purification of Soluble Human Growth Hormone Receptor from
E. coli
Cloned DNA sequences encoding the soluble human growth hormone receptor
shGHr (Leung, D. W., et al. (1987) Nature 330, 537) were subcloned into
pB0475 to form pJ1446 (see FIGS. 11 and 12).
The vector pClS.2 SHGHR (Leung, D. W., et al. (1987) Nature 330, 537) was
digested with XbaI and KpnI and the 1.0kb fragment containing the
secretion signal plus the 246 codon extracellular portion of the hGH
receptor was purified (Maniatis, T. et al. (1982) in Molecular Cloning,
Cold Springs Harbor Laboratory, New York). This fragment was ligated into
similarly cut M13-mp18 and single-stranded DNA for the recombinant gene
was purified (Messing, J. (1983) Methods in Enzymology, Vol. 101, p. 20).
Site-specific mutagenesis (Carter, P., et al. (1986) Nucleic Acids Res.
13, 4331) was carried out to introduce an NsiI site at codon +1 using the
18-mer digonucleotide, 5'-A-AGT-GAT-GCA-TTT-TCT-GG-3'. The mutant sequence
was verified by dideoxy sequence analysis (Sanger, F., et al. (1977) Proc.
Natl. Acad. Sci. USA 74, 5463). Double-stranded DNA for the mutant was
purified and cut with NsiI and SmaI. The 900bp fragment was isolated
containing the 246 codon extracellular portion of the hGH receptor. pB0475
was cut with NsiI and EcoRV and the 41kb fragment (missing the synthetic
hGH gene) was purified. The 900bp fragment for the receptor and the 4.1kb
vector fragment were ligated and the recombinant clone (pJ1446) was
verified by restriction mapping. This was transformed into the E. coli
KS303 (Strauch, K., et al. (1988) Proc. Natl. Acad. Sci. USA 85, 1576) and
grown in low-phosphate media (Chang, C. N. (1987) Gene 55, 189) at
30.degree. C. The receptor fragment protein was purified by hGH affinity
chromatography (Spencer, S. A., et al. (1988) J. Biol. Chem. 263, 7862;
Leung, D. W., et al. (1987) Nature M, 537). The sequence for pJ1446 is
shown in FIG. 12 together with the amino acid sequence of the cloned
receptor.
E. coli W3110, degp (Strauch, K. L., et al. (1988) PNAS USA 85, 1576) was
transformed with pJ1446 and grown in low-phosphate media (Chang, C. N.
(1987) Gene 55, 189) in a fermentor at 30.degree. C. The 246 amino acid
hGHr was used to generate preliminary data. A slightly shorter hGHr
containing amino acids 1 through 238 was used in the examples herein. The
results obtained with that receptor were indistinguishable from those
obtained with the 246 amino acid hGHr.
The plasmid phGHr(1-238) (Table X(A)) was constructed to generate a stop
codon after Gln238 to avoid the problem of carboxyl terminal
heterogeneity. The binding protein from KS330 cultures containing
phGHr(1-238) was produced in slightly higher yields and with much less
heterogeneity (data not shown) than from cultures containing phGHr(1-246).
Routinely, 20 to 40 mg of highly purified binding protein could be
isolated in 70 to 80 percent yield starting from 0.2 kg of wet cell paste
(-2 liters high cell density fermentation broth). Both N-terminal
sequencing and peptide mapping coupled to mass spectral analysis of the
C-terminal peptide confirmed that the product extended from residues 1 to
238.
Site-directed mutagenesis of the phGHr (1-246) template was performed
(Carter, et al. (1986) Nucleic Acids Res. 13, 4431-4443) to produce phGHr
(1-240, C241R) using the oligonucleotide
##STR1##
the asterisks are mismatches from the phGHr (1-246) template, underlined
is a new unique MluI site, and CGT-TAG directs the C241R mutation followed
by a stop codon (Table X(A)).
TABLE X(A)
__________________________________________________________________________
Sequences of amino- and carboxyl-termini of hGH binding protein
constructions
Plasmid
Termini
Protein/DNA sequence/Restriction sites
__________________________________________________________________________
-3-2-1+1+2+3
phGHr(1-246)
Amino
ALA--TYR--ALA--PHE--SER--GLY
GCC-TAT--GCA--TTT--TCT--GGA
NsiI
phGHr(1-246)
Carboxyl
238 239 240 241 242 243 244 245 246
GLN--PHE--THR--CYS--GLU--GLU--ASP--PHE--TYR--AM
CAA--TTT--ACA--TGT--GAA--GAA--GAT--TTC--TAC--TAG--CGGCCGC
NotI
phGHr Carboxyl
Gln--Phe--Thr--Arg--AM
(1-240,C241R)
*****
CAA--TTT--ACG--CGT--TAG--GAA--GAT--TTC--TAC--TAG--CGGCCGC
MluI NotI
phGHr(1-238)
Carboxyl
Gln--AM
******
CAA--TAG--ACA--CGT--TAG--GAA--GAT--TTC--TAC--TAG--CGGCCGC
NotI
__________________________________________________________________________
*Indicates mismatches from the wildtype template
The plasmid, phGHr (1-238) was produced by site-directed mutagenesis on the
phGHr (1-240, C241R) template using restriction-selection (Wells, et al.,
(1986) Phil. Trans. R. Soc. Lond. A, 317, 415-423) against the MluI site
(Table X(A)). Briefly, an oligonucleotide,
##STR2##
introduced a translation stop codon after Gln238 (CAA triplet) and altered
the MluI restriction-site (underlined). After growing up the pool of
duplex DNA from the initial transfection with heteroduplex, the DNA was
restricted with MluI and retransformed to enrich for the desired phGHr
(1-238) plasmid prior to DNA sequencing.
It was subsequently determined by DNA sequencing that the cloned hGH
binding proteins in phGHr(1-238) contained a T51, A mutation which arose
either as a cDNA variant or as a cloning artifact. The A51T revertant was
therefore to be identical to the published sequence (Leung, et al., (1987)
Nature (London) 330, 537-543. The purification and binding properties of
the proteins containing either Thr or Ala at position 51 were
indistinguishable (results not shown). The Ala51 binding protein variant
was selected for all subsequent analysis because it had been characterized
more thoroughly.
To compare the specificity of the recombinant hGH binding protein from E.
coli with the natural product isolated from human serum, the affinities
were determined for wild-type and various hGH mutants:
TABLE X(B)
__________________________________________________________________________
K.sub.d (nM) .+-.S.D..sup.a for hGH binding protein from:
K.sub.d (mut).sup.b
K.sub.d (mut).sup.b
K.sub.d (human serum).sup.b
hGH mutant
Human serum
K.sub.d (wt)
E. coli
K.sub.d (wt)
K.sub.d (E. coli)
__________________________________________________________________________
wt 0.55 .+-. 0.07
-- 0.40 .+-. 0.04
-- 1.4
I58A 21 .+-. 2
38 .+-. 6
14 .+-. 1
36 .+-. 5
1.5
R64A 12 .+-. 1
22 .+-. 4
11 .+-. 1
28 .+-. 5
1.1
E174A 0.27 .+-. 0.04
0.49 .+-. 0.11
0.16 .+-. 0.01
0.4 .+-. 0.1
1.7
F176A 71 .+-. 7
130 .+-. 20
48 .+-. 5
120 .+-. 20
1.5
__________________________________________________________________________
.sup.a Values of K.sub.d and corresponding standard deviations (SD) were
determined by competitive binding analysis (FIG. 24) with wildtype hGH
(wt) and a number of mutants of hGH.
.sup.b Reduction in binding affinity calculated from the ratio of
dissociation constants for the hGH mutant (mut) and wildtype hGH for each
hGH binding protein.
.sup.c Ratio of dissociation constants for the two hGH binding proteins
with a given hGH type.
Both proteins formed a specific stoichiometric complex with hGH (FIG. 24).
As can be seen, the affinities for wild-type and mutants of hGH are nearly
identical between the two binding proteins (right side column, supra). The
recombinant hGH binding protein has a marginally higher affinity compared
to the natural protein from human serum. This may reflect the greater
purity and homogeneity of the recombinant protein. Both proteins had
identical specificities as shown by the changes in binding affinities for
four alanine mutants of hGH that disrupt binding to the hGH binding
protein (K.sub.d (mut)/K.sub.d (wt) supra). The affinity of hGH for the
binding protein extending to Tyr246 (K.sub.d =0.36.+-.0.08 nM) was
virtually identical to that terminating after Gln238 (0.40.+-.0.03 nM)
indicating the last 8 residues (including the seventh cysteine in the
molecule) are not essential for binding hGH.
EXAMPLE 4
Receptor and Monoclonal Antibody Binding Assay
Purified hGH or hGH variants (over 95% pure) were assayed for binding to
the soluble hGH receptor of Example 3. Laser densitometric scanning of
Coomassie stained gels after SDS-PAGE was used to quantitate the
concentration of the purified hormones. These values were in close
agreement with concentrations determined from the absorbance at 280 nm
(.epsilon.2.sub.80.sup.0.1%= 0.93). The dissociation constants (K.sub.d)
were calculated from Scatchard analysis for competitive displacement of
›.sup.125 I! hGH binding to the soluble hGH receptor at 25.degree. C. The
.sup.125 I hGH was made according to the method of Spencer, S. A., et al.
(1988) J. Biochem. 263, 7862.
An enzyme-linked immunosorbent assay (ELISA) was used to assess the binding
of eight different monoclonal antibodies to various segment-substituted
and residue-substituted hGH variants. The following are the Mabs used:
______________________________________
Mab Identity Source/Method
______________________________________
1 MabA (*)
2 33.2 Hybritech, Inc.
3 Cat# H-299-01 Medix Biotech, Inc.
4 72.3 Hybritech, Inc.
5 Cat# H-299-02 Medix Biotech, Inc.
6 Mab 653 Chemicon
7 Mab D (*)
8 Mab B (*)
______________________________________
(*) Carbone, F. R., et al. (1985) J. Immunol. 135, 2609
Rabbit polyclonal antibodies to hGH were affinity purified and coated onto
microtiter plates (Nunc plates, InterMed, Denmark) at 2 .mu.g/mL (final)
in 0.005M sodium carbonate pH (10) at 24.degree. C. for 16-20 h. Plates
were reacted with 0.1 .mu.g/mL of each hGH variant in buffer B (50 mM TRIS
HCl Tris(hydroxymethyl)aminomethane hydrochloride; ›pH 7.5!, 0.15M NaCl, 2
mM EDTA, 5 mg/mL BSA, 0.05% TWEEN 20.TM. brand polyoxyethylene
sorbitanmonolaurate, 0.02% sodium azide) for two hours at 25.degree. C.
Plates were washed and then incubated with the indicated Mab which was
serially diluted from 150 to 0.002 nM in buffer B. After two hours plates
were washed, stained with horseradish peroxidase conjugated anti-mouse
antibody and assayed. Values obtained represent the concentrations (nM) of
each Mab necessary to produce half-maximal binding to the respective hGH
variant.
Competitive displacement of the hGH receptor from hGH by anti-hGH Mabs was
determined as follows. Assays were carried out by immobilization of
wild-type hGH in microtiter plates coated with anti-hGH rabbit polyclonal
antibodies as described. Receptor (fixed at 10 nM) and given anti-hGH Mab
(diluted over a range of 150 to 0.002 nM) were added to the hGH coated
microtiter plate for 16-20 hours at 25.degree. C., and unbound components
were washed away. The amount of bound receptor was quantified by adding an
anti-receptor Mab that was conjugated to horseradish peroxidase which did
not interfere with binding between hqH and the receptor. The normalized
displacement value was calculated from the ratio of the concentration of
Mab necessary to displace 50% of the receptor to the half-maximal
concentration of Mab necessary to saturate hGH on the plate. This value
was used to compare the relative ability of each Mab to displace the
receptor.
EXAMPLE 5
Active Domains for Somatogenic Receptor Binding
The 17 segment substituted hGH variants described in Example 1 and Example
2 were assayed for binding to the soluble somatogenic receptor of Example
3 and binding to the monoclonal antibodies as described in Example 4. The
results of the binding assay to the somatogenic receptor are shown in
Table III. As can be seen, the segment substitutions that are most
disruptive to binding are within regions A, C and F of FIGS. 4 and 5.
These regions were further directed into smaller segments to further
localize the active domains of the hGH molecule involved in binding to the
somatogenic receptor. The most significant results from Table III are
shown in FIG. 4, which is a bar graph showing the relative reduction in
binding to the soluble hGH receptor as a consequence of the substitution
of the indicated analogous sequences from the analogs hPRL, hPL and pGH as
shown. Three active domains were identified as regions A, C and F
comprising amino acid residues 12-19, 54-74 and 164-190, respectively.
These regions are identified in the three-dimensional representation of
the hGH molecule in FIG. 5.
As can be seen, the three active domains, A, C and F, although
discontinuous in the amino acid sequence of hGH, form a continuous region
in the folded molecule which defines the somatogenic binding site on hGH.
EXAMPLE 6
Epitope Mapping of hGH
The binding of the eight different monoclonal antibodies to specific
segment-substituted hGH variants is shown in Table XI.
TABLE XI
__________________________________________________________________________
Mab
1 2 3 4 5 6 7 8
Hybr
Medix
Hybr
Medix
hGH Variant
MCA 33.2
1 72.3
2 Chemicon
MCD MCB
__________________________________________________________________________
wt hGH 0.4 0.4 0.1 0.05
0.2 0.2 0.08
0.1
hPL (12-25)
0.4 0.4 >75 >50 0.2 0.2 0.08
0.1
pGH (11-33)
0.4 >100
1.5 0.05
0.2 0.2 0.08
0.1
hPRL (12-33)
0.4 >100
>75 >50 0.2 0.2 0.08
0.1
hPRL (12-19)
0.4 >12 >75 >50 0.2 0.2 0.08
0.1
hPRL (22-33)
0.4 0.4 0.1 0.05
0.2 0.2 0.08
0.1
hPL (46-52)
0.4 0.4 0.1 0.05
0.2 0.2 0.40
0.1
pGH (48-52)
0.4 0.4 0.1 0.05
0.2 0.2 0.08
0.1
hPL (56-64)
0.4 0.4 0.1 0.05
0.2 0.8 0.08
0.1
pGH (57-73)
0.4 0.4 0.1 0.05
>200
>200 0.08
0.1
hPRL (54-74)
0.4 0.4 0.1 0.05
0.2 0.6 0.08
0.1
hPRL (88-95)
>400
0.4 0.1 0.05
0.2 0.2 0.08
0.1
hPRL (97-104)
>400
>12 0.1 0.05
0.2 0.2 0.08
0.1
hPL (109-112)
>12 0.4 >75 15 0.2 0.2 0.08
0.1
hPRL (111-129)
>12 0.4 >75 >50 0.2 0.2 0.08
0.1
hPRL (126-136)
0.4 0.4 0.1 0.05
0.2 0.2 0.08
0.1
pGH (164-190)
0.4 0.4 0.5 0.3 >25 12.5 0.20
0.4
pGH (167-182
hGH (.DELTA.32-46)
0.4 0.4 0.1 0.05
0.2 0.2 >100
>100
N12A 0.4 0.4 >75 >50 0.2 0.2 0.08
0.1
C182A 0.4 0.4 0.1 0.05
2.0 0.2 0.08
0.1
__________________________________________________________________________
With the possible exception of the pGH (167-190) variant, disruption of
binding to each monoclonal antibody was dramatic and highly selective.
FIGS. 13 through 20 localize the epitope for each of the Mabs on the
three-dimensional structure of hGH. FIG. 6 comprises these epitopes to the
binding site for the somatogenic receptor.
For example, the hPRL (88-95), hPRL (97-104), hPL (109-112) and hPRL
(111-129) variants do not bind to Mab1 yet the other segment-substituted
hGHs outside of these regions bound as effectively as wild-type hGH.
Binding to Mabs 2, 3, 4, 5 and 6 was disrupted by mutations in
discontinuous regions in the primary sequence but in close proximity in
the folded hormone (see FIGS. 6 and 14 through 19). In contrast, Mabs 1, 7
and 8 were disrupted by mutations defined by a continuous sequence as
shown in FIGS. 13, 19 and 20.
The regions disrupting binding to a given monoclonal antibody were further
analyzed by dissecting specific segment-substituted hGH variants into
subdomains or by analyzing variants that had common substitutions that
still bound to the particular Mab. For example, pGH (11-33) retained tight
binding to Mab 4 yet hPRL (12-33) disrupted binding. Thus, the disruptive
mutations in the hPRL (12-33) variant can be confined to residues not
mutated in pGH (11-33): N12, L15, R16, D26 and E30. This set can be
further restricted to N12, L15 and R16 because the hPRL (12-19) variant
disrupts binding, but the hPRL (22-23) variant does not (see FIG. 16). The
N12H mutation in hPL (12-25) can entirely account for the disruption in
binding to Mab 4 because this is the only mutation not in common with pGH
(11-33). This was tested by substituting alanine for Asn-12. The binding
of Mabs 3 or 4 to the N12A residue-substituted hGH variant was reduced by
over 100-fold whereas binding to the other Mabs was unaffected.
Using this set of hGH variants, it was possible to resolve the epitopes
from all eight Mabs even though binding for most of these Mabs was blocked
by a common set of mutations. For example, although hPRL (12-19) disrupted
binding to Mabs 2, 3 and 4, other variants indicated that these Mabs
recognized different structures. Specifically, Mabs 2 and 3 were blocked
by pGH (11-33) yet Mab 4 was unaffected. Binding of Mabs 3 and 4 was
blocked by hPL (12-25) yet binding to Mab 2 was unaffected. Thus, the
eight antibodies may have epitopes that overlap but none superimposed.
Mutations that disrupt binding are present in both helices and loops and
are always in close proximity in the folded hormone.
Collectively, the epitopes with a set of eight Mabs cover most of the
hormone. However, there are still regions where these Mabs did not bind.
For example, three of the 20 variants did not significantly disrupt
binding to any of the Mabs tested (hPRL (22-33), pGH (48-52) and hPRL
(126-136)).
There are significant differences between the antibody epitopes and the
receptor binding site. Firstly, the patch defined by disruptive mutations
is larger for the receptor than for any of the Mabs. A second difference
is that the receptor has more tolerance to disruptive substitutions in the
hormone than do the Mabs. This is evidenced by the fact that the maximum
reduction in binding to the receptor for any of the mutants is about
70-fold, whereas almost every antibody has at least one variant that
causes more than a 1000-fold reduction in binding, some of which may be
the result of single substitutions such as N12A.
EXAMPLE 7
Competitive Binding of Mabs and shGHr
Many of the variants which cause disruption of receptor binding also
disrupt the binding of one or more of the Mabs. The ability of each of the
eight Mabs to block the binding of the hGH receptor to hGH was therefore
evaluated. Results of this assay are shown in Table XII.
TABLE XII
______________________________________
Normalized displacement
Mab 50% binding to hGH.dagger.
displace 50% of receptor
##STR3##
______________________________________
1 0.4 >150 >375
2 0.4 0.8 2
3 0.1 150 1500
4 0.05 150 3000
5 0.2 0.2 1
6 0.2 0.2 1
7 0.08 0.4 5
8 0.1 >150* >1500
______________________________________
*Binding of Mab 8 appears to slightly enhance binding of receptor to hGH.
.dagger.Data from Table X for binding of each Mab to hGH.
As can be seen Mabs 5 and 6 are the most efficient at blocking binding of
the hGH receptor. This is because these Mabs have antigenic determinants
located in the loop from residues 54 through 74 and in helix 4 closely
overlap determinants for the receptor (see FIGS. 5, 6, 17 and 18). Mab 2
was the next most competitive antibody and it too shared a common
disruptive mutation with the receptor (hPRL (12-9)). In contrast, Mabs 3
and 4 were roughly 1000-fold less competitive than Mab 2 yet they also
shared overlapping disruptive mutations with the receptor in helix 1. See
FIGS. 15 and 16. This apparent discrepancy may be easily reconciled if the
mutations in helix 1 that disrupt Mabs 3 and 4 differ from those residues
which disrupt binding to Mab 2 or the receptor. Indeed, one such mutant
(N12A) disrupts binding of either Mab 3 or 4 without affecting binding to
Mab 2 or the receptor.
Mab 7 competes relatively strongly with the receptor for hGH and it is
disrupted by segment-substituted hGH variants that cause a minor
disruption of receptor binding, e.g., hPL (46-52). Thus, it appears that
Mabs 2 and 7 sit on the border of the receptor binding site. Mabs 1 and 8
were unable to give detectable displacement of the receptor, and as
expected these contain no overlapping antigenic determinants with the
receptor. These competitive binding data taken together with the direct
epitope mapping and receptor binding data strongly support the general
location of the receptor binding site as shown in FIG. 5.
EXAMPLE 8
Receptor Active Amino Acid Residues
The analysis of hGH in Examples 5, 6 and 7 implicates the amino terminal
portion of helix 1 (residues 11-19) as being of moderate importance to
receptor binding. In addition, residues 54-74 and 167-191 were identified
as being important to receptor binding. Identification of which amino
acids in these domains are active in receptor binding was carried out by
analyzing a total of 63 single alanine variants. See Tables XIII, XIV and
XV.
TABLE XIII
______________________________________
Amino acid scanning of positions 2-19 in hGH
Variant K.sub.d (nM)
K.sub.d (variant)/K.sub.d (wt)
______________________________________
wt 0.34 1.0
P2A 0.31 0.90
T3A 0.31 0.90
I4A 0.68 2.0
P5A 0.71 2.1
L6A 0.95 2.8
S7A 0.61 1.8
R8A 0.48 1.4
L9A 0.32 0.95
F10A 2.0 5.9
D11A NE --
N12A 0.40 1.2
A13 (WT)
M14A 0.75 2.2
L15A 0.44 1.3
R16A 0.51 1.6
A17 (WT)
H18A 0.24 0.71
R19A 0.37 1.1
______________________________________
TABLE XIV
______________________________________
Amino acid scanning of positions 54-74 in hGH
Variant K.sub.d (nM)
K.sub.d variant/K.sub.d WT
______________________________________
WT 0.31 1.0
F54A 1.5 4.4
S55A 0.41 1.2
E56A 1.4 4.1
S57A 0.48 1.4
I58A 5.6 17.0
P59A 0.65 1.9
T60A NE --
S61A NE --
S62A 0.95 2.8
N63A 1.12 3.3
R64A 7.11 21.0
E65A 0.20 0.6
E66A 0.71 2.1
T67A NE --
Q68A 1.8 5.2
Q69A 0.31 0.9
K70A 0.82 2.4
S71A 0.68 2.0
N72A NE --
L73A 0.24 0.70
E74A NE --
______________________________________
TABLE XV
______________________________________
Amino acid scanning of positions 167-191 in hGH
Variant K.sub.d (nM)
K.sub.d variant/K.sub.d WT
______________________________________
WT 0.34 1
R167A 0.26 0.75
L168A 0.37 1.1
D169A NE --
N170A NE --
D171A 2.4 7.1
K172A 4.6 14
V173A NE --
E174A 0.075 0.22
T175A NE --
T175S 5.9 16
F176A 5.4 16
L177A NE --
R178A NE --
R178N 1.4 4.2
I179A 0.92 2.7
V180A 0.34 1.0
Q181A 0.54 1.6
C182A 1.9 5.7
R183A 0.71 2.1
S184A 0.31 0.90
V185A 1.5 4.5
E186A 0.27 0.80
G187A 0.61 1.8
S188A 0.24 0.7
C189A NE --
G190A NE --
F191A 0.20 0.60
______________________________________
The substitution of alanine was extended to include residues 2-19 because
of uncertainties in the position of the amino terminal residue
(Abdel-Meguid, S. S., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 5
6434). Indeed, the most pronounced reduction in binding occurred for F10A
(6-fold) followed by alanine substitutions at residues 4-6 at the
N-terminus of helix 1 (see FIG. 21). Substantially larger effects on
binding (greater than 20-fold) occurred for specific alanine substitutions
within the 54 to 74 loop and the carboxy terminal sequence 167-191. For
several alanine variants, binding was enhanced up to 4.5-fold. The most
dramatic example was E174A which was located in the midst of a number of
disruptive alanine mutations. See FIG. 4, 7 and 21.
The most disruptive alanine substitutions form a patch of about 25 .ANG. by
25 .ANG. on the hormone that extends from F10 to R64 and from D171 to V185
(see FIG. 21). Furthermore, these side chains appear to be facing in the
same direction on the molecule. For example, all of the alanine mutants
that most affect binding on helix 4 (D171A, K172A, E174A, F176A, I179A,
C182A and R183A) are confined to three and one-half turns of this helix,
and their side chains project from the same face of the helix (see FIG.
21). Based upon this model, it was predicted that T175 and R178 should be
involved in binding because they occupy a central position as shown in
FIG. 21.
Although the T175A mutant could not be expressed in high enough yields in
shake flasks to be assayed, a more conservative mutant (T175S) was.
Accordingly, the T175S mutant caused a 16-fold reduction in receptor
binding. Similarly, although R178A was poorly expressed, R178N could be
expressed in yields that permitted analysis. R178N exhibited a greater
than four-fold reduction in binding affinity.
The next most disruptive mutant in the carboxy terminus was V185A. Although
V185A is outside of helix 4, it is predicted by the model to face in the
same direction as the disruptive mutations within helix 4. In contrast,
alanine mutations outside the binding patch, or within it facing in the
opposite direction from those above (R167A, K168A, V180A, Q181A, S184A,
E186A, S188A), generally had no or little effect on receptor binding.
A similar analysis applied to alanine mutants in helix 1, albeit with more
moderate effects on binding. Within the helix, the alanine substitutions
that most disrupted binding were at residue 6, 10 and 14 which were
located on the same face of the helix. The least disruptive alanine
mutations (L9A, N12A and L15A) were located on the opposite face of helix
1. This is further confirmed by the fact that anti-hGH Mabs 3 and4, which
do not compete with the receptor for binding to hGH, both bind to Asn-12.
See Table XVI.
TABLE XVI
______________________________________
Binding of hGH and alanine variants to eight
different anti-hGH monoclonal antibodies (Mab).
Mab
Hormone 1 2 3 4 5 6 7 8
______________________________________
hGH 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
F10A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
N12A 0.4 0.4 >75 >50 0.2 0.2 0.08 0.1
I58A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
R64A 0.4 0.4 0.1 0.05 0.2 1.6 0.08 0.1
Q68A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
K168A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
D171A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
K172A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
E174A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
F176A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
C182A 0.4 0.4 0.1 0.05 2.0 0.2 0.08 0.1
V185A 0.4 0.4 0.1 0.05 0.2 0.2 0.08 0.1
______________________________________
The relative positions of side chains within the 54-74 loop cannot be fixed
in the model as they can be for those within helices 1 and 4. However,
there is a striking periodicity in the binding data in which mutations of
even-numbered residues cause large reductions in binding relative to
odd-numbered residues. This is especially true for the first part of this
region (54-59) and may reflect a structure in which even residues project
toward the receptor and odd ones away.
EXAMPLE 9
Conformational Integrity and Binding Energetics of Alanine Substituted hGH
Variants
Several lines of evidence indicate that the alanine substitutions that
disrupt the receptor binding do not do so by causing the molecule to be
misfolded. Firstly, the eight Mabs react as well with almost all of the
alanine mutants that disrupt binding to the receptor as they do with hGH.
See Table XII supra.
The exceptions are R64A and C182A which selectively disrupt binding to the
anti-hGH Mabs 6 and 5, respectively. These two Mabs as previously
indicated compete with the somatogenic receptor for binding to hGH. In
addition, two alanine variants were made which do not affect receptor
binding. One of these affects the binding of two Mabs (N12A) and the other
affects none of the Mabs (K168A). This data indicates that binding to
either the Mabs or receptors is disrupted by a very local perturbation in
the structure of the variant. Moreover, the far UV circular dichroic
spectra of all the hGH variants tested are virtually identical to
wild-type hGH.
About 20% of the alanine mutants (D11A, T60A, P61A, T67A, N72A, E74A,
D169A, M170A, V173A, T175A, L177A, K178A, C189A, G190A) were not secreted
at high enough levels in shake flasks to be isolated and analyzed. Since
genes encoding such variants were expressed in the same vector and
expression was independent of the specific alanine codon, variations in
steady-state expression levels most likely reflect differences in
secretion level and/or proteolytic degradation of the hGH variants.
Several of the non-expressing alanine variants in helix 4 are located on
its hydrophobic face (M170A, V173A and L177A) as shown in FIG. 21 wherein
the hydrophobic side of the helix is identified by open shading. However,
this is not a general effect because several alanine substitutions were
tolerated on the hydrophobic face of helix 1 (L6A, L9A and F10A) and helix
4 (F176A and V180A).
In addition, impaired expression of hGH variants was sometimes observed
when charged or neutral amino acids were replaced with alanine (D11A,
T60A, T67A, N72A, E74A, D169A, T175A, R178A). Mutations such as T175S and
R178N, which preserved the hydrogen bonding group at those sites, could be
expressed albeit at levels below wild-type. The non-expressing C189A
variant disrupts the carboxy-terminal disulfide and its counterpart
(C182A) was also expressed at levels far below wild-type. Several other
non-expressing alanine mutants (T60A, T61A and T67A) were located in a
loop structure. Thus, low levels of expression or non-expression can
result from a multitude of structural effects but can be obviated by
isosteric or isofunctional substitutions.
The substitutions that cause a ten fold or greater effect upon binding
(58A, R64A, K172A, T175S, F176A) are likely to be directly involved in
binding. The strengths of hydrogen bonds or salt bridges present in nature
(Fersht, A. R. (1972) J. Mol. Biol. 64, 497; Brown, L. R., et al. (1978)
Eur. J. Biochem. 88, 87; Malivor, R., et al. (1973) J. Mol. Biol. 76 123)
or engineered by site-directed mutagenesis experiments (Fersht, A. R., et
al. (1985) Nature 314, 225; Bryan, P., et al. (1986) Proc. Natl. Acad. Sci
USA 83, 3743; Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci USA 84,
5167; Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219;
Cronin, C. N.,et al. (1987) J. Am. Chem. Soc. 109 2222; Graf, L., et al.
(1988) Proc. Natl. Acad. Sci. USA 85 4961) overlap and range widely from 1
to 5 kcal/mole depending upon the microenvironment. For hGH, reductions in
binding base energy of 0.8, 1.0, 1.2, 1.6 and 1.8 kcal/mol
(.DELTA..DELTA.G.sub.binding =+RT 1n Kd (var)/Kd(wt)) occurred for alanine
substitutions at E56, Q68, D171, K172 and R64, respectively. The
energetics for burial of a hydrophobic side chain into a protein tends to
parallel its free energy of transfer into ethanol (Estell, D. A., et al.
(1986) Science 233, 659; Nozaki, Y. et al. (1980) in The Hydrophobic
Effect (Wiley, N.Y. pp. 4-21).
Accordingly, the reductions in binding free energies for F175A, F10A, F54A,
I58A, and V185A were 1.6, 1.0, 0.9, 1.7 and 0.9 kcal/mol, respectively.
These are slightly below the predicted change in hydrophobic free energy
in going from Phe, Ile or Val to Ala of 2.0, 2.4 and 1.0 kcal/mol,
respectively. By this analysis the effect of the T175S mutant
(.DELTA..DELTA.G.sub.binding =1.6 kcal/mol) is larger than expected for
loss of a .gamma. methyl group (.DELTA..DELTA.G.sub.hydrophobic =0.7
kcal/mol). To fully characterize the nature of the molecular contacts
between hGH and its somatogenic receptor requires direct structural
information. However, the energetics of binding of these alanine mutants
shows them to be in the range of previous measurements made on contact
residues in entirely different systems. In fact, the sum of binding free
energies for these alanine-substituted variants exclusive of C182A that
are most disruptive to receptor binding (-13.2 kcal/mol) is comparable to
the total free energy binding between hGH and its receptor (-13 kcal/mol).
EXAMPLE 10
Reactivity of hGH Variants with
Anti-hGH Polyclonal Antibodies
The hGH variants hPRL (22-33), E174A and hPRL (88-95) were tested in a rat
weight gain assay. The results of that assay are presented in FIG. 22. As
can be seen, all the variants except hPRL (22-33) have a reduced potency
after about 14 days of growth. The leveling off of growth is attributed to
the development of antibodies to the various growth hormones which
neutralize the biological effect. The fact that, the hPRL (22-33) variant
continues to induce growth suggests that it is not as immunogenic as
wild-type hGH or the other variants used.
A comparison of the reactivity of various hGH variants with human and
murine serum containing polyclonal antibodies to hGH is shown in Table
XVII.
TABLE XVII
______________________________________
Serum Anti-hGH Antibodies Binding to hGH Variants
Average % of Reduction
of Anti-Protropin .RTM. bond hGH
Binding .+-. SD % Incidence
Human Mouse Human Mouse
Sera N = 22
Sera (N = 6)
Sera Sera
______________________________________
hGH 0 0 100 100
pGH 11-33
86 .+-. 13 65 .+-. 16 100 100
hPRL 12-33
79 .+-. 19 52 .+-. 13 100 100
hPL 12-25
35 .+-. 19 16 .+-. 11 81 33
hPRL 12-19
29 .+-. 20 11 .+-. 12 71 33
hPRL 22-33
69 .+-. 15 38 .+-. 8 100 100
hPL 46-52
6 .+-. 8 2 .+-. 4 10 0
pGH 48-52
7 .+-. 8 4 .+-. 4 10 0
pGH 57-73
43 .+-. 15 39 .+-. 12 95 100
hPRL 54-74
14 .+-. 9 8 .+-. 7 24 0
D80 13 .+-. 15 7 .+-. 7 14 0
hPRL 88-95
14 .+-. 22 4 .+-. 5 19 0
hPL 109-112
10 .+-. 11 9 .+-. 9 24 17
hPRL 126-136
8 .+-. 12 2 .+-. 2 19 0
C182A 1 .+-. 5 1 .+-. 3 5 0
______________________________________
As can be seen, variants containing substitutions within the region from
residues 22 to 33 have substantially reduced binding activity, and in some
cases no activity, with individual human and mouse anti-serum for
wild-type hGH.
Except for the variant pGH 57-73, variants containing substitutions in the
other regions shown do not have a significant reduction in reactivity.
Since the segment substituted mutants between residues 11 and 10 33 retain
their ability to bind the somatogenic receptor, such variants demonstrate
the production of variants which maintain the ability to promote
somatogenesis but have another property which is modified, in this case
reactivity with anti-hGH polyclonal antibodies.
EXAMPLE 11
Relationship Between Kd and Potency
A semi-log plot of the ratio of Kd (variant)/Kd (wild type) for specific
hGH variants versus the potency of such variants in a rat weight gain
assay is shown in FIG. 23. As can be seen a linear relationship exists
which suggests that a decreased-binding affinity for the somatogenic
receptor will result in a decrease in potency.
As can be seen, the hGH variant E174A has a higher binding affinity for the
somatogenic receptor than the wild-type hGH. Its potency is also greater
than that of wild-type hGH by about 12%.
Further, the variant pPRL (97-104) has essentially the same binding
constant as wild-type hGH but about a 2.7-fold increase in potency.
EXAMPLE 12
Active Domains in hGH for Prolactin Receptor Binding
Human growth hormone (hGH) elicits a myriad of physiological effects
including linear growth, lactation, nitrogen retention, diabetogenic and
insulin-like effects, and macrophage activation. R. K. Chawla, J. S. Parks
and D. Rudman, Annu. Rev. Med. 34, 519-547 (1983); O. G. P. Isaksson, et
al. (1985) Annu. Rev. Physiol. 47, 483-499; C. K. Edwards, et al., (1988)
Science 239, 769-771. Each of these effects begins with the interaction of
hGH with specific cellular receptors. J. P. Hughs, et al. (1985) Annu.
Rev. Physiol. 47, 469-482. Thus far, the only cloned genes whose products
bind hGH are the hGH receptor from liver (D.W. Leung, et al., (1987)
Nature (London) 330, 537-543) and the human prolactin (hPRL) receptor from
mammary gland (J. M. Boutin, et al., (1988) Cell 5, 69-77). Receptor
"spillover" of hGH onto the hPRL receptor has clinical precedence in cases
where acromegalics, who produce high levels of hGH, develop a
hyperprolactinemic syndrome despite having normal levels of hPRL (J. E.
Fradkin, et al., (1989) New Engl. J. Med. 320, 640-644). However, other
receptors exist that bind hGH, including the placental lactogen (PL)
receptor (M. Freemark, et al., (1987) Endocrinology 120, 1865-1872). It
previously Was not known if the binding sites on hGH for these receptors
are identical or which receptor (or combination of receptors) is
responsible for which pharmacological effect. To begin to address these
issues the hGH and hPRL receptor binding sites on hGH were mapped. The
results obtained indicate that these receptor binding sites overlap but
are not identical. This has allowed the rational design of receptor
specific variants of hGH.
The hGH and hPRL receptors both contain a extracellular hormone binding
domain that share 32% sequence identity, single transmembrane domains, and
cytoplasmic domain which differs widely in sequence and length. The
extracellular binding domain of the hGH receptor has been expressed in E.
coli and has identical binding properties to that found naturally as a
soluble serum binding protein (S.A. Spencer, et al., (1988) J. Biol. Chem.
263, 7862-7867). Similarly, the extracellular domain of the hPRL receptor
has been expressed in E. coli and purified. The hPRL receptor fragment
extends from residues Glnl to Thr211 and terminates just before the single
transmembrane domain. It retains high binding affinity and specificity
that is virtually identical to its full-length receptor. The gene encoding
the hPRL receptor used in the experiments was kindly provided by Dr. P. A.
Kelly, Laboratory of Molecular Endocrinology, McGill University, Montreal,
Canada. This DNA sequence was obtained from a human mammary cDNA library
and identified with a probe covering known conserved regions amongst
cross-species members of the prolactin receptor family. See e.g., Davies,
J. A., et al., (1989) Mol. Endrocrinoloqy 3, 674-680; Edery, et al. (1989)
Proc. Natl. Acad. Sci. USA 86 2112-2116; Jolicoeur, et al. (1989) Mol.
Endocrinology 3, 895-900. These truncated and highly purified receptors
are extremely useful reagents for rapid and accurate assessment of binding
affinity for mutants of hGH.
Relationship Between hPRL and hGH Receptor Binding Sites.
To determine if the epitopes for the hGH and hPRL receptors overlapped we
analyzed whether or not the hPRL receptor fragment could displace the hGH
receptor fragment from hGH (results not shown). Indeed, the hPRL receptor
fragment competed for the hGH receptor binding site with an apparent Kd of
1 nM. This is virtually the same affinity as that measured by direct
binding of the hPRL receptor to hGH (results not shown).
Eleven of the segment-substituted hGH variants from Table III were used to
localize the epitope on hGH for the hPRL receptor. The hGH.DELTA.32-46
variant was also used in this experiment. The approach was similar to that
used to determine the epitope on hGH for the hGH receptor as previously
described, i.e. by the disruption in binding of variants of hGH except
that the receptor was hPRLr rather than hGHs. The results for the above
twelve segment-substituted hGH variants are summarized in Table XVIII.
TABLE XVIII
__________________________________________________________________________
Binding of hGH variants produced by homolog-scanning mutagenesis to the
extracellular domain of the hPRL receptor hPRLr. Mutants are named
according to the
extremes of segments substituted from the various hGH homologs: pGH, hPL,
or
hPRL. The exact description of the mutations introduced is given by the
series of
single mutants separated by commas. The component single mutants are
designated
by the single letter code for the wild-type residue followed by its codon
position in
mature hGH and then the mutant residue. Mutants of hGH were produced and
purified
as previously described herein. Binding to hPRLr was measured essentially
as described
for the hGHr (Spencer, S.A. et. al. (1988) J. Biol. Chem. 263, 7862-7867)
except
that affinity purified rabbit polyclonal antibodies raised against the
hPRLr were used
to precipitate the hPRLr complex with GIBCO .TM. bond BSA (crude) as
carrier protein.
Standard deviations in values of K.sub.D were typically at or below 20%
of the reported
value. The relative reduction in binding affinity (K.sub.D (mut)/K.sub.D
(hGH)) for the hGHr was
taken from Table III herein. The change in receptor preference was
calculated
from the ratios of the relative reductions in binding affinity for the
hGHr to the hPRLr.
WT = wild-type.
Change in receptor
hPRLr hGHr preference
Mutant
Mutations K.sub.D (mut)
K.sub.D (mut)
hGHr
Name Introduced
K.sub.D (nM)
K.sub.D (hGH)
K.sub.D (hGH)
hPRLr
__________________________________________________________________________
WT hGH
none 2.3 (1) (1) (1)
pGH
(11-33)
D11A, M14V,
852 370 3.4 110
H18Q, R19H,
F25A, Q29K, E33R
pGH
(48-52)
P48A, T50A, S51A,
2.0 0.9 2.8 0.32
L52F
pGH
(57-73)
S57T, T60A, S62T,
167 73 17 4.3
N63G, R64K, E65D,
T67A, K70R, N72D,
L73V
hGH
(.DELTA.32-46)
Deletion of
14 6.1 ND
residues 32 to 46
hPL
(46-52)
Q46H, N47D, P48S,
4.4 1.9 7.2 0.26
Q49E, L52F
hPL
(56-64)
E56D, R64M
4.1 1.8 30 0.06
hPRL
(12-19)
N12R, M14V, L15V,
3.2 1.4 17 0.08
R16L, R19Y
hPRL
(22-33)
Q22N, F25S, D26E,
168 73 0.85 85
Q29S, E30Q, E33K
hPRL
(54-74)
F54H, S55T, E56S,
2.5 1.1 69 0.02
I58L, P59A, S62E,
N63D, R64K, E66Q,
T67A, K70M, S71N,
N72Q, L73K, E74D
hPRL
(88-95)
E88G, Q91Y, F92H,
3.8 1.6 1.4 1.1
R94T, S95E
hPRL
(97-104)
F97R, A98G, N99M,
12.1
5.2 1.6 3.2
S100Q, L101D,
V102A, Y103P,
G104E
hPRL
(111-129)
Y111V, L113I,
2.6 1.1 1.5 0.73
K115E, D116Q,
E118K, E119R,
G120L, Q122E,
T123G, G126L,
R127I, E129S
WT hPRL
none 7.6 3.3 >100,000
--
__________________________________________________________________________
As can be seen pGH (11-33) and pGH (57-73) cause large disruptions in hPRL
receptor binding affinity, whereas pGH (48-52) has no effect. Unlike the
hGH receptor, the hPRL receptor will bind hPRL and hPL but not pGH. As
expected, virtually all of the substitutions tested from the
binding-competent hormones, hPRL or hPL, did not disrupt binding. The only
exception was hPRL (22-33) which caused a >70-fold reduction in binding
affinity for the hPRL receptor. Thus, the hPRL receptor is very sensitive
to mutations in hGH near the central portion of helix 1 and the loop
between residues 57 and 73.
The homolog-scan data also suggest that the hPRL and hGH receptor epitopes
are not identical because several segment substituted variants cause huge
changes in receptor binding preference (Table XVIII). For example, the
disruption in binding caused by the pGH (11-33) or hPRL (22-33) are about
100-fold greater for the hPRL receptor than for the hGH receptor. In
contrast, the hPL (56-64) and hPRL (54-74) have almost no effect on the
hPRL receptor, whereas they weaken binding to the hGH receptor by factors
of 17 and 69, respectively. These preferential binding effects (along with
binding of monoclonal antibodies as previously discussed) further
substantiate that reductions in receptor binding affinity are caused by
local and not global structural changes in the mutants of hGH.
The specific side-chains in hGH that strongly modulate binding to the hPRL
receptor were identified by alanine-scanning mutagenesis and homologous
substitutions. The hGH variants shown in Table XIX were prepared. The hPRL
substitutions, F25S and D26E, cause the largest reductions in binding
affinity (21 and 4.5-fold, respectively) in helix 1. These residues
project from the hydrophilic face of helix 1 (FIG. 25B) and are on the
same side as other mutations in helix 1 (notably H18A and F10A) that have
milder effects on binding.
Four residues in the loop region (54 to 68) known to affect binding of hGH
receptor as well as two residues (Q49A and T50A) preceding this region
that are nearby and do not affect hGH receptor binding were tested. The
most disruptive mutants are I58A and R64A, which reduced binding affinity
by 32 and 6-fold, respectively; the other four mutations have negligible
effects.
The fact that helix 1 and the loop region (58-64) contain strong binding
determinants for the hPRL receptor implicate helix 4 because this helix is
wedged between these two structures (FIG. 25B). Indeed, alanine-scanning
of the helix 4 region between a disulfide linked to C165 through V185
reveals strong binding determinants (Table XIX). The most disruptive
mutations extend nearly four helical turns, from R167 to R178, and are
located on the same hydrophilic face.
TABLE XIX
______________________________________
Binding of single mutants of hGH to hPRL or hGH receptor fragments
(hPRLr or hGHr). Mutants of hGH were prepared and purified as
previously described except for Q22N, F25S, D26E, Q29S and E33K
which were produced by site-directed mutagenesis (Cunningham, B. C.
and Wells, J. A. (1989) Science 244, 1330-1335; Zoller, M. J. and
Smith, M. (1982) Nucleic Acids Res. 10, 6487-6499). Receptor
binding assays and mutant nomenclature are described in Table XVIII.
Data for the reduction in binding affinity to the hGHr is taken from
Table III. ND indicates not determined.
Change in receptor
hPRLr hGHr preference
K.sub.D (mut)
K.sub.D (mut)
hGHr
Mutant K.sub.D (nM)
K.sub.D (hGH)
K.sub.D (hGH)
hPRLr
______________________________________
WT hGH 2.3 (1) (1) (1)
P2A 1.3 0.6 0.9 0.7
T3A 3.4 1.5 0.9 1.7
P5A 2.5 1.1 2.1 0.5
L6A 4.0 1.8 2.8 0.6
S7A 1.9 0.8 1.8 0.4
F10A 8.1 3.5 5.9 0.6
N12A 1.9 0.8 1.2 0.7
M14A 1.3 0.6 2.2 0.3
L15A 1.2 0.5 1.3 0.4
H18A 3.9 1.7 1.6 0.6
R19A 1.4 0.6 0.7 2.4
Q22N 2.1 0.9 ND --
F25S 48 21 ND --
D26E 10 4.5 ND --
Q29S 3.2 1.4 ND --
E33K 1.8 0.8 ND --
Q49A 1.5 0.7 ND --
T50A 1.9 0.8 ND --
F54A 1.8 0.8 4.4 0.2
I58A 73 32 17 1.9
R64A 13 5.7 21 0.3
Q68A 3.1 1.2 5.2 0.3
R167A 7.4 3.2 0.75 4.3
K168A 58 25 1.1 23
D171A 3.6 1.6 7.1 0.2
K172A 143 62 14 4.4
E174A 59 26 0.22 120
F176A 129 56 16 3.5
R178N 2.4 1.0 8.5 0.1
R178K 6.7 2.9 ND --
I179M 1.3 0.6 2.7 0.2
V185A 3.9 1.7 4.5 0.4
______________________________________
Functional contour maps were derived based upon the location of the
mutations in hGH that disrupt binding to the hGH and hPRL receptors (FIG.
28). The maximal extent of the epitope for the hPRL receptor (FIG. 25B) is
approximated by mutations having less than a two-fold reduction in binding
affinity. By this criteria the epitope for the hPRL receptor is
essentially confined to the front face of helix 1 from F10 to Q29, the
loop from F54 to Q68, and the hydrophilic face helix 4 from R167 to R178.
In contrast, the hGH receptor epitope (FIG. 25A) is comprised of residues
in the amino terminal region through the front face of helix 1 from I4
through M14, the loop region from F54 through S71, and the hydrophilic
face of helix 4 from D171 through V185. Although further mutagenic
analysis will be necessary to fill in remaining gaps in the hPRL epitope,
it is clear this epitope overlaps but is not identical to that for the hGH
receptor. These data suggest that not all of the binding determinants for
recognizing hGH are the same in the hGH and hPRL receptors despite them
sharing 32% sequence identity in their extracellular binding domains.
Residues that cause large changes in receptor binding affinity may do so by
indirect structural effects. However, it is believed that most of these
disruptive effects are due to local effects because all of the single
mutants tested retain full binding affinity to a panel of 8 hGH monoclonal
antibodies and often lead to changes in receptor preference (see Table XIX
and infra) and not uniform disruptions in receptor affinity.
Design of Receptor Specific Variants of hGH.
A number of the single hGH mutants cause enormous changes in receptor
binding preference (Table XIX). The most notable is E174A which causes a
4-fold strengthening in affinity for the hGH receptor while weakening
binding to the hPRL receptor by more than 20-fold. This represents a
120-fold shift in receptor preference. Other mutations (notably R178N and
I179M) cause hGH to preferentially bind to the hPRL receptor. Typically,
the variants that cause the greatest changes in receptor specificity are
located in the non-overlap regions of the two receptor epitopes.
It was reasoned that if the changes in receptor binding free energy were
additive, it could be possible to design highly specific variants of hGH
with only a few mutations. Indeed, when the two most
hGH-receptor-selective single mutants (K168A and E174A) are combined, the
double mutant exhibits a 2300-fold preference for binding to the hGH
receptor (Table XX). As previously indicated, the preference for binding
the hPRL receptor can be enhanced by nearly 20-fold by hPL (56-64) which
contains only two mutations, E56D and R64M (Table XIII). These hGH
variants (K168A,E174A or E56D,R64M) do not substantially reduce the
affinity for the preferred receptor, hGH or hPRL, respectively. It is also
possible to reduce binding to both receptors simultaneously.
TABLE XX
______________________________________
Binding of double mutants of hGH designed to discriminate between
the hGH and hPRL receptors (hGHr and hPRLr). Mutants of hGH were
prepared by site-directed mutagenesis, purified, and assayed for
binding to the hGHr or hPRLr as described in Table XIII. Standard
deviations in the determination of K.sub.D were at or below 20% of the
reported value except where the K.sub.d is above 10 .mu.M, in which case
they
were .+-. 100% of the reported value.
Change in
receptor
hPRLr hGHr preference
K.sub.D (mut) K.sub.D (mut)
hGHr
Mutant K.sub.D (nM)
K.sub.D (hGH)
K.sub.D (nM)
K.sub.D (hGH)
hPRLr
______________________________________
WThGH 2.3 (1) 0.34 (1) (1)
K168A, 1950 590 0.09 0.26 2300
E174A
R18N,
I179M ND -- ND -- --
K172A, .about.40,000
.about.20,000
190 50 .about.40
F176A
______________________________________
For example, combining K172A and F176A, which individually cause large
reductions in binding affinity to the hGH and hPRL receptors, produces
much larger disruptions in affinity of 550 -and 15,000-fold, respectively.
In all these instances the changes in the free energy of binding
(.DELTA..DELTA.G.sub.binding) are strikingly additive (Table XXI).
Additive effects of mutations have been observed in enzyme-substrate
interactions (P. J. Carter, et al. (1984) Cell 38, 835-840; J. A. Wells,
et al., (1987) Proc. Natl. Acad. Sci. USA 84, 5167-5171),
protease-protease inhibitor interactions (M. Laskowski, et al. in Protease
Inhibitors: Medical and Biological Aspects, (1983), eds. N. Katunuma,
Japan Sci. Soc. Press, Tokyo, pp. 55-68, and protein stability (D.
Shortle, et al., (1986) Proteins 1, 81-89 (1986); M. H. Hecht, J. M.
Sturtevant and R.T. Sauer. Proteins 1, 43-46) and, as disclosed in these
references, are most commonly found when the mutant residues function
independently and are in contact with each other. This suggests the
residues paired in the multiple mutants of hGH function independently.
Such additivity creates an extremely predictable situation for engineering
variants of hGH with desirable receptor binding affinity and specificity.
TABLE XXI
______________________________________
Additive effects of mutations in hGH upon binding to
the hGH or hPRL receptors (hGHr or hPRLr). the
change in the free energy of binding (.DELTA..DELTA.G.sub.binding)
for the variant relative to wild-type hGH was
calculated from the reduction in binding affinity
according to: .DELTA..DELTA.G.sub.binding = RT ln›K.sub.D (mut)/K.sub.D
(hGH)!.
The values of (K.sub.D (mut)/K.sub.D (hGH) for the single - or
multiple - mutant hormones were taken from Tables
XIII-XX.
Change in binding free
energy, .DELTA..DELTA.G.sub.binding (kcal/mol)
Mutation hGH hPRLr
______________________________________
K168A +0.04 +1.9
E174A -0.90 +1.9
K168A, E174 (expected)
-0.86 +3.8
(actual) -0.80 +3.8
K172A +2.5 +1.6
F176A +2.4 +1.6
K172A, F176A
(expected)
+4.9 +3.2
(actual) +5.7 +3.8
Q22N -0.06 ND
F25S +1.81 ND
D26E +0.89 ND
Q29S +0.20 ND
E30Q ND ND
E33K -0.13 ND
hPRL 22-33 (expected)
+2.7 --
(actual) +2.6 --
E56A ND +0.8
R64M ND +1.8
E56A, R64M (expected)
-- +2.6
hPL (56-64) (actual) -- +2.0
______________________________________
There are a number of other cases like hGH where two or more receptors or
receptor subtypes are known to exist such as for the adrenergic receptors
(for review see R. J. Lefkowitz and M.G. Caron (1988) J. Biol. Chem. 263,
4993-4996), IGF-I receptors (M. A. Cascieri, et al., (1989) J. Biol. Chem.
264, 2199-2202), IL-2 receptors (R. J. Robb, et al. (1984) J. Exp. Med.
160, 1126-1146; R. J. Robb, et al. (1988) Proc. Natl. Acad. Sci. USA 85,
5654-5658) and ANP receptors (D. Lowe and D. Goeddel, unpublished
results). In these situations it is difficult to link specific receptor
function to a specific pharmacological effect. However, the use of
receptor-specific hormone analogs can greatly simplify this task. For
example, catecholamine analogs were used to characterize .beta.-adrenergic
receptor subtypes and link receptor function to physiologic responses (for
review see R. J. Lefkowitz, et al. (1983) Annu. Rev. Biochem. 52,
159-186). By analogy, the receptor-specific variants of hGH should provide
a key tool for identifying other receptors for hGH, and for probing the
role of the hGH and hPRL receptors in the complex pharmacology of hGH.
This work represents a systematic approach to identifying receptor binding
sites in hormones that permits rational design of receptor specific
variants.
EXAMPLE 13
Engineering Human Prolactin to Bind to Human Growth Hormone
Prolactin (PRL) is a member of a large family of homologous hormones that
includes growth hormones (GH), placental lactogens (PL), and proliferins.
Nicoll, C.S. et. al. (1986) Endocrinol. Rev. 7, 169-203. Collectively,
this group of hormones regulates a vast array of physiological effects
involved in growth, differentiation, electrolyte balance, and others.
Chawla, R. K. et. al. (1983) Ann. Rev. Med. 34, 519-547: Isaksson, O. G.
P. et. al. (1985) Ann. Rev. Physiol. 47, 483-499. These pharmacological
effects begin with binding to specific cellular receptors. For instance,
hPRL binds to the lactogenic but not somatogenic receptor and stimulates
lactation but not bone growth; hGH can bind to both the lactogenic and
somatogenic receptors and stimulates both lactation and bone growth. The
molecular basis for the differences in receptor binding specificity is not
understood.
Cloning and Expression of hPRL.
The cDNA for hPRL was cloned from a human pituitary cDNA library in
.gamma.gt10 (Huynh, T. V. , et al. (1985) in DNA Cloning Techniques: A
Practical Approach, Vol. 1D. M. Glover, ed. (Oxford IRL Press) pp. 49-78)
by hybridization (Maniatis, T., et al., eds. (1982) Molecular Cloning A
Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.)) with oligonucleotide probes corresponding to 5' and 3' extremes of
the published DNA sequence (Cooke, N. E., et al. (1981) J. Biol. Chem.
256, 4007-4016). A near full-length cDNA clone was identified and the 720
-bp BstII-HindIII fragment, extending from codon 12 to 55 bp past the stop
codon, was subcloned into pUC118. The sequence was determined by the
dideoxy method (Sanger, F., et al. (1977) Proc. Natl. Acad. Sci. USA 74,
5463-5467) and matched exactly that previously reported (Cooke, N. E., et
al. (1981) J. Biol. Chem. 256, 4007-4016).
The intracellular expression vector, pBO760 (FIG. 26) was created in
several steps by standard methods (Maniatis, T., et al., eds. (1982)
Molecular Cloning A Laboratory Manual (Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.)). The E. coli trp promoter derived from pHGH207-1
(deBoer, H. A., et al. (1982) in Promoters Structure and Function, eds.
Rodriguez, R. L. & Chamberlin, M. J. (Praeger, New York) pp. 462-481) was
used to transcribe the hPRL gene. The hPRL coding sequence consisted of a
47 -bp XbaI-BstEII synthetic DNA cassette and the 720 -bp BstEII-HindIII
fragment derived from the hPRL cDNA. The synthetic DNA cassette had the
sequence
##STR4##
where the initiation codon is indicated by asterisks. The phage f1 origin,
pBR322 replication origin, and the pBR322 .beta.-lactamase gene were
derived from pBO475 (Cunningham, B. C., et al. (1989) Science 243,
1330-1335).
E. coli cells (MM 294) containing pBO760 were grown at 37.degree. C. for 4
hr (or early log phase; A.sub.550 =0.1 to 0.3) in 0.5 -L shake flasks
containing 100 ml of M9 Hycase media (Miller, J. H. (1972) Experiments in
Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.)) plus 15 .mu.g/ml carbenicillin. Indole acrylic acid was added (50
.mu.g/ml final) to induce the trp promoter. Cells were grown an additional
6-8 hr and harvested by centrifugation. Cell fractionation experiments
showed the hPRL was located almost exclusively in inclusion particles and
represented 2-5% of the total cell protein as analyzed by SDS-PAGE (not
shown).
Purification and Refolding of hPRL. Inclusion particles containing hPRL
were isolated essentially as described (Winkler, M. E., et al. (1986)
Biochemistry 25, 4041-4045). Briefly, 50 g of wet cell paste was suspended
in 0.25 liters, 10 mM TRIS HCl Tris(hydroxymethyl)aminomethane
hydrochloride; (pH 8. 0), 1 mm EDTA (TE buffer) and cells were lysed by
vigorous sonication. Insoluble material was collected by centrifugation
(10,000.times.g for 15 min) and resuspended in 25 ml of TE buffer. The
suspension was layered on a 0.2 liter cushion of 50% glycerol, and
centrifuged at 9,000.times.g for 25 min to pellet the hPRL inclusion
particles. The hPRL from the inclusion particles (about 20% pure) was
suspended in 5 ml of TE buffer.
The hPRL was refolded by solubilizing the inclusion particles in 156 ml of
8N GnHCl in TE buffer plus 0.3 g reduced glutathione (Sigma). After gentle
stirring at room temperature for 30 min, the mixture was chilled to
0.degree. C. and diluted with 844 ml of cold TE buffer plus 0.6 g oxidized
glutathione. The solution was stirred slowly overnight at 40.degree. C.,
and dialyzed with 4 liters of TE buffer that was changed three times over
24 hr. Insoluble material was removed by centrifugation (10,000.times.g
for 20 min).
The refolded and solubilized hPRL was further purified by precipitation
with (NH.sub.4).sub.2 SO.sub.4 to 45% saturation and stirred 2. 5 hr at
room temperature. The precipitate was collected by centrifugation
(12,000.times.g for 30 min) and redissolved in 5 ml of TE buffer. After 30
min at room temperature, the solution was clarified (10,000.times.g for 10
min) and filtered through a millipore filter (0.45 .mu.m). The solution
was dialyzed against 0.5 liters of TE buffer overnight at 4.degree. C. The
hPRL (85% pure) was finally purified to homogeneity (>95%) by FPLC using
DEAE fast-flow matrix essentially as described for purifying hGH
(Cunningham, B. C., et al. (1989) Science 243, 1330-1335).
Mutagenesis and Binding Properties of hGH and hPRL Variants.
Site-specific mutagenesis (Zoller, M. J., et al. (1982) Nucleic Acids Res.
10, 6487-6500) was carried out with the aid of a methylation repair
deficient strain of E. coli, Mut L (Kramer, B., et al. (1984) Cell 38,
879-887). Additional enrichment for mutant clones was obtained by
designing mutagenic oligonucleotides to either introduce or eliminate a
nearby unique restriction site so that restriction-purification or
restriction-selection (Wells, J. A., et al. (1986) Phil. Trans. R. Soc.
Lond. A 317, 415-423), respectively, could be applied to the first pool of
plasmid DNA obtained after transformation of the in vitro-generated
heteroduplex. All oligonucleotides were designed to have 12 bp of exact
match 5' to the most upstream mismatch and 10 bp 3' to the most downstream
mismatch. For mutagenesis of hGH, the previously described hGH synthetic
gene contained multiple restriction sites and was cloned into the plasmid,
pBO475. Variants of hGH were secreted into the periplasmic space of E.
coli (Chang, C. N., et al. (1987) Gene 55, 189-196) and purified as
previously described.
The K.sub.d of each analog was determined by competitive displacement of
›.sup.125 I! hGH bound to the purified recombinant hGH binding protein as
previously described herein and in Spencer, S. A., et al. (1988) J. Biol.
Chem. 263, 7862-7867. The previously described hGH binding protein
(containing residues 1 to 238 of the cloned human liver receptor) was
secreted and purified from E. coli as described in Fuh, G., et al. (1989)
(submitted). Displacement curves were generated in triplicate and the
standard deviations in the K.sub.d values were generally at or below 20%
of the reported values and did not exceed 50% of the reported value except
when K.sub.d values were greater than 10 .mu.M.
The concentrations of hPRL and hPRL mutants were determined by A.sub.280
using a calculated extinction coefficient of .-+.S(0.1%,280)=0.9
(Wetlaufer, D. B. (1962) Adv. in Prot. Chem. 17, 303-390). This was
adjusted accordingly when variants contained mutations in aromatic
residues. Concentration values determined by absorbance agreed to within
10% with those determined by laser densitometry of proteins run on
SDS-PAGE and stained with Coomassie blue for hGH. Circular dichroic
spectra were collected on an Aviv Cary 60 spectropolarimeter.
In order to-probe which of the divergent residues in hPRL were most
disruptive for binding to the hGH receptor (FIG. 27), a number of hPRL
residues were first introduced into hGH (Table XXII).
TABLE XXII
______________________________________
Comparison of hPRL and alanine substitutions
introduced into hGH
K.sub.d (mut)
hGH variant K.sub.d (nM)
K.sub.d (hGH)
______________________________________
WT 0.34 (1)
I58L 0.58 1.7
I58A 5.6 16
R64K 0.20 0.6
R64A 7.1 21
F176Y 2.9 8.6
F176A 5.4 16
R178K 1.7 5.1
R178N 2.9 8.5
______________________________________
Whereas single alanine substitutions in hGH at positions 58, 64, 176 and
178 strongly disrupted receptor binding, substitutions of hPRL residues
into hGH at these positions had less of an effect. The largest effects for
hPRL substitutions were in the helix 4 residues that included positions
176 and 178. These data suggested that residues in the helix 4 region of
hPRL could best account for the lack of binding to the hGH receptor.
The recombinant hPRL retained native-like structural and functional
properties. First, the near and far ultraviolet CD spectra (FIG. 28) are
identical to published spectra of natural hPRL (Bewley, T. A. (1979) in
Recent Progress in Hormone Research, vol. 35, pp. 155-213Acad. Press, New
York.). The far ultraviolet spectrum is similar to hGH, suggesting a
similar 4-helix bundle structure, although important differences in the
mean residue ellipticity at 208 and 224 nm have been noted (Id. ). These
hormones differ markedly in the near ultraviolet CD spectra which reflects
variation in number and microenvironment of the aromatic residues between
hGH and hPRL. In other studies (not shown), the recombinant hPRL retained
full immunological cross-reactivity in an hPRL ELISA, and was equipotent
with hGH in causing rat lymphoma Nb2 cells to proliferate (Tanaka, T., et
al. (1980) J. Clin. Endo. Metab. 51, 1058-1063). Upon reduction, the
purified hPRL showed a pronounced retardation in mobility by SDS-PAGE (as
seen for hGH) suggesting that disulfide bonds had formed (Pollitt, S., et
al. (1983) J. Bacteriol. 1, 27-32). Amino-terminal sequence analysis
showed that the intracellularly expressed hPRL retained the amino-terminal
methionine; however, as with methionyl-hGH (Olson, K. C., et al. (1981)
Nature (London) 293, 408-411), this does not apparently affect its
structure or function.
Binding of hPRL to the hGH binding protein is reduced by more than 10.sup.5
-fold compared to hGH (Table XXIII) which is below the detection limit of
our binding assay.
TABLE XXIII
______________________________________
Engineering residues in hPRL to permit binding to the
hGH binding protein.sup.1
Kd (mut)
hPRL Variant K.sub.d (nM).sup.2
K.sub.d (hGH)
______________________________________
hPRL WT >40,000 >100,000
A = H171DN175TY176F
4,900 14,000
B = A + K178R 220 660
B + hGH (184-188) 260 740
hGH (54-74) .sup..about. 25,000
.sup..about. 66,000
B + hGH (54-74) 2,000 5,800
B + H54FS56E:L58I: 36 110
E62S:D63N:Q66E
B + H54F:S56E:L58I 670 2,000
C = B + E174A 68 200
D = C + E62S:D63N:Q66E
2.1 6.2
D + H54F 4.4 13
D + S56E 2.5 7.4
D + L58I 3.6 11
D + A59P 2.5 7.4
D + N71S 3.6 11
D + L179I 2.1 6.2
______________________________________
.sup.1 Mutants of hPRL were generated, purified and analyzed as described
Multiple mutants are indicated by a series of single mutants (Table XXII)
separated by colons. Codon numbering is based upon the hGH sequency (FIG.
2).
.sup.2 Average standard errors are at or below 20% of the reported values
except in cases where the K.sub.d exceeds 1 .mu.M, where errors can be as
large as 50%, and errors are much larger still when K.sub.d exceeds 10
.mu.M.
A combination of three divergent residues in helix 4 from hGH (H171D,
N175T, and Y176F) were introduced into hPRl. Alanine scanning mutagenesis
and hPRL substitutions (Table XXII) had shown that these residues were
very important for binding hGH to the hGH receptor. This triple mutant of
hPRL exhibited detectable binding to the hGH binding protein albeit
14,000-fold weaker than hGH. Installation of another important helix 4
residue (K178R) to produce a tetramutant (called variant B in Table XIII)
further strengthened binding to a level now only 660-fold below wild-type
hGH. Additional incorporation of hGH residues 184 to 188 into hPRL variant
B did not enhance binding to the hGH binding protein. However,
introduction of E174A to give hPRL variant C (Table XXIII) caused an
additional 3.5-fold increase in binding affinity to the hGH binding
protein as was found when E174A was incorporated into hGH.
Having engineered binding with the helix 4 region, the loop region
containing residues 54 to 74 was analysed. Complete replacement of the
loop region in hPRL with the sequence from hGH (hGH (54-74) in Table XIII)
gave barely detectable binding to the hGH binding protein. When this
mutant was combined with variant B, the binding affinity increased
substantially. However, this new variant ›B plus hGH (54-74)! was reduced
in binding affinity by almost 10-fold from variant B alone. Thus, it
appeared that some of the hGH residues in the 54-74 loop were not
compatible with the hGH substitutions in helix 4. We then selected from
the 54 to 74 loop of hGH only those seven residues that were shown by
alanine-scanning mutagenesis to most greatly influence binding. Although
the R64A mutation in hGH caused more than a 20-fold reduction in binding
affinity, the R64K variant of hGH (which is an hPRL substitution) slightly
enhanced binding to the hGH binding protein (Table XXII). The Lys64 in
hPRL therefore was left unchanged. As a consequence, only six of the seven
substitutions from hGH were incorporated into hPRL that were most
disruptive when changed to alanine in hGH. This new mutant (B plus
H65F:S56E:L58I:E56S:D68N:Q66E) binds fifty-fold stronger than B plus hGH
(54-74) and was only 110-fold reduced in binding affinity from wild-type
hGH (Table XXIII). However, this represented only a modest improvement
(six-fold) over variant B alone, which was less than expected for strongly
favorable interactions previously observed in the loop region for hGH.
Therefore, the six mutations within the loop were further dissected and
revealed that the combination of H54F:S56E:L58I plus variant B bound
three-fold weaker than variant B alone. Finally, incorporating the
mutations E62S:D63N:Q66E into variant C (to give variant D) produced an
analog with highest affinity that was only 6-fold reduced in binding
affinity relative to hGH. Additional single mutations (H54F, S56E, L58I,
A59P, M71S and L179I) did not enhance the binding affinity of hPRL variant
D to the hGH binding protein. The conformation of variant D was virtually
indistinguishable from that of native hPRL by CD spectral analysis (FIG.
28) or by ELISA reactivity (not shown).
These studies demonstrate the feasibility of recruiting binding properties
for distantly related homologs using only functional information derived
from site-directed mutagenesis experiments. Alanine-scanning mutagenesis
of hGH provided a systematic analysis of side-chains that were important
for modulating binding of hGH to its receptor (FIG. 27).
This information highlighted a number of residues in hPRL that could
account for its inability to bind to the hGH receptor (FIG. 29). However,
further analysis showed that the alanine substitutions in hGH were more
disruptive than the hPRL substitutions in hGH (Table XXII). Furthermore,
some of the hPRL substitutions were considerably more disruptive than
others for binding affinity, especially when a larger side-chain was
present in hPRL. For example, the conservative (but larger) F176Y mutation
in hGH caused an eight-fold reduction in binding affinity with the hGH
receptor, whereas the smaller R64K substitution showed slightly enhanced
binding affinity. Thus, the analysis of disruptive hPRL substitutions in
hGH suggested the introduction of the cluster of divergent residues in
helix 4 to initially achieve binding affinity for hPRL. This was very
important because no binding to the hGH receptor with wild-type hPRL had
been observed, and it was necessary to introduce several hGH substitutions
simultaneously into hPRL in order to bring the binding affinity within the
range of the assay used (K.sub.d .ltoreq.50 .mu.M).
Readily detectable binding affinity was engineered into hPRL by
incorporating functionally important residues into helix 4. However,
engineering the loop region between 54-74 turned out to be more difficult.
Installing the entire loop from hGH into hPRL produced less enhancement in
binding than expected, and was disruptive to binding when combined with
the optimized helix 4 variant B. Our data suggest that the 54-74 loop
structure in hPRL is supported by other interactions in the protein. This
problem was solved in stages. First, only those six loop residues from hGH
that the alanine scan together with the hPRL substitutions in hGH had
identified to be important were introduced into hPRL. Although this
improved the situation, the combination of some of these hGH mutations
(narrowed down to H54F, S56E, and L58I) were disruptive to hPRL. These
data suggest that some of the residues in the loop are crucial for its
structure and are better off being left alone.
A number of iterative cycles of mutagenesis were necessary to converge upon
a combination of residues that permitted tight binding of hPRL to the hGH
receptor. This strategy relies on the assumption that the mutational
effects will be somewhat additive as was, in fact, observed. For example,
the E174A mutation enhanced the binding three to five-fold when added to
either hPRL variant C or hGH. Moreover, the product of the disruptive
effects of the H54F, S56E, and L58I single mutants to variant D (4.
4-fold) is about the same as the disruption caused by the combination of
all three mutations added to variant B (3-fold).
Even though variant D is only six-fold reduced in binding affinity, there
are several other residues that could be incorporated into variant D to
try to improve further on the binding, such as V14M and H185V; these are
sites where alanine substitutions in hGH cause two to five-fold reductions
in binding of hGH (FIG. 29). Although a high resolution structure would
have aided in the design process, it was clearly not essential. The
cumulative nature of the mutational effects allows one to converge upon
the binding property in much the same way as proteins evolve, by cycles of
natural variation and selection.
Previous protein engineering experiments have shown it is possible using
high-resolution structural analysis to virtually exchange the substrate
specificity of natural variant enzymes by site-directed mutagenesis of
substrate contact residues (Wells, J. A., et al. (1987) Proc. Natl. Acad.
Sci. USA 84, 5167-5171; Wilks, H. M., et al. (1988) Science 242,
1541-1544). Similarly, others have shown that binding properties can be
engineered by replacement of entire regions of secondary structure units
including antigen binding loops (Jones, P. T., et al. (1986) Nature 321,
522-525) or DNA recognition helices (Wharton, R. P., et al. (1985) Nature
316,601-605). However, to recruit the hGH receptor binding properties into
hPRL required selective residue replacements within the structural
scaffold of hPRL. Furthermore, the CD spectral data show that the overall
structure of the hPRL variant D resembles more closely the structure of
hPRL not hGH even though it attains binding properties like hGH.
The fact that the binding specificity for the hGH receptor could be
incorporated into hPRL confirms the functional importance of particular
residues for somatogenic receptor binding. These studies also provide
compelling proof for structural relatedness between hGH and hPRL despite
them having only 23% identity. This provides a rational approach to access
new receptor binding functions contained within this hormone family
starting with either a growth hormone, prolactin, proliferin or placental
lactogen scaffold. Such hybrid molecules should be useful for
distinguishing receptor binding and activation as well as the
pharmacological importance of receptor subtypes. These analogs could lead
to the design of new receptor-specific hormones having more useful
properties as agonists or antagonists.
EXAMPLE 14
Recruitment of Binding Properties of Human Growth Hormone into Human
Placental Lactogen.
Human placental lactogen (hPL) is reduced over thirty-fold in binding
affinity compared to hGH for the hGH receptor (G. Baumann, et al., (1986)
J. Clin. Endocrinol. Metab. 62, 134; A. C. Herington, et al. (1986) J.
Clin. Invest. 77, 1817). Previous mutagenic studies showed the binding
site on hGH for the hGH receptor is located primarily in two regions
(including residues 54-74 and 171-185) with some minor determinants near
the amino terminus (residues 4-14).
The overall sequence of hPL is 85% identical to hGH. Within the three
regions that broadly constitute the receptor binding epitope on hGH, hPL
differs at only seven positions and contains the following substitutions:
P2Q, I4V, N12H, R16Q, E56D, R64M, and I179M. (In this nomenclature the
residue for wild-type hGH is given in single letter code, followed by its
position in mature hGH and then the residue found in hPL; a similar
nomenclature is used to describe mutants of hGH.) Single alanine
substitutions have been produced in hGH at each of these seven positions.
Of these, four of the alanine substitutions were found to cause two-fold
or greater reductions in binding affinity including I4A, E56A, R64A, and
I179A. Generally, the alanine substitutions have a greater effect on
binding than homologous substitutions from human prolactin. Therefore, the
effect of some of the substitutions from hPL introduced into hGH were
investigated.
Whereas the I179A substitution caused a 2. 7-fold reduction in affinity the
I179M substitution caused only a slight 1. 7-fold effect. However, the
R64A and R64M substitutions caused identical and much larger reductions
(about 20-fold) in binding affinity. Moreover, the double mutant
(E56D:R64M) in hGH was even further reduced in affinity by a total of
30-fold (Table I). Thus, E56D and R64M primarily determine the differences
in receptor binding affinity between hGH and hPL. The double mutant D56E,
M64R in hPL therefore substantially enhances its binding affinity for the
hGH receptor. Additional modifications such as M179I and V4I also enhance
binding of hPL to the hGH receptor.
EXAMPLE 15
Effect of Amino Acid Replacement at Position 174 on Binding to the Human
Growth Hormone.
As previously indicated, replacement of Glu174 with Ala (E174A) resulted in
more than a 4-fold increase in the affinity of human growth hormone (hGH)
for its receptor. To determine the optimal replacement residue at position
174 hGH variants substituted with twelve other residues were made and
measured to determine their affinities with the hGH binding protein (Table
XXIV). Side-chain size, not charge, is the major factor determining
binding affinity. Alanine is the optimal replacement followed by Ser, Gly,
Gln, Asn, Glu, His, Lys, Leu, and Tyr.
TABLE XXIV
______________________________________
Side chain Kd (mut)
Mutant.sup.a
Charge Size (.ANG..sup.3).sup.b
Kd (nM).sup.c
Kd (wild type)
______________________________________
E174G 0 0 0.15 0.43
E174A 0 26 0.075 0.22
E174S 0 33 0.11 0.30
E174D - 59 NE --
E174N 0 69 0.26 0.70
E174V 0 76 0.28 0.80
wild-type
- 89 0.37 1.0
E174Q 0 95 0.21 0.60
E174H 0 101 0.43 1.2
E174L 0 102 2.36 6.4
E174K + 105 1.14 3.1
E174R + 136 NE --
E174Y 0 137 2.9 8.6
______________________________________
.sup.a Mutations were generated by sitedirected mutagenesis (Carter, P.,
et al. (1986) Nucleic Acid Res. 13, 4431-4443) on a variant of the hGH
gene that contains a KpnI site at position 178 cloned into pB0475.
Oligonucleotides used for mutagenesis had the sequence:
##STR5##
where NNN represents the new codon at position 174 and asterisks indicate
the mismatches to eliminate the KpnI site starting at codon 178. Mutant
codons were as follows: Gln, CAG; Asn, AAC; Ser, AGC; Lys, AAA; Arg, AGG;
His, CAC; Gly, GGG; Val, GTG; Leu, CTG. Following heteroduplex synthesis
the plasmid pool was enriched for the mutation by restriction with KpnI t
reduce the background of wildtype sequence. All mutant sequences were
confirmed by dideoxy sequence analysis (Sanger, F., et al. (1977) Proc.
Natl. Acad. Sci. USA 74, 5463-5467).
.sup.b Sidechain packing values are from C. Chothia (1984) Annu. Rev.
Biochem. 53, 537.
.sup.c Dissociation constants were measured by competitive diplacement of
›.sup.125 I!hGH from the hGH binding protein as previously described. NE
indicates that the mutant hormone was expressed at levels too low to be
isolated and assayed.
EXAMPLE 16
The hGH variants shown in Table XXV were constructed. Their relativity
potency as compared to wt-hGH are shown.
TABLE XXV
______________________________________
Relative potency in
hGH mutant rat weight gain assay
______________________________________
F97A 0.87
S100A 2.12
L101A 3.03
V102A 1.39
Y103A 1.73
T175S 1.21
______________________________________
Having described the preferred embodiments of the present invention, it
will appear to those ordinarily skilled in the art that various
modifications may be made to the disclosed embodiments, and that such
modifications are intended to be within the scope of the present
invention.
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