Growth hormone variants

Details Growth hormone variants Growth hormone variants

1995-06-07

2004-08-24

Saoud, Christine J. (Department: 1647)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S243000, C435S320100, C435S325000, C530S399000, C536S023510

Reexamination Certificate

active

06780613

ABSTRACT:

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
228, 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 immunologi

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