Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1991-01-22
1994-07-19
Hill, Jr., Robert J.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
435 697;320.1;252.33, 530399, 514 12, C12N 1500, A61K 3702
Patent
active
053309715
DESCRIPTION:
BRIEF SUMMARY
This invention relates to growth factors, their production as fusion proteins and use as well as to the production of other proteins as fusion proteins. As used herein, the term methionine porcine growth hormone (metpGH) means porcine growth hormone in which methionine has been substituted for the amino acid normally at the N-terminus.
Insulin-like growth factor-I (IGF-I) is a small protein that has been shown to stimulate the growth of a wide range of cells in culture. Animal growth is also stimulated in pituitary-deficient, normal and catabolic states.
Human, bovine and porcine IGF-I all share the identical sequence, shown here using the single letter amino acid code and numbering from the amino terminus: ##STR1## The animal and cell growth results have lead to the interpretation that IGF-I may usefully be applied: states such as following burns, infection or other trauma; enhance food conversion efficiency; and
There is accordingly a commercial demand for mammalian IGF-I for use in animal growth trials, clinical investigations and for cell culture. However, yields from natural sources and/or from recombinant DNA synthesis methods remain low.
Accordingly it is an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art.
Accordingly in a first aspect of the present invention there is provided a plasmid including
a suitable expression vector;
a first DNA sequence coding for a first polypeptide having growth hormone activity, or fragment thereof; and
a second DNA sequence joined to the 3' end of the first DNA sequence and coding for a second polypeptide.
It will be understood that the constructs so formed may express fusion proteins in inclusion bodies in high yields and exhibiting biological activity comparable to those in the prior art developed for growth hormone alone. Disruption of the organisms, isolation of the fusion protein from the inclusion bodies, oxidation to achieve correct disulphide bonds and purification may yield an extended polypeptide, for example a biologically active insulin-like growth factor. If sequences coding for a clearable sequence are introduced at the 3' end of the first DNA sequence, the addition of a cleavage step may yield a polypeptide that is not extended.
The suitable cloning vector may be selected from plasmid cloning vectors. The plasmid cloning vectors may be utilized in the process for production of insulin-like growth factor fusion proteins in high yield as discussed below. The plasmid cloning vector may be plasmid pGHXSC.4 that has a modified RBS/spacer region and strategic 5'-codon alterations downstream from the powerful trc promoter (J. Brosius et al. J. Biol. Chem. 260, 3539, 1985; P. D. Vize & J. R. E. Wells, FEBS Lett. 213, 155, 1987).
The first DNA sequence coding for a peptide having growth hormone activity or fragment thereof may code for a peptide having porcine growth hormone (pGH) activity. A methionine porcine growth hormone sequence (metpGH or MpGH) is preferred. The first DNA sequence may code for all 191 N-terminal amino acids of metpGH or any fragment thereof. The first DNA sequence may code for approximately the first 100 N-terminal amino acids of metpGH, more preferably approximately the first 46 N-terminal amino acids, most preferably the first 11 N-terminal amino acids of metpGH.
In a preferred aspect of the present invention, the first DNA sequence may include a clearable sequence at the 3' end thereof.
Specifically, in a preferred aspect of the present invention the first DNA sequence codes for an amino acid sequence selected from
These sequences represent, in order, the initiating methionine, the first 46 amino acids of pGH, valine, asparagine, phenylalanine, alanine, histidine and tyrosine.
In an alternate preferred aspect of the present invention the first DNA sequence codes for an amino acid sequence selected from:
These sequences represent, in order, the initiating methionine, the first 10 amino acids of pGH, valine, asparagine, glycine, phenylalanine, alanine, histidine a
REFERENCES:
patent: 5019500 (1991-05-01), Ueda et al.
patent: 5028531 (1991-07-01), Ueda et al.
patent: 5077276 (1991-12-01), Ballard et al.
Henderson, E., Henderson's Dictionary of Biological Terms, 10th Edition, Longman Scientific and Technical, UK, 1989, p. 216.
Ballard, F. J. et al. in Modern Concepts of Insulin-Like Growth Factors, Elsevier Science Publishing, 1991, pp. 617-627.
Read, L. C. et al. in Modern Concepts of Insulin-Like Growth Factors, Elsevier Science Publishing, 1991, pp. 225-234.
Tomas, F. M. et al., Biochem. J., 282:91-97, 1992.
Read, L. C. et al., Proceedings of the Nutrition Society of New Zealand, 17:136-143, 1992.
Read, L.C. et al., J. of Endocrinology, 133:421-31, 1992.
Ballard, F. J. et al., Growth Regulation, 3(1),:40-44, 1993.
Ballard, F. J. et al., Aust. J. Agric. Res., 44:1-11, 1992.
Tomas, F. M. et al., Biochem. J., 291:1-6, 1993.
Francis, G. L. et al., J. of Mol. Endocrinology, 8:213-223, 1992.
Sproat, B. S., NAR, 13(8):2959-2979, 1985.
Kadonaga, J. T. et al., Cell, 51:1079-1090, 1987.
D'Andrea, G. et al., NAR, 9(13):3119-3128, 1981.
Humbel, R., Eur. J. Biochem., 190:445-462, 1990.
Brosius et al., J. Biol. Chem., 260, 3539-3541 (1985).
Carter et al., Proteins: Structure, Function, and Genetics, 6, 240-248, (1989).
Amman et al., Gene, 40, 1983-190 (1985).
Messing, Recomb. DNA Tech. Bull., 2 43-48 (1979).
Francis et al., Biochem. J., 233, 207-213 (1986).
Rinderknecht et al., FEBS Lett., 89, 283-286 (1978).
Ballard et al., Aust. J. Agric. Res., 44, 1-11 (1992).
Cascieri, M. A. et al., Endocrinology, 122(4):1314-1320, 1988.
Misaka, F. et al., Biotechnology Letters, 11(12):839-44, 1989.
Moore, H. H. et al., Nature, 321:443-446, 1986.
Bayne, M. L. et al., PNAS, 84:2638-42, 1987.
Iwai, S. et al., Chem. Pharm. Bull., 34(11):4724-4730, 1986.
Bagley, C. J. et al., Biochem. J., 259:665-671, 1989.
Ballard, F. J. et al., Biochem. Biophys. Res. Comm., 149(2):398-404, 1987.
Vize, P. D. et al., FEBS Letters, 213(1):155-58, 1987.
Vize, P. D. et al., Gene, 55:339-44, 1987.
Nishikawa, S. et al., Protein Engineering, 1(6):487-92, 1987.
Francis Geoffrey L.
King Robert M.
Wells Julian R. E.
Allen Marianne Porta
GroPep Pty. Ltd.
Hill Jr. Robert J.
LandOfFree
Growth hormone fusion proteins, methods of production, and metho does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Growth hormone fusion proteins, methods of production, and metho, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Growth hormone fusion proteins, methods of production, and metho will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-520054