Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1989-08-18
1993-08-31
Wax, Robert A.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
43525233, 4353201, 536 2351, 536 241, 935 27, 935 29, 935 41, 935 42, C07H 2100, C12N 1518, C12N 1569, C12N 1570
Patent
active
052408370
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to the expression of heterologous polypeptides. More specifically, the invention discloses novel cDNAs encoding somatotropins which are expressed at high levels in E. coli. A generic expression system comprising a mutated E. coli host cell and an expression vector having a dual replicon arrangement also is disclosed herein.
INFORMATION DISCLOSURE
Expression of somatotropins from a variety of animals by transformed microorganisms is known (Goeddel, D. V. et al., "Direct Expression in Escherichia coli of a DNA sequence coding for human growth hormone", Nature, 281:544-548 (1979); and Seeburg, P. H. et al., "Efficient Bacterial Expression of Bovine and Porcine Growth Hormones", DNA, 2:37-45 (1983)).
Naturally occurring bovine somatotropin (BSt) is a mixture of heterogeneous proteins, the amino acid sequences of which are known (Paladini, A. C., et al., Molecular Biology of Growth Hormone, CRC Reviews in Biochem., 15(1):25-56 (1983)). The naturally occurring mixtures have been purified from pituitary glands of cattle. The commercial potential for the use of BSt for promoting growth and lactation is well recognized and documented by biological studies on both dairy and feed cattle (Eppard, P. J. and Bauman, D. E., The Effect of Long-Term Administration of Growth Hormone on Performance of Lactating Dairy Cows; and Bauman, D. E., Effect of Growth Hormone on Growth Rates and Mammary Development of Ruminants, Proc. 1984 Cornell Nutrition Conference for Feed Manufacturers, pp. 5-17, published by Cornell University, Ithaca, N.Y.).
Recobinant bovine somatotropin (rBSt) can be produced in transformed microorganisms using a variety of recombinant genetic plasmids (European Patent Application 47 600; United Kingdom Patent Application, GB 2073245A; and Schoner, B. E. et al., Role of mRNA Translational Efficiency in Bovine Growth Hormone Expression in Escherichia coli, Proc. Natl. Acad. Sci. USA, 81:5403-5407 (1984)).
Analogs of BSt are also known (European Patent Application 103 395; and Schoner, B. E., et al., supra). Unlike the present invention, these analogs of BSt relate to the insertion or deletion of bases at the 5' end of the BSt gene thereby creating a protein different from the naturally-occurring amino acid sequences. Modifications to rBSt cDNAs to maximize preferred codons include changing the first two native alanine codons of GCC to GCT (European Patent Application 111,814). However, EP 111,814 teaches that preferred codon substitution and reduction of secondary structure is critical towards optimizing expression. The instant invention demonstrates that many of these known changes are not necessary to achieve high levels of expression when the cDNAs are combined with a runaway-replicon-type plasmid.
Methods for culturing and fermenting transformed microorganisms expressing BSt are also known and fully described in the above-cited literature.
Purification of biologically active rBSt from transformed cells has also been described previously (U.S. Pat. Nos. 4,511,502; 4,511,503; 4,512,922; 4,518,526; European Patent Application 131 843; and, Schoner, R. G., et al., "Isolation and Purification of Protein Granules from E. coli Cells Overproducing BSt", Bio-Tech., 3:151-154 (1985)).
The instant invention discloses cDNAs encoding somatotropins and analogs of somatotropins that are expressed at high levels when compared to the native somatotropin cDNA. It is known that native cDNAs of porcine and bovine somatotropin are not expressed at commercially acceptable levels in most E. coli systems and that changes in the cDNA are required for such expression. Attempts to increase the percentage of preferred codons in the cDNA have had little affect and workers have had to resort to substantial modifications of the cDNA to reach acceptably high levels of expression. Seeburg, P. H. et al., DNA, 2:37-45 (1983) increased expression by eliminating the strong secondary structure resident after the condon for amino acid residue 11 in the native cDNA of bovine somatotropin (see als
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Wright, E. M. et al., "Dual-Origin Plasmids Containing an amplifiable ColElOri" Gene 49:311-321 (1986).
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B. E. Uhlin et al.: "New runaway replication-plasmid cloning vectors and suppression of runaway replication by novobiocin." Gene, 22, 255-65, (1983).
D. V. Goeddel et al., Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone, Nature, 281:544-548 (1979).
P. H. Seeburg et al., Efficient Bacterial Expression of Bovine and Porcine Growth Hormones, DNA, 2:37-45 (1983).
A. C. Paladini et al., Molecular Biology of Growth Homone, CRC Reviews in Biochem., 15(1):25-56 (1983).
P. J. Eppard and D. E. Bauman, The Effect of Long-Term Adminstration of Growth Hormone on Performance of Lactating Dairy Cows; and Effect of Growth Hormone on Growth Rates and Mammary Development of Ruminants, Proc. 1984 Cornell Nutrition Conference for Feed Manufacturers, pp. 5-17, published by Cornell University, Ithaca, New York.
B. E. Schoner et al., Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli, PNAS USA, 81:5403-5407 (1984).
R. G. Schoner et al., Isolation and Purification of Protein Granules from Escherichia Coli Cells Overproducing Bovine Growth Hormone, Bio-Tech., 3:151-154 (Feb. 1985).
Mott John E.
Olson Eric R.
Tomich Che-Shen C.
Darnley, Jr. James D.
Jacobsons Dian
Koivuniemi Paul J.
Steele Gregory W.
The Upjohn Company
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