Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1990-08-27
1993-12-07
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 694, 4353201, 536 2351, 536 241, 935 29, 935 39, 935 41, C07H 2100, C12N 1518, C12N 1570, C12N 1571
Patent
active
052682846
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
This invention relates to methods for enhancing expression of heterologous polypeptides. More specifically, the invention relates to the enhancement of expression of heterologous polypeptides by altering the ribosome binding sites operatively linked thereto.
BACKGROUND OF THE INVENTION
Some heterologous polypeptides, including bovine somatotropin (BSt), are difficult to express in most E. coli expression systems. Modifications within the first 4 codons of the CDNA encoding the BSt structural gene result in increased levels of BSt expression when these modified cDNAs are expressed in a pBR322-related vector. These modified cDNAs are expressed at even higher levels when placed in a runaway expression vector (U.S. patent application Ser. No. 016,294, filed 19 Feb. 1987 and incorporated herein by reference). The modified ribosome binding sites of the instant invention produce expression of heterologous polypeptides with a non-runaway vector, for example, a derivative of plYR322, equivalent to that achieved with a runaway vector.
Generally, methods for cloning and expressing heterologous polypeptides in transformed hosts are well known to those skilled in the art. Such heterologous polypeptides include, for example, human insulin, growth hormone, interferon and factor VIII, viral antigens, and other animal hormones.
The ribosome binding site is one of the elements involved in such expression. It covers the region about 20 nucleotides on both sides of the initiation codon and contains the Shine-Dalgarno sequence usually 6-10 nucleotides upstream from the initiation codon. The 20 nucleotides after the initiation codon includes the beginning of the gene sequence inserted for expression and often cannot be subjected to modifications without changing the amino acid sequence of the gene. Therefore, in a practical sense, manipulation of the ribosome binding site can only be carried out at the region upstream from the initiation codon. The ribosome binding site is known to function best if it contains certain bases but there is no known requirement for any particular base in a particular sits except that the Shine-Dalgarno sequence is generally purine-rich. The subtle differences among various known ribosome binding site sequences do not provide an obvious comparison to predict which genes will be highly and which will be poorly expressed when associated therewith. A preferred embodiment of the present invention utilizes sequences that are rich in A and T nucleotides to flank the Shine-Dalgarno sequence thereby producing ribosome binding sites that have an increased number of adenine and thymidine residues as compared to the naturally occurring ribosome binding sites from which they are derived.
Naturally occurring 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 using 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 Bausan, 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.).
Recombinant bovine somatotropin (rBSt) can be produced in transformed microorganisms using a variety of recombinant genetic plasmids (see, e.g., Seeburg, P. H., et al., "Efficient Bacterial Expression of Bovine and Porcine Growth Hormones," DNA, 2:37-45 (1983)); European Patent Application 47 600; United Kingdom Patent Application, GB 2073245A; Schoner, B. E., et al., Role of mRNA Translational Efficiency in Bovine Growth Hormone Expression in Escherichia coli, PNAS USA, 81:5403-5407 (1984); European Patent A
REFERENCES:
patent: 4582800 (1986-04-01), Crowl
Cho, K. et al. "Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA Translation and Decay," J. Mol. Biol. 204:51-60 (1988).
Seeburg, Peter H. "Efficient Bacterial Expression of Bovine and Porcine Growth Hormones", DNA 2:37-45, (1983).
George, Henry J. "High-level Expression in E. coli of Biologically Active Bovine Hormone," DNA 4:273-281 (1985).
Lewin, Benjamin Genes III pp. 193-200 (1987).
Schoner, B. E., et al., Role of mRNA Translational Efficiency in Bovine Growth Hormone Expression in Escherichia coli, PNAS USA, 81, pp. 5403-5407 (1984).
Gold, L., et al., Ann. Rev. Microbiol., 35, pp. 365-403 (1981).
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DeBoer, Herman A., et al., DNA, 2, pp. 231-235 (1983).
Olsen Mary K.
Tomich Che-Shen C.
Jacobson Dian C.
Patterson Jr. Charles L.
The Upjohn Company
Williams, Jr. Sidney B.
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