Introduction of Protein expression systems

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The present invention provides an improved expression system for the production of recombinant polypeptides utilizing auxotrophic selectable markers. In addition, the present invention provides improved recombinant protein production in host cells through the improved regulation of expression.

This application is a divisional of U.S. application Ser. No. 10/994,138, filed Nov. 19, 2004, now abandoned which claims the benefit of U.S. Provisional patent application Ser. No. 60/523,420, filed Nov. 19, 2003, entitled “Improved Pseudomonas Expression Systems with Auxotrophic Selection Markers,” and U.S. Provisional patent application 60/537,147 filed Jan. 16, 2004, and entitled “Bacterial Expression Systems with Improved Repression,” each of which is hereby incorporated in its entirety by reference herein.

The instant application contains a Sequence Listing which has been submitted as a text file via EFS-Web in compliance with the American Standard Code for Information Interchange (ASCII), and is incorporated herein by reference in its entirety. Said ASCII copy, created on Jan. 17, 2012, is named 38194705.txt and is 41,154 bytes in size.

The present invention provides an improved expression system for the production of recombinant polypeptides utilizing auxotrophic selectable markers. In addition, the present invention provides improved recombinant protein production in host cells through the improved regulation of expression.

The use of bacterial cells to produce protein based therapeutics is increasing in commercial importance. One of the goals in developing a bacterial expression system is the production of high quality target polypeptides quickly, efficiently, and abundantly. An ideal host cell for such an expression system would be able to efficiently utilize a carbon source for the production of a target polypeptide, quickly grow to high cell densities in a fermentation reaction, express the target polypeptide only when induced, and grow on a medium that is devoid of regulatory and environmental concerns.

There are many hurdles to the creation of a superior host cell. First, in order to produce a recombinant polypeptide, an expression vector encoding the target protein must be inserted into the host cell. Many bacteria are capable of reverting back into an untransformed state, wherein the expression vector is eliminated from the host. Such revertants can decrease the fermentation efficiency of the production of the desired recombinant polypeptide.

Expression vectors encoding a target peptide typically include a selection marker in the vector. Often, the selection marker is a gene whose product is required for survival during the fermentation process. Host cells lacking the selection marker, such as revertants, are unable to survive. The use of selection markers during the fermentation process is intended to ensure that only bacteria containing the expression vector survive, eliminating competition between the revertants and transformants and reducing the efficiency of fermentation.

As a result of these concerns, many governmental food, drug, health, and environmental regulatory agencies, as well as many end users, require that antibiotic resistance gene nucleic acid be removed from products or be absent from organisms for use in commerce. In addition, evidence demonstrating clearance of the selection antibiotics from the final product must be provided in order to secure regulatory clearance. The United Kingdom, Canada, France, the European Community, and the United States have all addressed the use of antibiotic resistance genes in foods, animal feeds, drugs and drug production, including recombinant protein drug production. Clearance of these agents, and especially demonstrating such clearance, is expensive, time consuming, and often only minimally effective.

Because of the concerns inherent in the use of antibiotic resistance genes for selection in the production of recombinant polypeptides, alternative selection methods have been examined.

Auxotrophic selection markers have been utilized as an alternative to antibiotic selection in some systems. For example, auxotrophic markers have been widely utilized in yeast, due largely to the inefficiency of antibiotic resistance selection markers in these host cells. See, for example, J T Pronk, (2002) “Auxotrophic yeast strains in fundamental and applied research,” App. & Envirn. Micro. 68(5): 2095-2100; Boeke et al., (1984) “A positive selection for mutants lacking orotodine-5′-phosphate decarboxylase activity in yeast; 5-fluoro-orotic acid resistance,” Mol. Gen. Genet. 197: 345-346; Botstein & Davis, (1982) “Principles and practice of recombinant DNA research with yeast,” p. 607-636, in J N Strathern, E W Jones. And J R Broach (ed.), The molecular biology of the yeast Saccharomyces cerevisiae, Metabolism and gene expression, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Cost & Boeke, (1996) “A useful colony color phenotype associated with the yeast selectable/counter selectable marker MET15,” Yeast 12: 939-941. However, yeast expression systems due not provide the potential speed and efficiency for producing target proteins that bacterial systems do.