Development of an escherichia coli culture and fermentation process for PL-regulated expression of HIV-1 protease.

Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.

Development of an escherichia coli culture and fermentation process for PL-regulated expression of HIV-1 protease.

Abstr Annu Meet Am Soc Microbiol. 1991 May 5-9;91:190 (abstract no. H-212). Unique Identifier : AIDSLINE ASM91/0140292
Strakalaitis N; Hoogerheide J; Mott J; Tomich CS; Brunner D; Vanzanten R; The Upjohn Co., Kalamazoo, MI.

Abstract: HIV-1 protease consists of 99 amino acids and shares activity and homology with aspartyl proteases, such as pepsin and renin. Specific protease inhibitors block viral replication and infection, and are potential therapeutic agents for the treatment of AIDS. To date, it has been difficult to appreciably express the protein. We cloned the protease gene into a pBR322-based vector containing the lambda PL promoter and synthetic ribosome-binding sites. Several potential expression cultures were evaluated, with RpoH-hosts proving to be superior. Use of the well-regulated expression system enabled evalutation of fermentation variables at the shake-flask and 10-liter scales. Protease production was optimized with strain DU479 in a chemically-defined medium supplemented with a casein hydrolysate and using a thermal induction form 28 degrees to 42 degrees C. Heterologous protein expression was proportional to the density at induction, reaching a maximum protein titer within two hours, and was stable at a level of ca. 20% total-cell protein through a seven-hour induction period. The rate of temperature shift to 42 degrees C profoundly influenced protease production.

Keywords: Acquired Immunodeficiency Syndrome/THERAPY Cloning, Molecular Escherichia coli/*METABOLISM Fermentation *Gene Expression Genetic Vectors HIV Protease/*GENETICS/METABOLISM HIV-1/*ENZYMOLOGY Promoter Regions (Genetics) Sequence Homology, Nucleic Acid ABSTRACT
920830
M9281069

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