Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space ofEscherichia coli

✍ Scribed by François Baneyx; Amanda Ayling; Terry Palumbo; Daniel Thomas; George Georgiou

• Publisher: Springer
• Year: 1991
• Tongue: English
• Weight: 861 KB
• Volume: 36
• Category: Article
• ISSN: 1432-0614
• DOI: 10.1007/bf00164691

✦ Synopsis

The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A-fl-lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5<pH<6.0) and at low temperatures. These conditions were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn 2÷ ions.