Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli.
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-beta-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 less than or equal to pH less than or equal to 6.0) and a 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 Zn2+ ions.
Baneyx
F
F
Department of Chemical Engineering, University of Texas, Austin 78712.
Ayling
A
A
Palumbo
T
T
Thomas
D
D
Georgiou
G
G
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001431:Bacteriological Techniques; D002712:Chlorides / Q000494:pharmacology; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013696:Temperature; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds
D001426:Bacterial Proteins; D002712:Chlorides; D011993:Recombinant Fusion Proteins; D017967:Zinc Compounds; C016837:zinc chloride; D010450:Endopeptidases; D015032:Zinc