An optimization study of a pH-inducible promoter system for high-level recombinant protein production in Escherichia coli.
The unique properties exhibited by the pH-inducible promoter system are clearly demonstrated by the plasmid construct, pSM552-545C-. Step changes of pH substantially increase the expression of beta-galactosidase. Very high expression, a level of around 40% of total cellular protein, can be achieved with superbroth. The high level of induction in rich media, typical of those commonly used to achieve high cell density, suggests the system is versatile enough to be adapted to many specific situations. The variable degree of induction by pH within the range of 8.0 and 5.5 makes possible a degree of expression control not easily accomplished with the existing systems. By precise monitoring of induction pH, a "fine tuning" of foreign gene expression and growth rate to optimum levels is possible. The effect of several operating parameters on recombinant protein production are evaluated. Our results show that operating environments play an extremely important role in achieving high recombinant protein expression levels in a dense culture. Under suboptimal conditions, as are shown in this study, only moderately high levels can be obtained. Even for suboptimal cases, an expression level of about 10 to 15% of total cellular protein while achieving an optical density higher than 25 is routinely obtained. Our results also show that a proper balance between cell growth and recombinant protein synthesis processes are critical in maintaining high expression levels in a dense culture. Any imbalance will most likely lead to more cell growth and poorer protein productivity. We have also demonstrated that reactor operating temperature can be a useful parameter to fine-tune this balance, resulting in significantly improved results.
Department of Chemical Engineering, Rice University, Houston, Texas 77251-1892.
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San
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Bennett
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Chou
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Aristidou
D001709:Biotechnology; D002455:Cell Division; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D013696:Temperature
D011994:Recombinant Proteins