San K Y, Bennett G N, Chou C H, Aristidou A A
Department of Chemical Engineering, Rice University, Houston, Texas 77251-1892.
Ann N Y Acad Sci. 1994 May 2;721:268-76. doi: 10.1111/j.1749-6632.1994.tb47400.x.
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.
pH诱导型启动子系统所展现的独特特性在质粒构建体pSM552 - 545C - 中得到了清晰的证明。pH的阶跃变化显著增加了β - 半乳糖苷酶的表达。使用超级肉汤可实现非常高的表达水平，达到细胞总蛋白的约40%。在常用于实现高细胞密度的丰富培养基中高水平的诱导表明，该系统具有足够的通用性，能够适应许多特定情况。在8.0至5.5范围内pH的可变诱导程度使得实现现有系统不易完成的一定程度的表达控制成为可能。通过精确监测诱导pH，可将外源基因表达和生长速率“微调”至最佳水平。评估了几个操作参数对重组蛋白生产的影响。我们的结果表明，操作环境在高密度培养中实现高重组蛋白表达水平方面起着极其重要的作用。在次优条件下，如本研究所示，只能获得中等偏高的水平。即使对于次优情况，在光密度高于25时，通常也能获得约占细胞总蛋白10%至15%的表达水平。我们的结果还表明，细胞生长与重组蛋白合成过程之间的适当平衡对于在高密度培养中维持高表达水平至关重要。任何失衡都很可能导致更多的细胞生长和更低的蛋白质生产力。我们还证明了反应器操作温度可以是微调这种平衡的一个有用参数，从而显著改善结果。
