Chen W, Kallio P T, Bailey J E
Department of Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Biotechnol Prog. 1995 Jul-Aug;11(4):397-402. doi: 10.1021/bp00034a006.
A novel cross-regulation expression system has been shown previously to be very effective for regulated recombinant protein production. Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors. Using production of cloned chloramphenicol acetyltransferase (CAT) as a model system, several factors determining the performance of this system were examined. Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT production were examined. The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase. Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level. CAT message levels were minimal preinduction and far above background postinduction, consistent with prior simulation results. Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10-fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate. Maintenance of significant CAT message levels with a concomitant 2-fold increase in CAT accumulation was achieved by extending cell growth in a fed-batch process.
先前已证明一种新型的交叉调控表达系统在调控重组蛋白生产方面非常有效。早期研究表明，与更传统的载体相比，该系统能更好地控制基础表达并实现更高的最大诱导表达。以克隆氯霉素乙酰转移酶（CAT）的生产作为模型系统，研究了决定该系统性能的几个因素。具体而言，研究了不同诱导时间和诱导剂（IPTG）浓度对细胞生长及CAT产生速率的影响。在指数生长中期添加至少0.5 mM IPTG时，CAT表达被最大程度诱导。特定的CAT含量（基于总蛋白）与CAT mRNA水平相关。诱导前CAT信息水平最低，诱导后远高于背景水平，这与先前的模拟结果一致。随着培养进入稳定期，CAT积累停止，同时CAT mRNA水平相应下降了10倍，这可能是由于mRNA降解速率增加所致。通过在补料分批培养过程中延长细胞生长，实现了维持显著的CAT信息水平，同时CAT积累增加了2倍。
