Process characterization of a novel cross-regulation system for cloned protein production in Escherichia coli.
Process characterization of a novel cross-regulation system for cloned protein production in Escherichia coli.
复制标题
大肠杆菌中克隆蛋白生产的新型交叉调节系统的过程表征。
DOI：
10.1021/bp00034a006
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Bailey,JE
中科院分区：
文献类型：
--
作者：
Chen,W;Kallio,PT;Bailey,JE
先前已证明一种新型交叉调节表达系统对于调节重组蛋白的生产非常有效。早期研究表明，该系统比更传统的载体能更好地控制基础表达和更高的最大诱导表达。使用克隆的氯霉素乙酰转移酶（CAT）的生产作为模型系统，检查了决定该系统性能的几个因素。具体来说，检查了不同诱导时间和诱导剂 (IPTG) 浓度对细胞生长和 CAT 产生速率的影响。在指数生长期添加至少 0.5 mM IPTG 可以最大限度地诱导 CAT 表达。特定 CAT 含量（以总蛋白为基础）与 CAT mRNA 水平相关。 CAT 消息水平在诱导前最低，远高于诱导后背景，与之前的模拟结果一致。当培养物进入稳定期时，CAT 积累停止，CAT mRNA 水平相应下降 10 倍，这可能是由 mRNA 降解率增加引起的。通过在分批补料过程中延长细胞生长时间，可以维持显着的 CAT 信息水平，同时 CAT 积累增加 2 倍。
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 erowth in a fed‐batch process.