Zhang Y, Liu N, Yang K
Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Chin J Biotechnol. 1991;7(3):169-75.
The expression plasmid pTaAC containing Tac promoter and calf prochymosin B gene was constructed and transformed into E.coli JM105. Addition of 0.1 mM IPTG into the culture at logarithmic phase induced the production of prochymosin markedly. The expression of prochymosin gene was regulated by temperature in addition to the inducer IPTG. At 30 degrees C in the presence of IPTG prochymosin was barely detected, whereas at 42 degrees C in the absence of IPTG a relatively high level of prochymosin was found. The expressed protein was estimated to be 12-19% of total cell proteins by electrophoresis analysis or 80-100 mg/l by ELISA. The yield of active chymosin was 14-20 mg/l after denaturation, renaturation and activation.
构建了含有Tac启动子和小牛凝乳酶原B基因的表达质粒pTaAC，并将其转化到大肠杆菌JM105中。在对数生长期向培养物中添加0.1 mM异丙基-β-D-硫代半乳糖苷（IPTG）可显著诱导凝乳酶原的产生。除了诱导剂IPTG外，凝乳酶原基因的表达还受温度调节。在30℃且存在IPTG的情况下几乎检测不到凝乳酶原，而在42℃且不存在IPTG的情况下发现了相对高水平的凝乳酶原。通过电泳分析估计表达的蛋白质占总细胞蛋白质的12 - 19%，通过酶联免疫吸附测定（ELISA）为80 - 100 mg/l。变性、复性和激活后，活性凝乳酶的产量为14 - 20 mg/l。
