Narayanan Niju, Xu Yali, Chou C Perry
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Biotechnol Prog. 2006 Nov-Dec;22(6):1518-23. doi: 10.1021/bp060135u.
The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the araB promoter (ParaB, also known as PBAD) in Escherichia coli (E. coli). The current ParaB expression system exhibited minimum leaking pac expression in the absence of arabinose as well as fast and high-level pac expression upon induction with arabinose in a wide concentration range. The production of PAC was limited by the accumulation of PAC precursors (i.e., proPAC in both soluble and insoluble forms) and various negative cellular responses, such as growth arrest and cell lysis. The culture performance could be improved by degP coexpression and the individual contribution of DegP protease and chaperone activities to the enhancement on the production of PAC was characterized. The study highlights the importance of identifying the step(s) limiting high-level gene expression and subsequent design and construction of the host/vector system for enhancing recombinant protein production in E. coli.
编码青霉素酰化酶（PAC）的pac基因在大肠杆菌（E. coli）中受阿拉伯糖操纵子启动子（ParaB，也称为PBAD）调控而过量表达。当前的ParaB表达系统在没有阿拉伯糖的情况下pac表达泄漏最少，并且在宽浓度范围内用阿拉伯糖诱导后pac能快速且高水平表达。PAC的产生受到PAC前体（即可溶性和不溶性形式的proPAC）积累以及各种负面细胞反应（如生长停滞和细胞裂解）的限制。通过共表达degP可改善培养性能，并对DegP蛋白酶和伴侣活性对提高PAC产量的各自贡献进行了表征。该研究突出了确定限制高水平基因表达步骤以及随后设计和构建宿主/载体系统以增强大肠杆菌中重组蛋白生产的重要性。
