Narayanan Niju, Hsieh Ming-Yi, Xu Yali, Chou C Perry
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Biotechnol Prog. 2006 May-Jun;22(3):617-25. doi: 10.1021/bp050367d.
Arabinose was shown to serve as an effective inducer for induction of the lac-derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-beta-d-thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by the trc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac-derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LL pac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression.
以青霉素酰化酶（PAC）作为模型蛋白，结果表明阿拉伯糖可作为诱导大肠杆菌中乳糖衍生启动子的有效诱导剂。在用传统诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）诱导pac过表达时（pac由trc或（DE3）/T7启动子调控），PAC的产生受到PAC前体（proPAC）以包涵体形式积累的限制。经常观察到负面的细胞反应，如生长抑制和细胞裂解，导致pac表达水平较低且培养性能较差。有趣的是，仅通过使用阿拉伯糖作为诱导剂就可以克服这些技术障碍。结果表明，阿拉伯糖不仅诱导用于pac（或LL pac）过表达的乳糖衍生启动子系统（即trc和（DE3）/T7），还促进proPAC的翻译后加工以实现成熟。然而，阿拉伯糖诱导性似乎依赖于宿主，在阿拉伯糖操纵子发生突变的菌株中不太明显。还在表达系统中研究了阿拉伯糖诱导性，该系统中调节pac表达的trc启动子系统与另一个调节degP共表达的araB阿拉伯糖诱导型启动子系统共存。
