Xu Yali, Weng Chiao-Ling, Narayanan Niju, Hsieh Ming-Yi, Anderson William A, Scharer Jeno M, Moo-Young Murray, Chou C Perry
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
Appl Environ Microbiol. 2005 Oct;71(10):6247-53. doi: 10.1128/AEM.71.10.6247-6253.2005.
Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The outcome of proPAC folding and PAC maturation could be affected by several factors, such as inducer type, proPAC formation rate, and chaperone availability. Misfolding of proPAC in the cytoplasm could be partially resolved through the coexpression of cytoplasmic chaperones, such as trigger factor, GroEL/ES, or DnaK/J-GrpE. The three chaperones tested showed different extents of the effect on proPAC solublization and PAC maturation, and trigger factor had the most prominent one. However, the chaperone-mediated solublization of proPAC did not guarantee its maturation, which is usually limited by the first autoproteolytic step. It was observed that arabinose could act as an effective inducer for the induction of LL pac expression regulated by the lac-derived promoter system of trc. In addition, PAC maturation could be highly facilitated by arabinose supplementation and coexpression of trigger factor, suggesting that the coordination of chaperone systems with proper culture conditions could dramatically impact recombinant protein production. This study suggests that folding/misfolding of proPAC could be a major step limiting the overproduction of PAC in E. coli and that the problem could be resolved through the search for appropriate chaperones for coexpression. It also demonstrates the analogy in the issues of proPAC misfolding as well as the expression bottleneck occurring in the cytoplasm (i.e., LL pac expression) and those occurring in the periplasm (i.e., wild-type pac expression).
在大肠杆菌中进行了无信号肽青霉素酰化酶（PAC）基因（LL pac）的表达研究。结果表明，PAC前体proPAC能够产生，甚至在细胞质中加工形成成熟的PAC，这表明PAC成熟的翻译后加工步骤可在大肠杆菌的周质和细胞质中发生。proPAC折叠和PAC成熟的结果可能受到多种因素的影响，如诱导剂类型、proPAC形成速率和伴侣蛋白可用性。通过共表达细胞质伴侣蛋白，如触发因子、GroEL/ES或DnaK/J-GrpE，可部分解决proPAC在细胞质中的错误折叠问题。所测试的三种伴侣蛋白对proPAC溶解和PAC成熟的影响程度不同，触发因子的作用最为显著。然而，伴侣蛋白介导的proPAC溶解并不能保证其成熟，成熟过程通常受第一步自蛋白水解步骤的限制。观察到阿拉伯糖可作为由trc的lac衍生启动子系统调控的LL pac表达诱导的有效诱导剂。此外，添加阿拉伯糖和共表达触发因子可极大地促进PAC成熟，这表明伴侣蛋白系统与适当培养条件的协调可显著影响重组蛋白的产生。本研究表明，proPAC的折叠/错误折叠可能是限制大肠杆菌中PAC过量生产的主要步骤，通过寻找合适的伴侣蛋白进行共表达可解决该问题。它还证明了proPAC错误折叠问题以及在细胞质中发生的表达瓶颈（即LL pac表达）与在周质中发生的问题（即野生型pac表达）之间的相似性。
