Wu Ming-Shen, Pan Kao-Lu, Chou C Perry
Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan.
Biotechnol Bioeng. 2007 Apr 1;96(5):956-66. doi: 10.1002/bit.21161.
High-level expression of recombinant penicillin acylase (PAC) using the strong trc promoter system in Escherichia coli is frequently limited by the processing and folding of PAC precursors (proPAC) in the periplasm, resulting in physiological stress and inclusion body formation in this compartment. Periplasmic heat-shock proteins with protease or chaperone activity potentially offer a promise for overcoming this technical hurdle. In this study, the effect of the two genes encoding periplasmic heat-shock proteins, that is degP and fkpA, on pac overexpression was investigated and manipulation of the two genes to enhance the production of recombinant PAC was demonstrated. Both DeltadegP and DeltafkpA mutants showed defective culture performance primarily due to growth arrest. However, pac expression level was not seriously affected by the mutations, indicating that the two proteins were not directly involved in the pathway for periplasmic processing of proPAC. The growth defect caused by the two mutations (i.e., DeltadegP and DeltafkpA) was complemented by either one of the wild-type proteins, implying that the function of the two proteins could partially overlap in cells overexpressing pac. The possible role that the two heat-shock proteins played for suppression of physiological stress caused by pac overexpression is discussed.
在大肠杆菌中使用强trc启动子系统对重组青霉素酰化酶（PAC）进行高水平表达，常常受到周质中PAC前体（proPAC）加工和折叠的限制，导致该区域出现生理应激和包涵体形成。具有蛋白酶或伴侣活性的周质热休克蛋白可能为克服这一技术障碍带来希望。在本研究中，研究了编码周质热休克蛋白的两个基因degP和fkpA对pac过表达的影响，并证明了通过操纵这两个基因可提高重组PAC的产量。DeltadegP和DeltafkpA突变体均表现出培养性能缺陷，主要原因是生长停滞。然而，pac表达水平并未受到这些突变的严重影响，这表明这两种蛋白并不直接参与proPAC周质加工途径。由这两种突变（即DeltadegP和DeltafkpA）导致的生长缺陷可被任一野生型蛋白互补，这意味着在过表达pac的细胞中，这两种蛋白的功能可能部分重叠。文中讨论了这两种热休克蛋白在抑制pac过表达引起的生理应激中可能发挥的作用。
