N端前肽介导的重组丝氨酸蛋白酶在大肠杆菌中跨外膜转运机制的研究

Eschericha coli is the most frequently used prokaryotic host for producing recombinant proteins. Extracellular production of recombinant proteins in E. coli has several advantages including rapid preparation and easy purification of the target protein, but remains problematic, mainly due to the presence of the outer membrane. Nevertheless, some recombinant proteins targeting to the periplasmic space of E. coli were found to leak into the culture medium. However, little is known about the mechanism for the "leakage" of recombinant proteins from the periplasmic space to the extracellular milieu. Recently, we found that two bacterial serine proteases, Als and WF146, had the ability to secrete into the culture medium when expressed in E. coli. Meanwhile, we noticed that the secretion efficiency of recombinant Als protease is significanly higher than that of recombinant WF146 protease. Interestingly, the N-terminal propeptide of Als was confirmed to mediate the transport of recombinant Als across the outer membrane of E. coli. It is well known that N-terminal propeptides of bacterial serine proteases usually act as an intramolecular chaperone and an inhibitor of its cognate enzyme, but so far there is no report on N-terminal propeptide-mediated transport of recombinant serine protease. In this research proposal, we are going to study the mechanism of the N-terminal propeptide-mediated transport of recombinant serine protease across the outer membrane of Escherichia coli. Firstly, we will probe possible transport pathway of the enzymes across the outer membrane, and will investigate the role of the N-terminal propeptide in this process. Then, a series of mutants and chimera of the two homologous Als and WF146 proteases will be constructed, in order to indentify the key structural elements involved in transport across the outer membrane. Finally, we will improve the extracellular production level of the recombinant enzymes by optimazing the key structural elements, and by overexpressing transport channel apparatus on the outer membrane. We hope this study could provide new insight into the molecular basis of transport of recombinant serine protease across the outer membrane of E. coli. In addition, the elucidation of the transport mechanism and the roles of the N-terminal propeptide is expected to provide a rational basis for developing a system for high-level extracellular production of recombinant proteins in E. coli.

重组蛋白需要同时跨越内膜和外膜两道障碍才能在大肠杆菌中实现胞外分泌。尽管一些重组蛋白由周质空间通过"渗漏"或未知方式实现了跨外膜转运，但机制不明。最近，我们发现两种丝氨酸蛋白酶在大肠杆菌中实现了胞外分泌，并且证明了N端前肽具有介导重组丝氨酸蛋白酶进行跨外膜转运的功能，这一现象和其中蕴含的机制尚无报道。本项目将利用这两种在大肠杆菌中具备不同胞外分泌效率的同源丝氨酸蛋白酶开展研究，在解析它们跨外膜转运的途径和方式的基础上，通过突变和构建嵌合体等手段寻找存在于N端前肽及其他结构区域的与跨外膜转运相关的关键结构特征，进而阐明重组丝氨酸蛋白酶跨外膜转运的分子机制。本项目的开展，有望为人们充分认识重组蛋白通过"渗漏" 或"机制不明"的方式实现胞外分泌这一现象中所蕴含的规律提供新见解。同时，对N端前肽介导重组酶跨外膜转运作用机制深入理解，在应用方面将为以大肠杆菌作为宿主高效分泌重组蛋白提供新的理论依据

本项目针对细菌来源的Als、WF146蛋白酶等丝氨酸蛋白酶在大肠杆菌中的跨外膜转运机制开展研究。通过构建信号肽和N端前肽缺失突变体和目标蛋白酶的定位分析等方法，确定重组蛋白酶先跨内膜分泌至周至空间后再转运至胞外。在缺失N端前肽的情况下，重组酶虽然可以由信号肽介导分泌至周至空间，但发生错误折叠并在周至空间积累而引起细胞破裂。因此，N端前肽在调节重组蛋白结构使其保持可转运状态方面发挥关键作用。构建了大肠杆菌周质空间蛋白质质量控制系统（DegQ、DegP和DegS）的基因敲除菌株，当重组酶在这些敲除菌株中进行表达时，跨外膜转运效率显著提高。这一方面是因为重组酶进入周至空间后被降解程度降低，另一方面是因为蛋白质质量控制系统相关基因的敲除对外膜蛋白的转运与定位造成影响，使得膜稳定性下降而通透性增强，进而有利于可转运状态的重组酶跨外膜转运。对大肠杆菌的多种外膜通道蛋白（如OmpF、OmpA、GspD、FimD、CsgG、YbgQ、HtrE、SfmD）的基因进行了敲除，重组酶在这些通道蛋白基因敲除菌株中表达时的跨外膜转运并未减弱，说明这些通道蛋白并非重组酶跨外膜转运所必需；相反，重组酶在这些菌株中的胞外组分中的含量显著上升，并伴随着大肠杆菌细胞周质空间蛋白向胞外释放，表明上述外膜通道蛋白的缺失导致外膜通透性提高。同时，野生型丝氨酸蛋白酶Als可在周质空间成熟并且其在胞外组分中的含量高于活性位点突变体Als-SA；表达Als的大肠杆菌菌株对溶菌酶处理的敏感性提高并发生细胞形态改变，暗示Als成熟酶释放的蛋白酶活性可攻击外膜蛋白而提高外膜通透性。另外，我们发现Als具有良好的热稳定性和低温活性，并且具有降解羽毛角蛋白的能力。总体而言，本项目对N端前肽介导重组丝氨酸蛋白酶在大肠杆菌中跨外膜转运的方式与机制进行了阐释，在理论上为蛋白质跨膜转运这一基本生物学过程提供了新信息，在应用上为实现包括蛋白酶在内的重组蛋白在大肠杆菌中的高效胞外分泌提供新的理论依据。