温度诱导表达体系中应激反应对目标蛋白的影响机制

The temperature inducible expression system has been widely use to produce recombinant proteins in prokariotic cells since the use of special media, toxic or expensive chemical inducers is avoided and culture handling is minimized and easily scalable. In this system, an increase in temperature induces the heterologous protein production and activates complex responses. The same responses are activated just by the overproduction of recombinant protein. All such responses result in a metabolic burden to the cells and can alter the quantity and quality of the produced foreign protein. Investigation of stress responses by proteomic and transcriptomic technology has been the hot spot of recombinant protein production and almost all relevant study focused on aggregation-prone heterologous proteins. In this study, we shall focused on stress response during fed-batch culture of recombinant Escherichia coli containing human-like collagen (that is soluble in vivo) cDNA using the temperature inducible expression. The stress responses that occur after heat induction under different culture operation variables and induction strategies will be analyzed using 2-dimensional gel electrophoresis combined with LC-MS/MS, RNA-Seq and qRT-PCR. To elucidate the effects of stress response protein on recombinant protein production, we shall construct coexpressing identified molecular chaperones system or delete identified proteases gene from the strain for human-like collagen production. The results obtained in this study would be useful for further understanding the integral effects of stress responses of E. coli, and would provide valuable information for enhanced soluble recombinant production, especially for developing improved strategies for high yield human-like collagen production and recovery.

温度诱导型原核表达体系由于不需使用诱导剂，易于调控放大，被广泛用于重组蛋白工业化生产中。该体系高密度发酵生产过程中，细胞受到高温和蛋白表达的双重胁迫，代谢负担加重，并发生复杂的应激反应，进而影响目标蛋白生产过程。应用组学技术研究细胞的应激反应是目前重组蛋白生产体系的研究热点之一，但通常局限于对易聚集蛋白生产体系的研究。本研究拟采用不易聚集的类人胶原蛋白温度诱导型原核表达体系为研究对象，采用2-D-PAGE（结合蛋白质谱）和RNA-Seq技术，结合qRT-PCR定量分析，研究重组大肠杆菌在不同高密度培养调控工艺下的应激反应特征，识别出特征应激蛋白并进行功能分析；通过共表达分子伴侣或敲除蛋白酶基因，揭示特征应激蛋白对目标蛋白的影响机制。研究结果将有助于深入理解应激反应对重组蛋白生产的影响机制，揭示影响目标蛋白可溶性表达的分子机制，同时可为类人胶原蛋白生产体系的进一步优化提供理论依据。

温度诱导型原核表达体系被广泛用于重组蛋白工业化生产中，本研究针对该体系高密度发酵过程中的应激反应及其对目标蛋白的影响机制进行了较为系统的研究：采用TMT全蛋白定量组学分析和Illumina HiSeq2500转录组高通量测序识别了温度诱导型原核表达类人胶原蛋白（human-like collogen，HLC）高密度发酵生产过程中细胞的关键应激蛋白，并采用qRT-PCR技术对关键经典热激因子进行了时序变化分析；构建了热激蛋白和类人胶原蛋白共表达体系；分别在摇瓶水平、30L发酵罐水平、125L发酵罐水平比较了构建体系的生产能力；基于应激反应，优化设计了类人胶原蛋白诱导表达策略。主要研究结果如下：1）HLC高密度发酵过程中温度诱导操作导致的上调关键应激相关蛋白共26个（AI/BI Ratio大于1.5倍，p<0.05），其中经典热激蛋白有clpB，dnaK，dnaJ，ibpA，ibpB，rpoH，rpoS；2）在宿主细胞E. coli BL21 3.7中，伴侣蛋白GroEL-GroES单独表达时可促使水溶性HLC表达水平提高31.8%，在伴侣蛋白TF的辅助下可使水溶性HLC表达水平提高29.3%；在宿主细胞E. coli BL21 3.7 ∆ptsG中，伴侣蛋白GroEL-GroES与TF的协同表达可使水溶性HLC表达量提高23.7%；DnaK:DnaJ:GrpE共表达体系对水溶性HLC的表达没有促进作用；3）构建菌株在30L、125L发酵罐上生产能力稳定；4）预诱导策略通过应激交叉保护作用，可以有效提高HLC表达量。42℃预诱导3小时，HLC表达增加了21.4%。