表达GLP-1融合蛋白的重组大肠杆菌分批补料培养工艺优化  

Optimization of Fed-batch Culture of Recombinant E.coli. for High Expression of Glucagon Like Peptide-1

作　　者：胡爽[1] 蔡海波[1] 蒋加庆[1] 谭文松[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《高校化学工程学报》2010年第3期468-475,共8页Journal of Chemical Engineering of Chinese Universities

基　　金："重大新药创制"科技重大专项(2009ZX09102-253)

摘　　要：人胰高血糖素样肽-1(Glucagon Like Peptide-1,GLP-1)是一种有潜在应用价值的治疗糖尿病的药物。今以一株表达GLP-1融合蛋白的重组大肠杆菌为研究对象,在分批培养条件下,通过流加甘油并在诱导前0.5h添加氮源使菌体密度、融合蛋白表达水平、融合蛋白体积得率和融合蛋白细胞得率分别提高了138.0%、29.5%、216.9%和29.6%。在此基础上,进一步考察了甘油补加策略、氮源补料方式以及补氮液浓度等过程因素对菌体生长以及GLP-1融合蛋白表达的影响,发现氮源流加过程是提高融合蛋白表达水平的关键因素。进而通过对补料过程的优化,建立了高密度高表达的分批补料培养工艺,最终使菌体密度、融合蛋白表达水平、融合蛋白体积得率和融合蛋白细胞得率分别达到51.93g·L-1、34.6%、2.937g·L-1和0.057g·g-1cell,较分批培养分别提高了314.5%、100.0%、784.6%和111.1%。研究结果对GLP-1融合蛋白的产业化开发有一定的指导意义。Glucagons Like Peptide-1 （GLP-1） is a gastrointestinal and incretion hormone released from the enteroglucagon L-cell in the small intestine of human, and its physiological features play important roles in regulation of postprandial insulin release. Therefore, GLP-1 is considered as a great potential reagent for pharmacotherapy of type Ⅱ diabetes in the clinic. The recombinant E.coli strain named HT02 was applied to produce GLP-1 in this study. Under the fermentation conditions of batch culture, the cell density, GLP-1 fusion protein expression level, the volume yield of GLP-1 fusion protein and the cell yield of GLP-1 fusion protein can increase 138.0%, 29.5%, 216.9% and 29.6%, respectively, by using the means of feeding glycerol and adding nitrogen source before induction. On the basis of above, the effects of feeding strategies of glycerol and nitrogen source were further investigated in a 5 L fermentor, and it was found that the key factor for obtaining high GLP-1 fusion protein expression is the feeding strategy of nitrogen source. Furthermore, the initial feeding time of glycerol, the mode and amount of feeding nitrogen source were optimized, and the proper fed-batch culture process was established subsequently. The cell density, GLP-1 fusion protein expression level, the volume yield of GLP-1 fusion protein and the cell yield of GLP-1 fusion protein can reach 51.93 g·L^-1, 34.6%, 2.937 g·L^-11 and 0.057 g·gcell^-1, respectively, by using the proposed fed-batch culture process. Comparing with batch culture, they increase 314.5%, 100%, 784.6% and 111.1%, correspondingly. Finally, the results of this study have a guiding significance for the industrial production of GLP-1.

关 键 词：重组大肠杆菌 人胰高血糖素样肽-1 分批补料培养 高密度发酵 

分 类 号：TQ936.16[化学工程]