大孔离子交换树脂在重组大肠杆菌生产hEGF中的原位分离作用

引　言乙酸是重组大肠杆菌培养过程中的主要代谢副产物1,2 ] ,乙酸的产生和积累对重组菌的生长和外源蛋白表达都有严重的抑制作用3,4 ] .许多研究者对此开展了多方面的研究 ,如通过代谢工程手段构建乙酸酶缺陷菌株作为宿主菌来减少乙酸的产生5] 、采用发酵 /分离耦合技术除去或降低乙酸量6～ 8] 、透析培养技术9,10 ] 、控制菌体的比生长速率及限制基质葡萄糖浓度等方法 .　　　本实验室开展了重组大肠杆菌分泌性高效表达人表皮生长因子 (humanepidermalgrowthfactor ,hEGF)的研究 ,发现在重组菌培养过程中乙酸的积累对重组菌生长和hEGF表达有严重抑制作用 .本文开展了高效吸附乙酸的离子交换树脂筛选 ,并在摇瓶和发酵罐规模两个水平上研究离子交换树脂在重组大肠杆菌发酵生产hEGF过程中对乙酸的原位分离作用 .1　材料与方法1 1　菌株与质粒E coliJM10 1/pSP10 3,本研究所保存的高效分泌型hEGF的重组大肠杆菌 .1 2　培养基及培养方法种子培养基和发酵培养基 ,种子培养、摇瓶培养及 2 5LB Braun发酵罐培养的条件参照文献11],其中发酵培养基中...

IN-SITU SEPARATION BY MACROSPORE-ANIONIC EXCHANGE RESIN DURING RECOMBINANT E. coli CULTIVATION FOR hEGF PRODUCTION

Production and accumulation of toxic by-products such as acetic acid can inhibit the growth of recombinant cells and the expression of exogenous gene in E.coli.An anionic exchange resin, A-D3-1, which is high in adsorption selectivity and capability for acetic acid, was screened from a variety of resins based on its physical and chemical properties.On the scale of shake flask culture, the addition of 1.0g resin per 30ml medium was insignificant for the cell growth, however,it could improve the hEGF expression significantly. The batch culture in 2.5L fermentor showed that in-situ adsorption of acetic acid by anionic exchange resin could enhance the expression level of interested protein and reduce the fermentation period by 2 hours. And up to 10％ improvement of hEGF (human epidermal growth factor)volumetric productivity (225.0mg•L^-1) could be achieved by supplementing 3.3g resin per 100ml medium.