文献类型：期刊文献

中文题名：HIV-TATm-Survivin(T34A)高表达大肠杆菌BL21(DE3)菌株的自适应进化筛选及其特性

英文题名：Screening and Characterization of E.coli BL21(DE3) Mutant Hosts Overexpressing HIV-TATm-Survivin(T34A) through Adaptive Evolution

作者：姚雪凌[1];王恒伟[1];王玮[1];王风清[1];魏东芝[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,鲁华生物技术研究所,上海200287

年份：2010

期号：2

起止页码：203

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

基金：国家科技重大专项“生物技术药物中试放大及分离纯化技术平台”(2009ZX09306-001)

语种：中文

中文关键词：大肠杆菌;重组蛋白;自适应进化;菌种保存;发酵放大;HIV-TATm-Survivin(T34A)

外文关键词：Escherichia coli; recombinant protein; adaptive evolution; bacteria preservation; fermentation scale up; HIV-TATm-Survivin（T34A）

摘要：表达蛋白HIV-TATm-Survivin(T34A)的重组大肠杆菌BL21(DE3)在长时间保存后其表达水平明显下降,发酵罐放大难以顺利进行。利用该蛋白的本底表达对宿主菌产生的生长压力进行了实验室自适应进化工作,得到携带相同质粒但表达水平呈两极分化的两类菌株HTS1和HTS2。摇瓶培养时,HTS1菌株生长较迅速,但重组蛋白几乎无表达;HTS2生长较缓慢,却可以高效积累重组蛋白。因此HTS1菌株的出现,可能是原始菌株长时间保存后蛋白表达能力下降的原因之一。另外,HTS2菌株应用上的优势,外源添加高达300 mmol/L的乙酸钠对HIV-TATm-Survivin(T34A)的表达水平没有较明显负的影响,4 L和30 L发酵罐表达验证了摇瓶培养的结果。
The expression of recombinant protein HIV-TATm-Survivin（T34A） by E.coli BL21（DE3） decreased significantly in long-time preservation,which usually led to difficulty in scale up and process improvements.In this study,adaptive evolution experiments of the host cells were conducted under the stress from background expression,and two distinct strains,E.coli BL21（DE3） HTS1 and HTS2 with the same plasmid pRSET-B-TAT-Survivin（T34A）,were finally obtained.In shake flask cultivations,the strain HTS1 showed a higher growth rate but a poor capacity of protein expression;in contrast,strain HTS2 grew a little bit slowly but could overexpress the target protein.We proposed that the appearance of strain HTS1 was responsible partly for the protein expression decrease during the preservation.Furthermore,strain HTS2 was advantageous in the scale-up process,the overexpression of HIV-TATm-Survivin（T34A） by it did not negatively affected by up to 300 mmol/L sodium acetate,and such results were further confirmed in the（4 L） and 30 L fermentors.