具有抗血管新生活性的K5突变体GST-K5M5基因的构建、表达及纯化  

Construction, Expression, and Purification of Active Human Plasminogen Mutant GST-K5M5 from E.coli

作　　者：黄莉钧[1] 石丁波[1] 姚亚超[1] 李磊[1] 戴智育[1] 张婷[1] 高国全[1,2] 杨霞[1,3] 

机构地区：[1]中山大学中山医学院生物化学教研室,广东广州510080 [2]热带病防治研究教育部重点实验室,广东广州510080 [3]海洋微生物功能分子广东省高校重点实验室,广东广州510080

出　　处：《热带医学杂志》2013年第2期133-139,161,共8页Journal of Tropical Medicine

基　　金：科技部新药创制重大专项(2013ZX09102-053);国家自然科学基金(30971208;30973449;81172163;81272515;81272338);(教育部)高等学校博士学科点专项科研基金(20100171110049;201201171110053);广东省自然科学基金(10251008901000009;S2012010009250);广东省科技计划项目(2011B031200006);广州市科技计划项目(12A52061519;2011Y1-00017-8);教育部高校基本科研业务费中山大学青年教师培育项目(10YKPY28)

摘　　要：目的构建可溶性的人纤溶酶原Kringle 5(K5)突变体GST-K5M5基因,并在单位体积的原核系统中获得高表达、高纯度、可水溶的融合GST-K5M5蛋白粉针剂。方法 K5M5突变体基因(模板已经对K5N端5个带负电的酸性氨基酸进行中性氨基酸丝氨酸的中性突变),通过PCR从本实验室保存的pET22b(+)/K5mut5 cDNA模板中扩增得到,再克隆到pGEX-4T-1/BL21(DE3)原核表达系统中。用自动诱导培养系统诱导其表达,提高产量。通过GST树脂亲和层析柱分离,双蒸水透析纯化,再制成冻干粉剂保存。结果该重组GST-K5M5蛋白的分子量为37000Mr,通过SDS-PAGE和Western-blot可以检测到其存在。冻干粉复溶的GST-K5M5呈剂量依赖抑制内皮细胞的增殖。结论在以前的研究基础上设计并用自动诱导培养的方法在大肠杆菌中表达纯化出了GST-K5M5融合蛋白,得到的GST-K5M5蛋白产量高、纯度高、可溶性好、生物学效应强,且方法简便、经济,为工业大规模发酵提供了基本参数。Objective The purpose of this study was to construct human plasminogen Kringle 5 GST-Mutant 5 gene, and to obtain the soluble recombinant GST-KSM5 protein. Methods The gene of human plasminogen kringle 5 GST-Mutant 5 （neutral mutation of the template was accomplished by replacing 5 zincative amino acids with 5 neutral serine）was obtained by amplifying the pET22b （＋）/KSmut5 cDNA template with PCR.The amplified fragment was cloned to the pGEX-4T-1/BL21 （DE3） prokaryotic system, and the recombinant plasmid was expressed by auto inductive culture system.Recombinant protein was purified using GST-resin affinity chromatography.The purified protein was preserved using the freeze-drying method. Results The recombinant GST-KSM5 protein was found to have an apparent molecular weight of 37 000 Mr in the SDS-PAGE.The GST-K5M5 protein could inhibit the proliferation of HUVEC in a dose dependent manner. Conclusion We have designed, expressed and purified an active soluble GST-K5M5 fussion protein with the auto inductive culture system.This study provides basic parameter for the further large scale production of this recombinant protein.

关 键 词：K5突变体 融合蛋白GST—K5M5 自动诱导培养 水溶性 冻干粉 

分 类 号：R392.116[医药卫生—免疫学]