酿酒酵母RAVE复合物的155kD亚基Rav1p的克隆及其在大肠杆菌中的表达纯化  

Expression and Purification of Ravlp, a 155 kD Subunit of RAVE Complex from Saccharomyces cerevisiae

作　　者：徐灿[1] 张震宇[1] 

机构地区：[1]江南大学生物工程学院、江南大学工业生物技术教育部重点实验室,无锡214122

出　　处：《生物技术通报》2012年第5期99-104,共6页Biotechnology Bulletin

基　　金：高等学校博士学科点专项科研基金，国家自然科学基金，工业生物技术教育部重点实验室主任基金

摘　　要：以酿酒酵母基因组DNA为模板，根据GenBank上公布的酿酒酵母Rav1p基因（ray1）序列和表达载体特性设计特异性引物，PCR扩增得到4074bp的DNA片段，将PCR产物和原核表达载体pET28a（＋）同时进行双酶切；双酶切后的PCR产物和表达载体进行连接，构建成重组质粒pET28a．rav1。再将pET28a．rav1转化到BL21（DE3）感受态细胞中。经IPTG16℃低温诱导40h表达His-tag融合的Rav1p。诱导后的菌体进行超声波破碎，然后用GEheahhcare公司的AKTA蛋白纯化仪和HisTrapHP1mL亲和层析柱纯化目的蛋白。SDS—PAGE电泳分析和Westernblot分析显示在155kD有明显的条带，成功实现了Rav1p在大肠杆菌中的表达纯化。The ravlp gene was amplified from Saccharomyces cerevisiae genome DNA using PCR technique, and the PCR product was approximately 4 074 bp DNA segment. Then the PCR product and the pET28a （ ＋ ） vector were double digested by EcoR I and Sal I . The PCR product was conjuncted to the pET28a （ ＋ ） vector and the recombined plasmid was identified by enzyme cutting analysis and DNA sequencing. After that, the recombined plasmid pET28a-ravl was transferred to BL21 （ DE3 ） competent cell. The recombinants was induced in the condition of 1 mmol/L IPTG, 16℃, 80 r/min for 40 h. Then the cells was broken by ultrasonication and the target protein was purified using the affinity chromatography column of His Trap HP 1 mL. The his-tag fusion protein about 155 kD was shown in SDS-PAGE gel after electrophoresis as well as PVDF film after Western blot.

关 键 词：酿酒酵母RAVE复合物Ravlp克隆原核表达蛋白纯化 

分 类 号：Q492.4[生物学—生理学]