GST-NDPK-A融合蛋白的原核表达及体外互作蛋白的检测  

The Purification of GST-NDPK-A Fusion Protein and Detection of its Interacting Proteins in vitro

作　　者：吕芬[1] 刘忠[1] 银兴峰 赵振岭[1] 陈妙娟[2] 张嘉萱[1] 陈伟[1] 钱垂文[1] 熊盛[1] 

机构地区：[1]暨南大学生命科学技术学院生物医药研究开发基地,广州510632 [2]暨南大学生命科学技术学院生命与健康工程研究院,广州510632

出　　处：《生物技术通报》2013年第5期137-143,共7页Biotechnology Bulletin

基　　金："重大新药创制"科技重大专项(2012ZX09103301-033);暨南大学科研培育与创新基金杰出人才培育项目(11611206)

摘　　要：旨在了解核苷二磷酸激酶A(NDPK-A)的功能,通过GST-Pull down技术寻找与其存在体外相互作用的蛋白。以含有目的片段的质粒pBV-NDPK-A为模板,扩增出相应的目的基因片段,将其插入带有GST标签的原核表达载体pGEX-4T-2,构建重组表达质粒。双酶切及测序鉴定正确后,转化至大肠杆菌BL21,经IPTG诱导,Glutathione Sepharose 4B亲和纯化,通过SDS-PAGE电泳及Western blot确定融合蛋白的表达,并与高表达细胞系A549孵育,进行GST-Pull down试验,并通过LC-MS/MS质谱鉴定体外与NDPK-A相互作用的蛋白。结果显示,成功构建GST-NDPK-A的原核表达载体;在大肠杆菌BL21中诱导表达出可溶性融合蛋白;经Glutathione Sepharose 4B纯化后,获得了有生物活性的GST-NDPK-A融合蛋白,GST-Pull down试验和质谱鉴定结果发现NDPK-A与Fussel-18、Rrp12体外存在相互作用。It was to understand the function of nucleoside diphosphate kinase A（NDPK-A）and explore its interacting proteins in vitro through GST-Pull down technique.The target genes were amplified from the templates pBV-NDPK-A and inserted into the prokaryotic expression vector pGEX-4T-2 with glutathione-S-transferase（GST）tag to construct the expression plasmid.After identified by restriction endonuclease digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21 strain and exogenous protein expression was induced by IPTG.After purification using Glutathione Sepharose 4B affinity chromatography,the GST-NDPK-A fusion protein was identified by SDS-PAGE and Western blot then incubated with A549.GST-Pull down assay was performed to explore the interacting proteins in vitro followed by LCMS/MS identification.Results showed that the prokaryotic expression vector of GST-NDPK-A fusion protein was successfully constructed and expressed effectively in E.coli BL21 strain.The fusion protein with bioactivity was purified using Glutathione Sepharose 4B beads.Furthermore,GST-Pull down assay indicated that NDPK-A could bind to Fussel-18 and Rrp12 in vitro.

关 键 词：NDPK-A Fussel-18 Rrp12 表达纯化 相互作用 

分 类 号：Q51[生物学—生物化学]