小鼠TAp63γ及两种突变体的原核表达、纯化和DNA结合活性  

Soluble Expression,Purification,DNA Binding Activity of Mouse TAp63γ and Two Deltion Mutants Fusion Proteins

作　　者：严君喜[1] 黄新河[1] 杜林方[1,2] Zhi-Xiong Jim Xiao 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610064 [2]四川大学纳米生物医学技术与膜生物学研究所,成都610041 [3]School of Medicine, Boston University, Boston MA02118, USA

出　　处：《中国生物工程杂志》2008年第9期39-45,共7页China Biotechnology

基　　金：教育部新世纪人才资助计划(NCET-04-0861)

摘　　要：采用PCR扩增法得到小鼠TAp63γ野生型及两种缺失突变体的cDNA,3种cDNA与表达载体pGEX-2TK重组构建成GST融合表达质粒并转化感受态E.coli BL21(DE3),经IPTG诱导了小鼠TAp63γ野生型及两种缺失突变体的可溶性表达。诱导表达的菌液经离心收集菌体、超声破碎及Triton X-100增溶后获得可溶性表达蛋白粗提液。利用Glutathione Sepharose 4 Fast Flow亲合层析纯化出电泳均一的3种GST融合蛋白。凝胶滞留分析证实仅野生型小鼠TAp63γ蛋白能特异结合p53靶序列,经序列比对及同源建模分析,表明小鼠TAp63γDBD结合区的完整性、关键氨基酸的保守性及三维结构的相似性可能是其DNA结合活性所必需的。2TK expression The cDNAs encoding mouse TAp63γ and its two deletion mutants were inserted into the pGEX vector and transformed into competent cell of E. coli BL21 （ DE3 ）, respectively. recombinant plasmids expressed effectively soluble GST fusion proteins in E. coli BL21 （ DE3 ） when The three induced by IPTG under the most optimal expression conditions. The three soluble expressed crude protein lysates were achieved by harvesting cell pellet through centrifugation, breaking the cell wall through sonication and dissolving with Triton X-100. The three near-homogeneous purified GST fusion proteins judged by SDS-PAGE were obtained by Glutathione-Sepharose affinity chromatography. Electrophoretic mobility shift assay （EMSA） indicated only wild type GST-TAp633, protein can bind to the p63 DNA-binding consensus motif, which is also the p53 consensus sequence. Furthermore, sequence alignment and homology modeling accounted for to some content the conservation of important residues and the high similarity of three dimensional structure of the intact DNA binding domain are necessary for the DNA binding activity of mouse TAp63γ protein.

关 键 词：融合蛋白 缺失突变 原核表达 凝胶滞留 序列比对 同源建模 

分 类 号：Q7[生物学—分子生物学]