定点突变对Procaspase-8 DEDs蛋白溶解性的影响  

Effect of Site-Directed Mutagenesis on Soluble Expression of Procaspase-8 DEDs

作　　者：庞威威 孔庆宏[1] 王冠林[1] 张宽仁[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500

出　　处：《昆明理工大学学报（自然科学版）》2017年第6期75-80,共6页Journal of Kunming University of Science and Technology(Natural Science)

基　　金：国家自然科学基金项目(81360162;81260351)

摘　　要：在进行人源Procaspase-8 DEDs(也称为Caspase-8 DEDs)蛋白研究时,采用定点突变的方法对野生型(WT)Procaspase-8 DEDs蛋白编码基因的三个位点F122、L123、I128进行突变,并将获得的突变体基因片段构建入表达载体p ET-28a(+)和p CMV6-AC-GFP中;在原核细胞中转化入E.Coli Transetta(DE3),获得具有三突变(F122G/L123G/I128D)的表达菌株,当以IPTG诱导目的基因表达时,发现突变蛋白(Procaspase-8 DEDsF122G/L123G/I128D)的可溶性表达显著提高;当真核质粒转染细胞HCT116时,与野生型Procaspase-8 DEDs蛋白相比,突变型蛋白不利于死亡效应纤丝(Death Effector Filaments,DEFs)的形成.结果表明:定点突变能提高Procaspase-8DEDsF122G/L123G/I128D蛋白在大肠杆菌中的可溶性表达,并经Ni^(2+)蛋白纯化柱和蛋白浓缩柱作用可富集可溶性目的蛋白;同时,在真核细胞中改善了死亡效应纤丝形成问题.本文研究有助于Procaspase-8 DEDs蛋白的体外功能研究以及相关细胞系构建.In the study of human Procaspase - 8 DEDs （ also known as Caspase - 8 DEDs）, three sites F^122, L^123 and I^128 of the wild - type （WT） Procaspase - 8 DEDs protein were subjected to simultaneous site - directed mu- tagenesis, and the mutant gene fragments were constructed into expression vectors pET -28a （ ＋ ） and pCMV6 -AC -GFP. Transforming prokaryotic plasmid into E. coli Transetta （DE3）, prokaryotic expression strain was successfully acquired. The soluble expression of Procaspase - 8 DEDs^F122G/L123c/I12SD protein was significantly high- er than that of wild - type Procaspase - 8 DEDs by induced expression with IPTG. When recombinant eukaryotic plasmid was transfected into HCT1 16 cells, mutant protein did not tend to form death effector filaments （DEFs） compared with the expression of Proeaspase - 8 DEDs protein. It is concluded that soluble expression of Pro- caspase - 8 DEDs^F122G/L123G/I128D protein inE. coli could be enhanced by site - directed mutagenesis, and the soluble target protein could be assembled by Ni2＋ protein purification column and protein concentration column. Meanwhile, in eukaryotic cells, Procaspase -8 DEOs^F122G/L123G/I128D protein improves the phenomenon of death ef- fector filaments. These findings will provide helpful insights into the functional study of Procaspase - 8 DEDs in vitro and the construction of related cell lines.

关 键 词：半胱氨酸蛋白酶-8 定点突变 诱导表达 纯化 死亡效应纤丝 

分 类 号：O629.73[理学—有机化学]