胰岛素降解酶突变体T142A的原核表达及其活性检测  

Prokaryotic expression and activity detection of insulin-degrading enzyme mutant T142A

作　　者：肖树 黄晨[1] 马树涛 张凯[1] 李依萌 李真 陆昌瑞 陈婷[1] XIAO Shu;HUANG Chen;MA Shutao;ZHANG Kai;LI Yimeng;LI Zhen;LU Changrui;CHEN Ting(College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《中国生物制品学杂志》2023年第8期924-929,共6页Chinese Journal of Biologicals

基　　金：上海市科学技术委员会国际合作计划(19410711000);国家大学生创新计划项目(105-03-0178010/025)。

摘　　要：目的 原核表达胰岛素降解酶(insulin-degrading enzyme,IDE)突变体T142A,并检测其活性。方法 结合多序列比对结果和IDE底物共晶体结构,预测IDE的β6-strand结构中的活性残基。采用点突变技术将IDE的第142位苏氨酸替换为丙氨酸,构建重组质粒ppSUMO-T142A,通过E.coli系统表达后,经镍离子柱亲和层析、离子交换层析、凝胶过滤层析纯化,获得突变体T142A,荧光法测定其活性。结果 IDE氨基酸序列在16个物种间高度保守,T142直接参与底物结合,并与底物剪切位点相互作用,且靠近催化活性中心和门亚结构域等重要结构。经测序鉴定,证明重组质粒ppSUMO-T142A突变正确。表达的融合蛋白His-SUMO-T142A相对分子质量约131 000,主要以可溶形式存在于上清中,浓度为18 mg/mL;经3步纯化获得突变体T142A的纯度达86%。T142A催化降解荧光底物Substrate V的最大反应速率(Vmax)为501.06 min^(-1),米氏常数(Km)为9.01μmol/L,与野生型IDE(V_(max)为2 814.32 min^(-1),K_(m)为11.93μmol/L)比较,活性大幅下降。结论 本研究表达的IDE突变体T142A活性大幅降低,T142是IDE发挥活性功能的重要残基,为新型IDE活性调控分子的研发提供了实验依据。Objective To express insulin-degrading enzyme(IDE)mutant T142A in prokaryotic cells and detect its activity.Methods According to the results of multi-sequence alignment and IDE substrate co-crystal structure,an active residue in β6-strand structure of IDE were predicted.The recombinant plasmid ppSUMO-T142A,with the site mutation of threonine 142 to alanine,was constructed by point mutation technique and expressed by E.coli prokaryotic expression system.After purification by nickel ion column affinity chromatography,ion exchange chromatography and gel filtration chromatography,the mutant T142A was obtained and determined for the activity by fluorescence method.Results IDE amino acid sequence is highly conserved among 16 species.T142 directly participates in substrate binding,interacts with substrate shear sites,and is close to important structures such as catalytic active sites and door-subdomains.The mutation of recombinant plasmid ppSUMO-T142A was proved to be correct by sequencing.The expressed fusion protein His-SUMO-T142A was mainly existed in soluble form in the supernatant at a concentration of 18 mg/mL,with a relative molecular mass of about 131 000;After three steps of purification,the purity of mutant T142A reached 86%.The maximum reaction rate(V_(max))of T142A catalytic degradation of fluorescent substrate V was 501.06 min^(-1) and the Michaelis constant(K_(m)) was 9.01μmol/L.Compared with wild-type IDE(V_(max) was 2 814.32 min~(-1),K_(m) was 11.93μmol/L),the activity of T142A decreased significantly.Conclusion The activity of IDE mutant T142A expressed in this study greatly decreases,while T142 is an important residue for IDE to play its enzymatic function,which provides an experimental basis for the development of new IDE activity regulatory molecules.

关 键 词：胰岛素降解酶 点突变 突变体T142A 原核表达 酶活 活性残基 

分 类 号：Q51[生物学—生物化学]