原癌蛋白KRas的表达和纯化及其与小分子抑制剂ZCL1688的分子对接分析  

Expression and Purification of Proto-oncoprotein KRas and Its Molecular Docoking with Small Molecule Inhibitor ZCL1688

作　　者：牟榕梓 孙暄 吴更[1] MU Rong-Zi;SUN Xuan;WU Geng(School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学生命科学技术学院,上海200240

出　　处：《分析化学》2022年第2期263-270,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.31670106)资助。

摘　　要：经过特殊设计的小分子抑制剂可共价结合KRas蛋白的催化结构域,从而抑制KRas与下游效应蛋白的结合。本研究采用镍柱亲和层析、酶切标签、凝胶过滤层析等步骤制备了高纯度的KRas催化结构域(氨基酸残基1-169)蛋白;等温滴定量热(ITC)实验证明了KRas蛋白通过K42残基与小分子抑制剂ZCL1688进行结合;通过分子对接得到KRas蛋白与ZCL1688的复合物的建模结构,发现两者的结合对KRas与下游蛋白丝氨酸/苏氨酸激酶RAF的结合有明显竞争作用。本研究建立了一种简便的KRas蛋白催化结构域(氨基酸残基1-169)高效表达与高纯度制备方法,模拟了此蛋白与ZCL1688的结合模式,并分析了该结合模式下的相互作用,为后续筛选此蛋白与小分子抑制剂的共结晶以及复合物结构解析奠定了良好基础。Specially designed small molecule compounds can covalently bind to the catalytic domain of KRas,thereby inhibiting the effect of downstream effector molecules.In this work,a high-purity KRas protein catalytic domain(AA 1-169)was prepared by nickel column affinity chromatography,enzyme cleavage labeling,gel chromatography and other steps.The results showed that KRas protein bound to the small molecule inhibitor ZCL1688 through K42 residue by isothermal titration calorimetry(ITC)experiments.Next,the modeled structure of the complex of KRas and ZCL1688 was obtained through molecular docking,and it was found that the combination of the two molecules had a significant competitive effect on the binding of KRas to down stream protein,serine/threonine kinase RAF.A simple method for high-efficiency expression and high-purity preparation of the KRas protein catalytic domain(AA 1-169)were thus established.The binding mode and weak interaction between KRas and ZCL1688 were analyzed.It laid a good foundation for the subsequent screening of the binding conditions of the protein with small molecule inhibitors and analysis of complex structure.

关 键 词：KRAS 丝氨酸/苏氨酸激酶RAF 小分子抑制剂 蛋白的纯化 分子对接 弱相互作用 

分 类 号：TQ460.1[化学工程—制药化工]