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目的通过体外结合实验验证EHEC O157:H7内膜素受体偶联细胞骨架蛋白重复片段与人肠上皮细胞IRTKS蛋白SH3结构域之间的相互作用,并制备二者蛋白复合体,为利用晶体学方法研究其相互作用奠定基础。方法将IRTKS蛋白的SH3结构域cDNA序列克隆至pET30a表达质粒,转化BL21(DE3),诱导表达纯化SH3(IRTKS)蛋白;制备TCCP重复片段TC-CP(3R)蛋白;将SH3(IRTKS)与TCCP(3R)按照不同比例混合后,非变性丙烯酰胺凝胶电泳鉴定两者相互结合后产物;利用分子筛层析收集标准蛋白、TCCP(3R)、SH3(IRTKS)和TCCP(3R)-SH3(IRTKS)复合体的出峰体积,回归计算复合物的分子量,分析蛋白相互结合的比例。结果成功克隆表达了SH3(IRTKS)重组质粒;并制备了高纯度的SH3(IRTKS)和TCCP(3R)蛋白;非变性丙烯酰胺凝胶电泳结果证实TCCP(3R)与SH3(IRTKS)发生相互作用形成了蛋白质复合体;复合体的分子量约为33 000,二者相互结合的比例约为1:1。结论本研究为TCCP重复片段与SH3(IRTKS)相互作用提供了直接证据,同时复合体的制备,为利用晶体学研究两者的相互作用奠定了基础。
Objective To verify the interaction between the repeat fragment of EHEC O157: H7 neointiin receptor coupled cytoskeletal protein and the SHT domain of IRTKS protein in human intestinal epithelial cells by in vitro binding assay and to prepare the two protein complexes. Methods to study the basis for their interaction. Methods The cDNA sequence of SHT domain of IRTKS was cloned into pET30a expression vector and transformed into BL21 (DE3) to express and express purified SH3 (IRTKS) protein. TCCP repeat fragment TC-CP (3R) protein was prepared. (3R), SH3 (IRTKS) and TCCP (3R) -SH3 (IRTKS) were collected by molecular sieve chromatography after being mixed with different proportions of 3R (3R) and non-denaturing acrylamide gel electrophoresis. ) Complex of the peak volume, regression calculation of the molecular weight of the complex, the protein analysis of the proportion of each other. Results High purity SH3 (IRTKS) and TCCP (3R) proteins were successfully cloned and expressed by SH3 (IRTKS). Non-denaturing acrylamide gel electrophoresis confirmed the interaction between TCCP (3R) and SH3 (IRTKS) Formed a protein complex; the molecular weight of the complex is about 33 000, the ratio of the two combined with each other is about 1: 1. Conclusions This study provides direct evidence for the interaction between the TCCP repeat fragment and SH3 (IRTKS), and the preparation of the complex provides the basis for the study of the interaction between TCCP repeat fragment and IRTKS.