小鼠RHOX5蛋白两种截短型突变体的原核表达及其与MDFIC互作的鉴定

Prokaryotic Expression,Purification of Two Truncated Mutants of RHOX5 and Their Interaction with MDFIC Protein

目的:表达和纯化两种小鼠RHOX5蛋白的截短型突变体,确定完整的RHOX5蛋白及其两种截短型突变体与MDFIC蛋白结合的能力。方法:生物信息学分析小鼠同源异型框蛋白RHOX5的cDNA序列,分别对RHOX5的两种截短型片段RHOX5 N和RHOX5 C进行PCR、分别扩增RHOX5的两种截短型片段RHOX5 N和RHOX5 C并将其克隆至pGEX4T3原核表达载体,构建重组表达质粒。用重组表达质粒分别转化大肠杆菌RosettaTM2(DE3)菌株,经IPTG诱导后,使用Glutathione-Sepherase 4B颗粒对融合蛋白进行小批量亲和纯化,通过SDS-PAGE电泳分离目标蛋白,确定融合蛋白的表达。进行GST-pull down实验,检测完整的RHOX5蛋白及其两种截短型突变体与MDFIC蛋白结合的能力。结果:在大肠杆菌RosettaTM2(DE3)中有效地实现了GST-RHOX5、GST-RHOX5 N和GST-RHOX5 C 3种融合蛋白的可溶性表达;经Glutathione Sepherase 4B颗粒亲和纯化后,获得了纯化后GST融合蛋白;GST-pull down实验证实,含有homeodomain的RHOX5蛋白和RHOX5C截短型突变体可以与MDFIC蛋白相结合,而RHOX5N截短型突变体则丧失了与MDFIC蛋白结合的能力。结论:实现了RHOX5及其两种截短型突变体的原核表达和纯化,证实RHOX5蛋白的homeodomain结构域是其与MDFIC结合的关键部位。

Objective： To express and purify two truncated mutants of mouse RHOX5 protein and explore the ability of the intact RHOX5 and its two truncated mutants to bind MDFIC protein. Methods： The cDNA sequences encoding two truncated mutants of mouse RHOX5 protein RHOX5 N and RHOX5 C were amplified and then subcloned into the pGEX4T3 vector to construct the prokaryotic expression plasmids. The recombinant plasmids were transformed into E. coli RosettaTU2 （DE3）strain respectively in order to overcome the codon usage bias problem, and the exogenous protein expression was induced by IPTG. After purification using Glutathione- Sepharose 4B affinity chromatography, the GST fusion proteins were separated by SDS-PAGE. GST pull-down assay was used to determine the ability of the intact RHOX5 and its two truncated mutants to bind MDFIC protein. Results： GST-RHOX5, GST-RHOX5N and GST-RHOX5C fusion protein were expressed effectively in E. coli RosettaTM2 （ DE3 ） strain and purified using Glutathione-Sepharose 4B beads. Furthermore, GST pull-down assay indicated that both RHOX5 and RHOX5 C could bind MDFIC in vitro, while the RHOX5 N truncated mutant lost the ability of interacting with MDFIC. Conclusion ： GST-fused RHOX5 and its truncated mutants were expressed and purified successfully. In addition,it suggested that the homeodomain of RHOX5 protein is critical for its interaction with MDFIC protein.