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| Prokaryotic Expression,Purification of Two Truncated Mutants of RHOX5 and Their Interaction with MDFIC Protein |
| GUO Fen1,2,LI Shi-qian1,OU Shu-fang1,CHU Yan-hui1,3,LI Yue-qin1,ZHANG Xin1,ZHOU Tian-hong1 |
1.National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China
2.College of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006,China
3.Mu Dan Jiang Medical University,Mudanjiang 157011, China |
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Abstract 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 RosettaTM2(DE3)strain respectively in order to overcome the codon usage bias problem, and the exogenous protein expression was induced by IPTG. After purification using GlutathioneSepharose 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 GlutathioneSepharose 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.
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Received: 06 July 2009
Published: 21 December 2009
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