关键词: 类泛素化蛋白3；GST融合蛋白；蛋白纯化

Abstract:

Abstract:Objective
 To construct pGEX-4T-1/SUMO3 recombinant plasmid in order to express the full length of small ubiquitin-like modifier 3 (SUMO3) in E.coli and purify the GST-SUMO3 fusion protein. Methods The DNA sequence of SUMO3 (full length: 312 bp) was amplified by PCR using the template plasmid pcDNA3.1/SUMO3 and was then cloned into the expression vector pGEX-4T-1.After identified by restriction enzyme digestion and sequencing,the recombinant clone was transformed into the competent expression cells of E.coli BL21.GST-SUMO3 fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain a fusion protein with molecular weight of 38　000.Results It was identified that the recombinant expression vector of pGEX-4T-1/SUMO3 contained a 312 bp insert fragment by BamHⅠ and XhoⅠ double digestion and the insert fragment showed exactly consistant sequence with SUMO3.The fusion protein of SUMO3 combined with GST was successfully expressed and purified with 0.1 mmol·L^-1 IPTG induction.Conclusion The SUMO3 protein combined with GST-tag is gained successfully,which provides the basis for the preparation of SUMO3 antibody and the further functional research of SUMO3.

Key words: SUMO3;GST fusion protein;protein purification