Construction of small ubiquitin-like modifiers SUMO3/pGEX-4T-1 plasmid and expression of GST/SUMO3 fusion protein in E. coli

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 BamH I and Xho I 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.