Efficient expression of the soluble murine IL-1β with SUMO expression system

Objective:To clone mouse interleukin-1β from thymus that stimulated by LPS,and obtain the soluble and biologically active mouse IL-1β protein by prokaryotic expression for further study of the IL-1β protein.Methods:Total RNA was isolated from mouse thymus that stimulated by LPS,and reverse-transcripted into cDNA,which was used as the template for PCR reaction.The nested PCR primers for cloning mIL-1β were designed according to the published sequence.The cDNA coding for mIL-1β was cloned and constructed into the prokaryotic expressing vector pHisSUMO express-mIL-1β.The vector was transformed into E.coli Rosetta (DE3),the mIL-1β/SUMO fusion protein was induced by IPTG and purified by Ni-NTA Agarose.The expressing product was evaluated by SDS-PAGE and Western blot.After purification,the SUMO tag was removed by protease-1 cleavage to obtain the mature mIL-1β protein.The biological activity of the target protein was determined by the MTT method in L929 cells.Results:The mIL-1β cDNA was confirmed by DNA sequencing.The recombinant protein was mainly expressed in soluble form and the molecular weight of the fusion protein was 37 kD,the untagged protein was 17 kD in SDS-PAGE gel,consistent with the theoretical value.The mIL-1β protein was verified by Western blot analysis.The purity of the untagged mature protein is more than 95% after purification procedure.The mature mIL-1β protein could stimulate proliferation of L929 cells.Conclusion:Sumoylation enhances the expression level and solubility of the recombinant protein expressed from the E.coli system,and the recombinant protein is bioactive.