High-level Production Of Yeast SUMO And SUMO Protease In E.Coli

Yeast SUMO(small ubiquitin-related modifier)as a fusion tag is widely used for improving soluble expression of heterologous protein in E.coli,and the SUMO protease ULP can be used to remove the SUMO tag efficiently.At present,yeast SUMO and ULP expressed in E.coli have yielded low soluble levels,limit the applications of SUMO fusion system.In this study,we synthesized the genes of yeast SMT3 and ULP using codon optimization strategy,analyzed the effects of codon optimization on synthetic genes' expression and characterization,and the influence of promoter on ULP synthetic gene's expression.Get the following results: 1.Constructed the prokaryotic expression vectors of SUMO,ULP synthetic genes(sumo(S),ulp(S))fused His6 tag.Realized the soluble expressions of synthetic genes in E.coli.2.Analyzed the effects of codon optimization on the expression of SUMO fusion proteins.Codon optimization enhanced the expression level of the SUMO fusion proteins,but not necessarily could improve protein folding.3.Analyzed the effects of promoter element on the expression of ULP synthetic gene.The gene expression products are mainly as inclusion bodies when ULP(S)expressed in pET28 b vector while presentation of soluble state in pMF vector.But the wild type ULP gene expression productsdo not affected by promoter.4.Qualitatively and quantitatively analyzed the expression of SUMO,ULP synthetic genes in E.coli.The results showed that the expression level of synthetic genes in BL21(DE3)were significantly higher than wild type in BL21(DE3)and RosettaTM(DE3).Meanwhile,the mobility of SUMO(S),ULP(S)in SDS-PAGE gel are changed compared with wild type proteins.5.Analyzed the effects of seven kinds of protein tags on the expression and enzyme activity of ULP(S),the results showed that protein tags cannot improve the expression level and activity.6.Analyzed the activities of two forms of ULPs cleaved SUMO fusion substrates,the results showed that wild type SUMO fusion substrate can be cleaved completely by two forms of ULPs,but synthetic SUMO fusion substrates couldn't be cleaved completely.7.Purified ULP(S)protease by Ni-NTA affinity chromatography.The products with high purity,yielded 3 folds as compared to earlier published reports about wild type ULP production.In conclusion,the synthetic genes of yeast SUMO and SUMO protease have achieved high expression in E.coli,promoted the production of ULP two folds than wild type production,but still maintain a high enzyme activity.This study is aimed at realizing the high level expression of yeast SUMO and ULP in E.coli,extended the applications of SUMO fusion system.