Non-fusion soluble expression of broad-spectrum antivirus protein in Escherichia coli by translational-coupling with SUMO
10.7644/j.issn.1674-9960.2015.08.006
- VernacularTitle:利用翻译偶联实现广谱抗病毒蛋白在大肠杆菌中的可溶性表达
- Author:
Lingyue XING
;
Dejian XIE
;
Bingyu YE
;
Zhang ZHANG
;
Ping LI
;
Wenlong SHEN
;
Minglei SHI
;
Yan ZHANG
;
Zhihu ZHAO
- Publication Type:Journal Article
- Keywords:
small ubiquitin-related modifier proteins;
pTIG-mSUMO;
prokaryotic expression;
solubility;
translation coupling;
non-fused
- From:
Military Medical Sciences
2015;(8):597-601
- CountryChina
- Language:Chinese
-
Abstract:
Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO(small ubiq-uitin-related modifier) using the widely used solubility promoting protein SUMO and based on the translational coupling phenomenon in order to enable the non-fusion soluble expression of the broad-spectrum antiviral protein RA in Escherichia coli by pTIG-mSUMO.Methods The smt3 gene coding for SUMO protein was cloned from yeast genome DNA by PCR. After directed-site silent mutation to eliminate the EcoRⅠsite, the mutant mSUMO was inserted into pET-22b to obtain the translational coupling expression vector pTIG-mSUMO.The RA was subject to PCR amplification and cloned into the pTIG-mSUMO to obtain the expression plasmid pTIG-mSUMO/RA which was supposed to direct the soluble expression of RA by the translational coupling with mSUMO.Results A translational coupling expression vector pTIG-mSUMO which could di-rect/drive the SUMO and heterogonous protein non-fusion expression simultaneously was designed and constructed.The Western blotting result indicated that pTIG-mSUMO could direct the high-level expression of RA, around 40%of which was soluble.Conclusion A translational coupling expression vector pTIG-mSUMO is obtained.After coupling with SUMO, RA is highly expressed in E.coli and both the expression level and solubility are greatly improved.pTIG-mSUMO might contrib-ute to soluble expression of other proteins.
