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

Non-fusion soluble expression of broad-spectrum antivirus protein in Escherichia coli by translational-coupling with SUMO

目的以具有广泛助溶活性的小泛素相关修饰蛋白(small ubiquitin-related modifier,SUMO)为辅助蛋白,利用翻译偶联,构建一种新型非融合可溶原核表达载体,实现广谱抗病毒蛋白RA在大肠杆菌中的非融合可溶表达。方法以PCR方法从酵母基因组中克隆SUMO蛋白基因smt3并进行定点突变,去除其中的EcoRⅠ酶切位点,获得同义突变体m SUMO。通过翻译偶联Linker序列的设计、合成,构建以m SUMO蛋白为辅助蛋白的翻译偶联表达载体p TIG-m SUMO。PCR扩增获得带有相应酶切位点的广谱抗病毒蛋白RA的基因,将其克隆至p TIG-m SUMO中,获得表达载体p TIG-m SUMO/RA,实现RA在大肠杆菌中的非融合可溶性表达。结果 IPTG诱导蛋白表达以后,SDSPAGE和Western印迹检测证实,目的蛋白RA的表达量明显增加,且可溶比例约占50%。结论以优化后的SUMO为辅助蛋白,成功构建了一种非融合的可溶原核表达载体p TIG-m SUMO,实现了RA蛋白在大肠杆菌中的高效非融合可溶表达,既提高了表达水平,也提高了可溶性。p TIG-m SUMO作为一种可溶的非融合表达载体,在实现外源蛋白的高效可溶表达方面,很可能具有一定的普遍性。

Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO （small ubiquitin-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 EcoR I site, 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 contribute to soluble expression of other proteins.