Construction Of The Chaperones Coexpression Plasmids And Functional Analysis Of Tobacco Etch Virus Protease Mutant

Escherichia coli is the most extensively used host for production of homologous proteins. However, the recombinant proteins in E. coli often fail to properly fold and overexpressed as inclusion bodies. The several chaperones were determined to help the recombinant proteins folding independently or as the fusion tag at N terminus. Removal of the tag requires the high efficient specific proteinase. In this study, we constructed two helper vectors expressing six chaperone genes under T7 promoter and analyzed the effects on soluble expression of three maize proteins. We mutated TEV protease based on the structure and characterized this mutant. The main results are listed as below:1. Six chaperone genes were amplified by PCR using Escherichia coli DH5a genomic DNA as the template. All PCR products were identified by the sequential analysis.2. Six chaperone genes digested with two restrictive enzymes were inserted sequentially into the expression vector pRSFDuet-1 and pCDFDuet-1 treated with the same manner to generate the resultant plasmid, named pR-GESP with groEL, groES and grpE gene, and pC-DJCL with dnaK,dnaJ and clpB gene.3. The SDS-PAGE analysis from the induced crude extract showed that groEL, groES, grpE dnaK, and clpB were expressed obviously, but the expression level of dnaJ was not observed clearly.4. Co-overexpression chaperones in E. coli improved the solubility of the recombinant maize sirohydrochlorin ferrochelatase, and partly improved the folding of the recombinant maize glutamate semiadhyde aminotransferase, but has not effect on the folding of the recombinant maize uroporphyrinogen III decarboxylase obviously, as shown by SDS-PAGE.5. Using the TEV protease S219V as the model, the new mutant named TEVF and TEVQ were created using site-directed mutagenesis. The production of the purified recombinant protein was up to 108 mg/L for TEVF and 165 mg/L for TEVQ.6. The TEVQ mutant protease activity is more than TEVQ using the purified recombinant GST fusion within TEV protease cleavage site as the substrate, as shown by SDS-PAGE. The efficient cleavage in vivo of GST fusion by coexpression TEVQ mutant protease was also detected.In conclusion, we constructed two helper coexpression vectors and analyzed molecular chaperones expression level. The effect of both helper vectors on the enhancement of the solubility of three maize enzymes involved in the biosynthesis of tetrapyrroles in E. coli is dependent of the intrinsic folding character of the enzyme. The high soluble TEV protease mutant with the increased activity was obtained and provided a platform for the use of various fusion tags in protein expression.