CLONING OVEREXPRESSION AND REFOLDING OF MUTANT ESCHERICHIA COLI TARTRATE DEHYDRATASE BETA SUBUNIT

Objective To obtain the mutant Tartrate Dehydratase Beta Subunit so as to reseach protein cross-linking.Methods By using the Polymerase Chain Reaction(PCR) point mutation technique,the mutant TtdB was amplified.The fragment was inserted into plasmid PGM-T by the sites of two restriction enzyme BamHI and HindⅢ,and the target gene was confirmed by DNA sequencing.Then the target gene was subcloned into the expression plasmid pTrcHisC.The recombinant protein expression was induced by IPTG in Escherichia coli BL21.The recombinant protein was purified by immobilized metal affinity chromatography(IMAC).Results The expression was associated with formation of inclusion bodies with SDS-PAGE electrophoresis and refolded by dialysis which resulted in a recovery rate exceeding 70 %.Conclusion Purity and activity of the mutant target protein satisfy next research on protein cross-linking.