Protein Cross-linking In Vitro During Thermal Unfolding Of Recombinant Escherichia Coli Tartrate Dehydratase Beta Subunit (TtdB)

Escherichia coli tartrate dehydratase beta subunit(TtdB) is studyed to make clear in vitro protein cross-linking. By using the Polymerase Chain Reaction (PCR) technique, TtdB gene was amplified from the Escherichia coli BL21 cell. The fragment was inserted into plasmid PGM-T and confirmed by DNA sequencing .Then the target gene was subcloned into the expression plasmid pTrcHisC by the sites of two restriction enzyme BamHI and Hindâ…¢. The recombinant protein was induced by IPTG in Eschehchia coli BL21 , and the expression was associated with formation of inclusion bodies with SDS-PAGE electrophoresis. The recombinant protein was purified by immobilized metal affinity chromatography (IMAC), and refolded by dialysis. Thermal unfolding and oxidative refolding of lysozyme and TtdB led to the formation of cross-linked dimmers/oligomers as revealed by SDS-PAGE gel electrophoresis; no dimmers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol(DTT). Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of lysozyme and TtdB. In addition,the Cys/Ser point mutation in protein TtdB abrogated its ability to cross-link into homodimers.There results provide further evidence that in vitro protein cross-linking can be accomplished in three concerted steps:(1)a change in protein conformation; (2)formation of interchain disulfide bonds;and(3)formation of interchain isopeptide cross-links.