[In vitro cross-linking of Escherichia coli tartrate dehydratase beta subunit].
To test the hypothesis that in vitro protein cross-linking could 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, we amplified wild and Cys/Ser mutant genes with PCR technique from E. coli BL21 cells and subcloned them into expression plasmid pTrcHisC. Recombinant proteins, which were associated with formation of inclusion bodies induced by IPTG, were purified by immobilized metal affinity chromatography (IMAC) and refolded by dialysis. In thermal unfolding and oxidative refolding experiment, wild TtdB was proved to form cross-linked dimmers/oligomers as revealed by SDS-PAGE; cross-linking intensity was obviously weakened when the loading buffer contained the reducing agent dithiothreitol (DTT). The residual cross-linking was isopeptide bonds; no dimmers/oligomers were detected when the refolding and unfolding solution contained DTT. In addition, Cys/Ser point mutation abrogated its ability to cross-link into homodimers, which showed disulfide bonds could facilitate the following formation of isopeptide bonds.
Xu
Yan
Y
Biochemistry Research Division, Shandong Medical College, Linyi 276002, China.
Gao
Yin
Y
D003432:Cross-Linking Reagents / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D017510:Protein Folding
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D006836:Hydro-Lyases; C083087:L(+)-tartrate dehydratase; D019704:Protein Disulfide-Isomerases