大肠杆菌L-酒石酸脱氢酶b亚基体外分子交联

In vitro Cross-linking of Escherichia coli Tartrate Dehydratase Beta Subunit

摘要:

通过PCR技术扩增大肠杆菌L-酒石酸脱氢酶b亚基(L-tartrate dehydratase beta subunit, TtdB)野生型与Cys/Ser突变型目的基因, 构建带6×His标签的诱导型表达载体pTrcHisC-TtdB。重组蛋白以包含体形式存在, 应用TALON固定化金属亲和树脂(Immobilized metal affinity chromatography, IMAC)以变性的方法纯化, 通过分步透析逐步去除变性剂的方法复性, 复性率可达70%。将复性后的两种蛋白通过热诱导去折叠和氧化重折叠方法进行体外蛋白质分子交联实验。SDS-PAGE分析表明: 野生型TtdB在其变性的临界温度反应时, 出现交联二聚体和多聚体; 在氧化重折叠后SDS-PAGE前加入100 mmol/L DTT时, 交联强度明显减弱。这种DTT打不开的交联即为异肽键交联; 若在其氧化重折叠反应液中加入DTT则没有任何交联。突变型TtdB在与野生型TtdB相同的热诱导去折叠条件下, 完全没有二聚体和多聚体的形成。

Abstract:

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 studied Escherichia coli tartrate dehydratase beta subunit (TtdB). With polymerase chain reaction (PCR) technique, wild and Cys/Ser mutant genes were amplified from E. coli BL21 cells and subcloned 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.