共价交联捕获聚酮合成中的酮基还原酶和酰基载体蛋白结构域的瞬时复合物

【背景】在模块化聚酮合成酶(polyketidesynthase,PKS)的催化过程中,催化结构域与同源酰基载体蛋白(acyl-carrierprotein,ACP)之间的蛋白质-蛋白质相互作用起重要作用,但这种瞬时可逆的相互作用难以捕捉分析。【目的】获得ACP和酮基还原酶(ketoreductase,KR)相互作用的蛋白复合物。【方法】在KR和ACP之间的Linker上插入烟草蚀纹病毒(tobacco etch virus,TEV)蛋白酶切位点,通过双功能马来酰亚胺试剂BMH将KR和ACP共价交联,随后TEV酶切检测交联结果。调整反应条件,使交联效率最大化。根据KR-ACP交联复合物与体系内其他蛋白标签和分子量的差异,通过亲和层析和凝胶过滤等纯化手段,获得纯度较高的KR-ACP稳定交联复合物。【结果】单独表达的KR和ACP结构域交联不成功,融合表达的KR+ACP双结构域可以有效交联,结合使用亲和层析和凝胶过滤等纯化手段成功获得纯度较高的复合物。该策略可运用于多个KR和ACP的共价交联。【结论】建立了捕获并纯化KR和ACP瞬时相互作用复合物的有效方法,为后期晶体结构的解析、KR与ACP相互作用机理的揭示及参与相互作用关键氨基酸的鉴定提供了实验基础。

Trapping transient complex of ketoreductase and acyl carrier protein domains of modular polyketide synthases by covalently cross-linking

[Background] The protein-protein interaction between catalytic domains and the cognate acyl carrier protein (ACP) of modular polyketide synthases (PKSs) are essential for PKSs to function normally, but difficult to trapping due to the transient nature. [Objective] In this research, we tried to obtain stable protein complex of ketoreductase and ACP domains of modular PKSs. [Methods] Bifunctional maleimide agents, BMH, were used to cross-link KR and ACP domains while a tobacco etch virus (TEV) protease was inserted into the linker between KR and ACP to help the confirmation of the cross-linked complex. Reaction conditions were optimized to increase the cross-linker efficiency. Stable pure KR-ACP complex was obtained by affinity and size exclusion chromatography according to the tags of proteins and their molecular weight. [Results] The cross-linking of isolated KR and ACP was unsuccessful. However, the fused di-domain of KR and ACP could be cross-linked efficiently and generated stable pure complex following affinity and size exclusion chromatography. This strategy is useful for KR and ACP domains from different modular PKSs. [Conclusion] A method for capturing and purifying stable KR and ACP complexes was established.