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

Abstract:

[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.

Key words:Acyl carrier protein, Ketoreductase, Chemical cross-linking, Protein-Protein interaction, Polyketide synthase