Genome duplication in E.coli is carried out by DNA polymeraseⅢ,an enzyme complex consisting of ten subunits.Investigations of the biochemical and structural properties of DNA polymeraseⅢrequire the expression and pu...Genome duplication in E.coli is carried out by DNA polymeraseⅢ,an enzyme complex consisting of ten subunits.Investigations of the biochemical and structural properties of DNA polymeraseⅢrequire the expression and purification of subunits includingα,ε,θ,γ,δ′,δ,andβseparately followed by in vitro reconstitution of the polⅢcore and clamp loader.Here we propose a new method for expressing and purifying DNA polymeraseⅢcomponents by utilizing a protein coexpression strategy.Our results show that the subunits of the polⅢcore and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits.The resulting polⅢcore,clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization.Our strategy considerably simplifies the expression and purification of DNA polymeraseⅢand provides a feasible and convenient method for exploring other multi-subunit systems.展开更多
基金the National Basic Research Program of China(Grant Nos.2009CB522605 and 2011CB910302)the Key Project Specialized for Infectious Diseases from the Ministry of Health of China(No.2008ZX10003-005)+1 种基金the Chinese Academy of Sciences(Grant No.KSCX2-YW-R-164)the National Natural Science Foundation of China(Grant No.30970590).
文摘Genome duplication in E.coli is carried out by DNA polymeraseⅢ,an enzyme complex consisting of ten subunits.Investigations of the biochemical and structural properties of DNA polymeraseⅢrequire the expression and purification of subunits includingα,ε,θ,γ,δ′,δ,andβseparately followed by in vitro reconstitution of the polⅢcore and clamp loader.Here we propose a new method for expressing and purifying DNA polymeraseⅢcomponents by utilizing a protein coexpression strategy.Our results show that the subunits of the polⅢcore and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits.The resulting polⅢcore,clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization.Our strategy considerably simplifies the expression and purification of DNA polymeraseⅢand provides a feasible and convenient method for exploring other multi-subunit systems.
