A simplified method for reconstituting active E. coli DNA polymerase III

Genome duplication in E. coli is carried out by DNA polymerase Ⅲ, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase Ⅲ require the expression and purification of subunits including α, ε, θ, γ, δ', δ, and β separately followed by in vitro reconstitution of the pol Ⅲ core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase Ⅲ components by utilizing a protein coexpression strategy. Our results show that the subunits of the pol Ⅲ core and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits. The resulting pol Ⅲ core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase Ⅲ and provides a feasible and convenient method for exploring other multi-subunit systems.