title: A simplified method for reconstituting active E. coli DNA polymerase III.
abstract: 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, ge, 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 co-expression 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.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Shi-Qiang
authors.initials: SQ
authors.lastname: Lin
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Bi
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D058105:Polymerization; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D021122:Protein Subunits; D011994:Recombinant Proteins; D004258:DNA Polymerase III