title: Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits.
abstract: The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB. RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.
authors.affiliation: Department of Biochemistry and Genetics, University of Newcastle upon Tyne, Medical School, U.K.
authors.forename: P E
authors.initials: PE
authors.lastname: Boehmer
authors.forename: P T
authors.initials: PT
authors.lastname: Emmerson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli