title: Coexpression of the subunits of T7 DNA polymerase from an artificial operon allows one-step purification of active gp5/Trx complex.
abstract: T7 DNA polymerase expression was performed from an artificial operon by cloning its cofactor, thioredoxin, downstream of a N-terminal 9xHis-tagged T7 gene 5 (gp5). Up to 90% of gp5 was soluble in the presence, but not in the absence of thioredoxin coexpression suggesting that free-form thioredoxin assisted solubilization of gp5. Expression and single-step nickel-agarose affinity purification resulted in recovery of an enzyme that was 97% pure. Copurification of thioredoxin was observed and the estimated molar ratio of thioredoxin to gp5 was 1:1 in the purified DNA polymerase complex. Purified T7 DNA polymerase exhibited full polymerase activity compared to the commercial enzyme and required no exogenous thioredoxin for activity.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Joyce
authors.initials: J
authors.lastname: Chiu
authors.forename: Daniel
authors.initials: D
authors.lastname: Tillett
authors.forename: Paul E
authors.initials: PE
authors.lastname: March
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009876:Operon / Q000235:genetics*; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D021122:Protein Subunits; C491203:TXN protein, human; D013879:Thioredoxins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase