title: Expression and purification of active recombinant T7 RNA polymerase from E. coli.
abstract: For large-scale transcription reactions or for cost savings, a laboratory may want to prepare its own recombinant T7-, SP6-, or T3-phage RNA polymerases. It is convenient to perform this preparation every 2-3 years and have a consistent and reliable source of phage RNA polymerase for many in vitro transcription reactions. In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-d-1-thio-galactopyranoside (IPTG)-inducible promoter. The bacteria are lysed by sonication, the his6-tagged protein in the bacterial lysate is purified by binding to Ni-NTA agarose, and the resin is then extensively washed and eluted with imidazole. The purified enzyme is dialyzed against a glycerol-containing storage buffer and can then be stored for months or years at -20°C.
authors.forename: Donald C
authors.initials: DC
authors.lastname: Rio
mesh_terms: D002846:Chromatography, Affinity; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases