Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase.
A modular gene that encodes T7 RNA polymerase (T7 RNAP) and consists of cassettes delimited by unique restriction sites was constructed. The modular and wild-type genes of T7 RNAP were cloned into a vector designed to express His-tagged proteins. The modular and wild-type genes provided the same level of protein expression (i.e., T7 RNAP represented up to 30% of the total protein in Escherichia coli strain BL21). Purification of both proteins by immobilized metal ion affinity chromatography (IMAC) resulted in similar yields (700-800 microg of enzyme per 20 ml of culture) and purity (>95%) as indicated by Coomassie blue staining, Western blotting and the absence of detectable contaminating nuclease activities. Both proteins exhibited identical efficiency in transcription assays, and their specific activities (about 200 U/microg) were close to that of a commercial T7 RNAP preparation. The modular gene provides a useful tool for cassette directed mutagenesis of T7 RNAP.
Arnaud
N
N
Unité Mixte 103 CNRS-bioMérieux, Ecole Normale Supérieure de Lyon, France.
Cheynet
V
V
Oriol
G
G
Mandrand
B
B
Mallet
F
F
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases