Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase
1997; Elsevier BV; Volume: 199; Issue: 1-2 Linguagem: Inglês
10.1016/s0378-1119(97)00362-4
ISSN1879-0038
AutoresNadège Arnaud, Valérie Cheynet, Guy Oriol, Bernard Mandrand, François Mallet,
Tópico(s)RNA and protein synthesis mechanisms
ResumoA 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.
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