| RRC ID |
33420
|
| 著者 |
Arnaud N, Cheynet V, Oriol G, Mandrand B, Mallet F.
|
| タイトル |
Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase.
|
| ジャーナル |
Gene
|
| Abstract |
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.
|
| 巻・号 |
199(1-2)
|
| ページ |
149-56
|
| 公開日 |
1997-10-15
|
| DOI |
10.1016/s0378-1119(97)00362-4
|
| PII |
S0378-1119(97)00362-4
|
| PMID |
9358051
|
| MeSH |
Bacteriophage T7 / enzymology
Bacteriophage T7 / genetics*
Chromatography, Affinity / methods
DNA-Directed RNA Polymerases / genetics*
DNA-Directed RNA Polymerases / isolation & purification
DNA-Directed RNA Polymerases / metabolism
Escherichia coli / genetics
Gene Expression
Genes, Viral / genetics*
Genetic Vectors / genetics
Mutagenesis, Insertional / methods*
Recombinant Fusion Proteins / isolation & purification
Recombinant Fusion Proteins / metabolism
Transcription, Genetic
Viral Proteins
Viral Structural Proteins / genetics*
|
| IF |
2.984
|
| 引用数 |
21
|
|
WOS 分野
|
GENETICS & HEREDITY
|
| リソース情報 |
| 原核生物(大腸菌) |
pMR-7wt |
