論文 - 詳細
| 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 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 8 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 原核生物(大腸菌) | pMR-7wt |