Reference - Detail
| RRC ID | 43470 |
|---|---|
| Author | Mikami S, Kobayashi T, Masutani M, Yokoyama S, Imataka H. |
| Title | A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. |
| Journal | Protein Expr Purif |
| Abstract |
The aim of this study was to develop an efficient cell-free protein expression system derived from mammalian cells. We established a HeLa cell-based in vitro coupled transcription/translation system with T7 RNA polymerase and a plasmid that harbored a T7 promoter/terminator unit. To enhance protein synthesis in the coupled system, we placed the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or the hepatitis C virus (HCV) IRES between the T7 promoter and the coding region of the plasmid. Remarkably, we found that these IRES-dependent systems were able to produce large proteins including GCN2 (160 kD), Dicer (200 kD) and mTOR (260 kD) to levels detectable on SDS-PAGE by Comassie Brilliant Blue-staining. We purified the synthesized proteins to near homogeneity, and validated their functionalities in the appropriate biochemical assays. In conclusion, the HeLa cell-based in vitro coupled transcription/translation system using the EMCV or HCV IRES is a convenient tool, particularly for the production of large recombinant proteins. |
| Volume | 62(2) |
| Pages | 190-8 |
| Published | 2008-12-1 |
| DOI | 10.1016/j.pep.2008.09.002 |
| PII | S1046-5928(08)00227-1 |
| PMID | 18814849 |
| MeSH | 5' Untranslated Regions / genetics Encephalomyocarditis virus / genetics HeLa Cells Hepacivirus / genetics Humans Protein Biosynthesis* Recombinant Proteins / biosynthesis* Regulatory Sequences, Ribonucleic Acid Transcription, Genetic* |
| IF | 1.513 |
| Times Cited | 40 |
| WOS Category | BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY |
| Altmetric score |
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| The most frequently cited source | Patent(IFI CLAIMS) |
| Total number of mentions | 7 |
| Altmetric score changes over past 6months | 9.0 |
| Resource | |
| Human and Animal Cells | |