| 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
|
| Resource |
| Human and Animal Cells |
|
