A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins.
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.
Mikami
Satoshi
S
RIKEN Systems and Structural Biology Center, Yokohama, Japan.
Kobayashi
Tominari
T
Masutani
Mamiko
M
Yokoyama
Shigeyuki
S
Imataka
Hiroaki
H
D020121:5' Untranslated Regions / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D038621:Regulatory Sequences, Ribonucleic Acid; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D011994:Recombinant Proteins; D038621:Regulatory Sequences, Ribonucleic Acid