Journal
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
Volume 84, Issue 10, Pages 2028-2036Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1080/09168451.2020.1779024
Keywords
Cell-free translation; in vitroprotein synthesis; callus; rice; Oryza sativa
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Funding
- Ministry of Education, Culture, Sports, Science and Technology, Japan [16K14665, 15H03768]
- CREST program of the Japan Science and Technology Agency [JPMJCR14F3]
- Grants-in-Aid for Scientific Research [16K14665, 15H03768] Funding Source: KAKEN
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Eukaryotic in vitro translation systems require large numbers of protein and RNA components and thereby rely on the use of cell extracts. Here we established a new in vitro translation system based on rice callus extract (RCE). We confirmed that RCE maintains its initial activity even after five freeze-thaw cycles and that the optimum temperature for translation is around 20 degrees C. We demonstrated that the RCE system allows the synthesis of hERG, a large membrane protein, in the presence of liposomes. We also showed that the introduction of a bicistronic mRNA based on 2A peptide to RCE allowed the production of two distinct proteins from a single mRNA. Our new method thus facilitates laboratory-scale production of cell extracts, making it a useful tool for the in vitro synthesis of proteins for biochemical studies. A novel method for preparing extracts of rice cultured cells for the establishment of a cell-free protein expression system
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