Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display.
Biochem Biophys Res Commun
; 373(1): 48-52, 2008 Aug 15.
Article
em En
| MEDLINE
| ID: mdl-18544339
ABSTRACT
The 5'-untranslated region (5'-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5'-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5'-UTRs with high translation efficiency using a ribosome display technique. A 5'-UTR random library, comprised of 5'-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5'-UTR with high translation efficiency was obtained from random 5'-UTR library.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Ribossomos
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Biossíntese de Proteínas
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Elementos Facilitadores Genéticos
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Evolução Molecular Direcionada
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Regiões 5' não Traduzidas
Limite:
Animals
Idioma:
En
Revista:
Biochem Biophys Res Commun
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Japão