Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display.
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.
Mie
Masayasu
M
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-28 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
Shimizu
Shun
S
Takahashi
Fumio
F
Kobatake
Eiry
E
D020121:5' Untranslated Regions / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D001483:Base Sequence; D019020:Directed Molecular Evolution / Q000379:methods*; D004742:Enhancer Elements, Genetic; D015723:Gene Library; D049410:Luciferases, Renilla / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes; D012270:Ribosomes / Q000378:metabolism*
D020121:5' Untranslated Regions; D049410:Luciferases, Renilla