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A Trimmed Viral Cap‐Independent Translation Enhancing Sequence for Rapid in Vitro Gene Expression
Author(s) -
Kawarasaki Yasuaki,
Kasahara Satoru,
Kodera Natsuko,
Shinbata Tomoya,
Sekiguchi Satoshi,
Nakano Hideo,
Yamane Tsuneo
Publication year - 2000
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp000021x
Subject(s) - biology , start codon , microbiology and biotechnology , gene , ribosomal binding site , eukaryotic translation , primer (cosmetics) , untranslated region , translation (biology) , messenger rna , five prime untranslated region , in vitro , protein biosynthesis , gene expression , genetics , chemistry , organic chemistry
We prepared a short (29 nucleotides) 5′ UTR that enhanced cap‐independent translation in a wheat germ translation system by trimming the tobacco etch virus 5′ UTR. The trimmed sequence, designated as TE(37−65), was obtained from a conserved region among several potyviruses. The productivities of uncapped reporter mRNAs carrying the TE(37−65) sequence were comparable to those of capped counterparts, in that 5−20 μg of proteins were synthesized per 1 mL of translation reaction mixture. The ribosome that entered onto the TE(37−65) sequence precisely initiated polypeptide synthesis at the defined initiation codon, which ensures rapid and efficient protein truncation analyses. Moreover, the TE(37−65) sequence is short enough to be involved in a PCR primer, which allows a simple method for rapid gene expression, i.e., PCR amplification of a target gene and succeeding in vitro transcription and translation. As a demonstration, the rapid in vitro expression of rice cDNAs using the TE(37−65) sequence was also performed.