A Trimmed Viral Cap‐Independent Translation Enhancing Sequence for Rapid in Vitro Gene Expression

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.