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Journal of Biochemistry 2004 136(5):601-606; doi:10.1093/jb/mvh167
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© 2004 The Japanese Biochemical Society

MOLECULAR BIOLOGY

Cell-Free Synthesis of Polypeptides Lacking an Amino-Terminal Methionine by Using a Dicistroviral Intergenic Internal Ribosome Entry Site

Norihiro Shibuya, Takashi Nishiyama and Nobuhiko Nakashima*

National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305-8634

An amino-terminal methionine corresponding to a recombinant AUG initiation codon sometimes affects the functions of proteins. To test the performance of translation mediated by a dicistroviral internal ribosome entry site (IRES), which initiates protein synthesis with elongator tRNAs, we optimized the conditions for cell-free translation. Although the IRES is 188 nucleotides long, a further 50 nucleotides of the upstream sequence stabilized translation efficiency. Optimal ion concentrations were affected by the sequences of the constructs. In a wheat-germ system, IRES-mediated translation produced 78 µg/ml of firefly luciferase from the AUG-deleted sequence, suggesting that dicistroviral IRESs will be able to yield polypeptides with a specific N-terminal amino acid other than methionine.

* To whom correspondence should be addressed. Phone: +81-29-838-6109, Fax: +81-29-838-6028, E-mail: nakaji{at}affrc.go.jp


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