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

Abstract

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 microg/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.