Cell-free synthesis of polypeptides lacking an amino-terminal methionine by using a dicistroviral intergenic internal ribosome entry site.
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.
Shibuya
Norihiro
N
National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Nishiyama
Takashi
T
Nakashima
Nobuhiko
N
D002474:Cell-Free System; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D014176:Protein Biosynthesis; D012328:RNA Viruses / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D010455:Peptides; D008715:Methionine; D008274:Magnesium; D011188:Potassium