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Nucleic Acids Symposium Series 2005 49(1):267-268; doi:10.1093/nass/49.1.267
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© 2005 Oxford University Press

In vitro read-through polysome/ribosome display of full-length protein ORF and it's applications

Atsushi Ogawa, Shinsuke Sando and Yasuhiro Aoyama

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engeneering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

Combination of nonsense suppression and protein-ribosome-mRNA (PRM) complexation techniques leads to a new strategy "read-through polysome/ribosome display", which is designed to display full-length open reading frame (ORF) domain of the protein on the natural mRNA templates. The optimised conditions are to use the anticodon-adjusted tRNA for Leu as a nonsense suppressor in a reconstituted translation system containing diminished amounts of release factors (RFs). When applied to pseudo-natural mRNAs of Escherichia coli dihydrofolate reductase (E. coli DHFR), the input mRNA was recovered as a polysome complex displaying full-length DHFR.


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