In vitro read-through polysome/ribosome display of full-length protein ORF and it's applications.
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.
Ogawa
Atsushi
A
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engeneering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Sando
Shinsuke
S
Aoyama
Yasuhiro
Y
D020413:3' Untranslated Regions / Q000737:chemistry; D000926:Anticodon; D001483:Base Sequence; D018388:Codon, Terminator / Q000737:chemistry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019151:Peptide Library; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012356:RNA, Transfer, Leu / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics
D020413:3' Untranslated Regions; D000926:Anticodon; D018388:Codon, Terminator; D019151:Peptide Library; D012356:RNA, Transfer, Leu; D013762:Tetrahydrofolate Dehydrogenase