title: High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system.
abstract: Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.
authors.affiliation: Department of Biosciences and Informatics, Keio University, Yokohama, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kawahashi
authors.forename: Nobuhide
authors.initials: N
authors.lastname: Doi
authors.forename: Yo
authors.initials: Y
authors.lastname: Oishi
authors.forename: Chizuru
authors.initials: C
authors.lastname: Tsuda
authors.forename: Hideaki
authors.initials: H
authors.lastname: Takashima
authors.forename: Tomoya
authors.initials: T
authors.lastname: Baba
authors.forename: Hirotada
authors.initials: H
authors.lastname: Mori
authors.forename: Takashi
authors.initials: T
authors.lastname: Ito
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D002474:Cell-Free System / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D040901:Proteomics / Q000379:methods; D011691:Puromycin / Q000031:analogs & derivatives*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D002232:Carbocyanines; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; C089187:cyanine dye 3; D011691:Puromycin; C073439:PUP2 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex