In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins.
Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.
Kawahashi
Yuko
Y
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama 223-8522, Japan.
Doi
Nobuhide
N
Takashima
Hideaki
H
Tsuda
Chizuru
C
Oishi
Yuko
Y
Oyama
Rieko
R
Yonezawa
Masato
M
Miyamoto-Sato
Etsuko
E
Yanagawa
Hiroshi
H
D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000737:chemistry; D005898:Glass; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D008956:Models, Chemical; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011691:Puromycin / Q000737:chemistry / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D011691:Puromycin; D005982:Glutathione Transferase; D005978:Glutathione