Rapid interactome profiling by massive sequencing.
We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.
Department of Life Sciences, University of Trieste, Trieste, Italy.
Roberto
R
Di Niro
Ana-Marija
AM
Sulic
Flavio
F
Mignone
Sara
S
D'Angelo
Roberta
R
Bordoni
Michele
M
Iacono
Roberto
R
Marzari
Tiziano
T
Gaiotto
Miha
M
Lavric
Andrew R M
AR
Bradbury
Luigi
L
Biancone
Dina
D
Zevin-Sonkin
Gianluca
G
De Bellis
Claudio
C
Santoro
Daniele
D
Sblattero
D018076:DNA, Complementary / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D016366:Open Reading Frames; D019151:Peptide Library; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D011503:Transglutaminases / Q000378:metabolism
D018076:DNA, Complementary; D019151:Peptide Library; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins