Rapid interactome profiling by massive sequencing

被引：48

作者：

Di Niro, Roberto ^{[4
]}

Sulic, Ana-Marija ^{[4
]}

Mignone, Flavio ^{[3
]}

D'Angelo, Sara ^{[1
,2
]}

Bordoni, Roberta ^{[5
]}

Iacono, Michele ^{[5
]}

Marzari, Roberto ^{[4
]}

Gaiotto, Tiziano ^{[4
]}

Lavric, Miha ^{[4
]}

Bradbury, Andrew R. M. ^{[6
]}

Biancone, Luigi ^{[7
]}

Zevin-Sonkin, Dina ^{[8
]}

De Bellis, Gianluca ^{[5
]}

Santoro, Claudio ^{[1
,2
]}

Sblattero, Daniele ^{[1
,2
]}

机构：

[1] Univ Piemonte Orientale, Dept Med Sci, Novara, Italy

[2] Univ Piemonte Orientale, IRCAD, Novara, Italy

[3] Univ Milan, Sch Pharm, Dept Struct Chem & Inorgan Stereochem, Milan, Italy

[4] Univ Trieste, Dept Life Sci, Trieste, Italy

[5] Natl Res Council ITB CNR, Inst Biomed Technol, Milan, Italy

[6] Los Alamos Natl Lab, Los Alamos, NM USA

[7] Univ Turin, CERMS, Turin, Italy

[8] Quark Pharmaceut Inc, QBI Enterprises Inc, Ness Ziona, Israel

来源：

NUCLEIC ACIDS RESEARCH | 2010年 / 38卷 / 09期

基金：

美国国家卫生研究院;

关键词：

OPEN READING FRAMES; TISSUE TRANSGLUTAMINASE; BINDING-PROTEINS; EFFICIENT IDENTIFICATION; CDNA LIBRARIES; PHAGE DISPLAY; IN-VIVO; SELECTION; EXPRESSION; COMPLEMENTATION;

D O I：

10.1093/nar/gkq052

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：e110 / e110

页数：10

共 40 条

[1] Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin [J].