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.

[1]  N. Caberoy,et al.  Efficient identification of tubby‐binding proteins by an improved system of T7 phage display , 2009, Journal of molecular recognition : JMR.

[2]  G Meier,et al.  Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data. , 2000, Biophysical journal.

[3]  S. L. Wong,et al.  Towards a proteome-scale map of the human protein–protein interaction network , 2005, Nature.

[4]  T. Terwilliger,et al.  Rapid protein-folding assay using green fluorescent protein , 1999, Nature Biotechnology.

[5]  A. Baird,et al.  Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection. , 2004, BioTechniques.

[6]  Tarcisio Not,et al.  Characterizing monoclonal antibody epitopes by filtered gene fragment phage display. , 2005, The Biochemical journal.

[7]  B. Suter,et al.  Interactive proteomics: what lies ahead? , 2008, BioTechniques.

[8]  A. Mittermaier,et al.  A simple in vivo assay for increased protein solubility , 1999, Protein science : a publication of the Protein Society.

[9]  Mauro Piacentini,et al.  Transglutaminase 2: an enigmatic enzyme with diverse functions. , 2002, Trends in biochemical sciences.

[10]  E. Tongiorgi,et al.  Molecular Dissection of the Tissue Transglutaminase Autoantibody Response in Celiac Disease1 , 2001, The Journal of Immunology.

[11]  G. Waldo,et al.  Genetic screens and directed evolution for protein solubility. , 2003, Current opinion in chemical biology.

[12]  John F. Hunt,et al.  Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein , 2001, Nature Biotechnology.

[13]  S. Akimov,et al.  Tissue Transglutaminase Is an Integrin-Binding Adhesion Coreceptor for Fibronectin , 2000, The Journal of cell biology.

[14]  Suresh Mishra,et al.  Tissue Transglutaminase Has Intrinsic Kinase Activity , 2004, Journal of Biological Chemistry.

[15]  Andrea S. Llera,et al.  The role of the matricellular protein SPARC in the dynamic interaction between the tumor and the host , 2008, Cancer and Metastasis Reviews.

[16]  A. Brunger,et al.  Transglutaminase 2 Undergoes a Large Conformational Change upon Activation , 2007, PLoS biology.

[17]  C. Esposito,et al.  Mammalian transglutaminases , 2005, The FEBS journal.

[18]  U. Dianzani,et al.  Antibody library selection by the {beta}-lactamase protein fragment complementation assay. , 2009, Protein engineering, design & selection : PEDS.

[19]  Xinchun Shen,et al.  Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome. , 2008, Cell calcium.

[20]  Bertalan Meskó,et al.  Transdab wiki: the interactive transglutaminase substrate database on web 2.0 surface , 2009, Amino Acids.

[21]  Piero Carninci,et al.  Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. , 2000, Genome research.

[22]  F. Facchiano,et al.  The role of transglutaminase-2 and its substrates in human diseases. , 2006, Frontiers in bioscience : a journal and virtual library.

[23]  P. Davies,et al.  Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin. , 1997, Journal of cell science.

[24]  M. Maki,et al.  Screening for the Preferred Substrate Sequence of Transglutaminase Using a Phage-displayed Peptide Library , 2006, Journal of Biological Chemistry.

[25]  S. Dübel,et al.  Enrichment of open reading frames presented on bacteriophage M13 using hyperphage. , 2006, BioTechniques.

[26]  H R Hoogenboom,et al.  By-passing immunization. Human antibodies from V-gene libraries displayed on phage. , 1991, Journal of molecular biology.

[27]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[28]  Masashi Kikuchi,et al.  A novel function of tissue-type transglutaminase: protein disulphide isomerase. , 2003, The Biochemical journal.

[29]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[30]  M. Taussig,et al.  Detection of protein-protein interactions by ribosome display and protein in situ immobilisation. , 2009, New biotechnology.

[31]  Joseph W. Carlson,et al.  Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP) , 2005, Nucleic acids research.

[32]  M. Sheng,et al.  Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries. , 2001, Journal of immunological methods.

[33]  M. Piacentini,et al.  Identification of ‘tissue’ transglutaminase binding proteins in neural cells committed to apoptosis , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[34]  Rita Casadio,et al.  Transglutaminases: nature's biological glues. , 2002, The Biochemical journal.

[35]  R. Marzari,et al.  Selecting open reading frames from DNA. , 2003, Genome research.

[36]  Xianqun Fan,et al.  Efficient identification of phosphatidylserine-binding proteins by ORF phage display. , 2009, Biochemical and biophysical research communications.

[37]  R. Cortese,et al.  Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands. , 2002, Nucleic acids research.

[38]  László Fésüs,et al.  Phage display selection of efficient glutamine‐donor substrate peptides for transglutaminase 2 , 2006, Protein science : a publication of the Protein Society.