title: Protocols for Screening for Binding Partners of CCN Proteins: Yeast Two-Hybrid System.
abstract: Yeast two-hybrid screening is a powerful method to identify proteins that interact with a protein of interest. CCN2 consists of four domains, and identification of new proteins that bind to individual domains of CCN2 may reveal a variety of CCN2 functions. To identify CCN2-interactive proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8, with CCN2 cDNA used as a bait. In this chapter, we describe our methods for screening for CCN2 binding partners by this system in detail. This protocol may be applied to other CCN proteins as well.
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Hoshijima
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Hattori
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. takigawa@md.okayama-u.ac.jp.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
mesh_terms: D055511:CCN Intercellular Signaling Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D055511:CCN Intercellular Signaling Proteins; D002352:Carrier Proteins
keywords: Bait protein; CCN2 interactive proteins; Prey protein; Reporter gene; Yeast two-hybrid system; cDNA library