RRC ID 17481
著者 Barnard E, Timson DJ.
タイトル Split-EGFP screens for the detection and localisation of protein-protein interactions in living yeast cells.
ジャーナル Methods Mol Biol
Abstract Proteomics aims to identify and classify the proteins present in a particular cell or tissue. However, we know that proteins rarely function alone and knowledge of which proteins interact with which other proteins is vital if we wish to understand how cells work. The budding yeast, Saccharomyces cerevisiae, is a well-established model for studying protein-protein interactions, and a number of methods have been developed to do this. A method for the in vivo detection and localisation of interacting pairs of proteins in living yeast cells is presented. The method relies on the ability of fragments of enhanced green fluorescent protein (EGFP) to reassemble if brought into close proximity. The reassembled EGFP regains the ability to fluoresce, and this fluorescence can be detected providing evidence of interaction and information about its location. S. cerevisiae is an ideal organism to apply this method to due to the relative ease with which its genome can be manipulated. The method described enables the modification of S. cerevisiae genes at the 3'-end with DNA encoding fragments of EGFP. Consequently, the expression levels of the proteins are unlikely to be affected and thus the method is unlikely to result in false positives. In addition to the protocol for labelling and detection of interacting pairs of yeast proteins, methods for simple tests for the effects of the labelling on the organism's function are presented.
巻・号 638
ページ 303-17
公開日 2010-1-1
DOI 10.1007/978-1-60761-611-5_23
PMID 20238279
MeSH Fluorescence Genome, Fungal Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism* Microscopy, Fluorescence / methods* Protein Interaction Mapping / methods* Recombinant Fusion Proteins / analysis* Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Saccharomyces cerevisiae / genetics Saccharomyces cerevisiae / growth & development Saccharomyces cerevisiae / metabolism* Saccharomyces cerevisiae Proteins / analysis* Saccharomyces cerevisiae Proteins / genetics Saccharomyces cerevisiae Proteins / metabolism Transformation, Genetic
引用数 3
WOS 分野 BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
酵母 BYP6335 BYP6336