Molecular and Cell Biology Methods for Fungi pp 303-317

Part of the Methods in Molecular Biology book series (MIMB, volume 638)

Split-EGFP Screens for the Detection and Localisation of Protein–Protein Interactions in Living Yeast Cells

Protocol

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.

Keywords

Bimolecular fluorescence complementation assay BiFC Protein-fragment comple-mentation assay PCA Enhanced green fluorescent protein EGFP Genomic modification 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.School of Biological Sciences, Queen’s UniversityBelfastUK

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