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Unformatted text preview: Lecture 26: Proteomics and Systems Biology § Protein-protein Interactions (continued) • Yeast two-hybrid • Protein and Antibody arrays § Yeast Systems Biology • Gene Deletion project • Genetic Interaction screening • Protein Localization screening Fuse two proteins to activation and binding domain of a transcriptional activator Express both in yeast Interaction between two proteins turns on transcription Gene transcription can yield a detectable signal (reporter gene) Fusion proteins made at gene level and expressed in yeast Positive colonies are then isolated and DNA sequenced to ID interacting proteins Yeast Two-hybrid Slide originally created by Dr. James Bruce Yeast two-hybrid Experimental Strategy § Express two fusion proteins (X and Y) in yeast, one fused to a DNA binding domain (DBD) and one fused to an activation domain (AD) § Activation of the reporter gene happens only if the two proteins interact § Screen library for interacting genes by selecting for the expression of the reporter gene (e.g. nutritional marker or LacZ) Reporter Gene DBD P r o t e i n Y AD P r o t e i n X Activate Transcription Protein-protein interaction Example of Yeast Two-Hybrid Interaction Screen Image from Uetz et al. (2000) Nature 403:623-627 Nonselective media Selective media for protein interaction Array of 6000 different yeast strains each with different gene fused to the Gal4 activation domain. Activation domain clones positive for interaction with the DNA binding domain-fusion protein Pcf11 • Pcf11 functions in pre-mRNA cleavage and polyadenylation Yeast two-hybrid Screening for Human Protein Interactions Image from Rual et al. (2005) Nature 437:1173-1178 Human Protein-Protein Interaction Network • Nodes are proteins • Edges represent protein- protein interactions Image from Rual et al. (2005) Nature 437:1173-1178 Yeast Two-hybrid Advantages “in vivo” Can screen thousands of interactions in parallel Disadvantages False positives – proteins may never co-localize in vivo Unsuitable for proteins that can itself activate transcription If interaction is PTM-dependent, interaction may not be detected If co-factor is required, interaction may not be detected Slide originally created by Dr. James Bruce Protein and Antibody Arrays § High-throughput method for measuring protein interactions or enzymatic activities § In protein arrays, recombinant proteins are expressed in...
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This note was uploaded on 01/20/2012 for the course MBIOS 478 taught by Professor Staff during the Fall '11 term at Washington State University .
- Fall '11