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Affinity chromatography/mass spec
Bait proteinGST
Add yeast extractProtein complexes bindMost proteins do not bind
Page 252
Evaluation of affinity chromatography/mass spec
Advantages:Thousands of protein complexes identifiedFunctions can be assigned to proteins
Disadvantages:False negative resultsFalse positive results
Page 253-254
Affinity chromatography/mass spec
False negatives:• Bait must be properly localized and in its native condition• Affinity tag may interfere with function• Transient protein interactions may be missed• Highly specific physiological conditions may be required• Bias against hydrophobic, and small proteins
Bait proteinGST
Page 253
The yeast two-hybrid system
Reporter gene
Prey proteinDNA activation
Prey proteinDNA activation
Prey proteinDNA activation Prey protein
DNA activation
Box 8-3Page 255
The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
Prey proteinDNA activation
Box 8-3Page 255
The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
Prey proteinDNA activation
Isolate and sequence the cDNAof the binding partner you have found
Box 8-3Page 255
Evaluation of the yeast two-hybrid system
Advantages:Thousands of protein complexes identifiedFunctions can be assigned to proteins
Disadvantages:Detects only pairwise protein interactionsFalse-negative results (as for affinity chromatography)
-- bait may be mislocalized-- transient interactions may be missed-- some complexes require special conditions-- bias against hydrophobic proteins
False-positive results-- some proteins may be sticky-- bait protein may auto-activate a reporter
Page 256
The Rosetta Stone approach
Page 258
Marcotte et al. (1999) and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes, but occasionally theyare fused into a single gene.
By scanning many genomes for examples of “fusedgenes,” several thousand protein-protein predictionshave been made.
Pathway maps
A pathway is a linked set of biochemical reactions
ExPASyProNetEcoCyc: E. coli pathwaysMetaCyc: 600 pathways, 300 organismsKEGG: Kyoto Encyclopedia of Genes & Genomes
Issues:Is the extrapolation between species valid?Have orthologs been identified accurately?False positive, false negative findings
Page 258