What is Tandem affinity purification (TAP)?
"Dystrophin and Duchenne Muscular Dystrophy." Protein-protein Interactions -. N.p., n.d. Web. 11 Apr. 2015.
"Erwin Schrödinger Prize 2008 Goes to Researchers at the Max Delbrück Center First Map Showing Human Protein
Interactions." Erwin Schrödinger Prize 2008 Goes to Researchers at the Max Delbrück Center First Map Showing
Human Protein Interactions. N.p., n.d. Web. 11 Apr. 2015.
Why is it important to identify interacting proteins?
proteins can have multiple
functions and multiple
interacting partners
Millan, Pablo P. "Train Online." Protein Interactions and Their Importance. EMBL-EBI, n.d. Web. 14 Apr. 2015.
How can you identify interacting proteins?
3
What are ways can you identify interacting proteins?
Co-IP: Co-immunoprecipitation
Millan, Pablo P. "Train Online." Protein Interactions and Their Importance. EMBL-EBI, n.d. Web. 14 Apr. 2015.
4
What ways can you identify interacting proteins?
TAP tagging
"Developmental Biology." Utrecht University. N.p., n.d. Web. 14 Apr. 2015.
What does a TAP tag look like?
ProtA
Angers, Stephanie. "Identification of the KLHL12–Cullin3 E3 Ligase as a Dishevelled Interacting
Complex." Nature.com. Nature Publishing Group, 19 Mar. 2006. Web. 14 Apr. 2015.
6
Step 1: Bind tag to beads and cleave with TEV protease
IgG beads
ProtA
Angers, Stephanie. "Identification of the KLHL12–Cullin3 E3 Ligase as a Dishevelled Interacting
Complex." Nature.com. Nature Publishing Group, 19 Mar. 2006. Web. 14 Apr. 2015.
What does ProtA screen for? What does TEV protease screen for?
Step 2: Calmodulin affinity binding & EGTA elution
ProtA
Angers, Stephanie. "Identification of the KLHL12–Cullin3 E3 Ligase as a Dishevelled Interacting
Complex." Nature.com. Nature Publishing Group, 19 Mar. 2006. Web. 14 Apr. 2015.
What does Calmodulin screen for? What does EGTA screen for?
Step 3: Digest with trypsin and send to tandem mass spec
Angers, Stephanie. "Identification of the KLHL12–Cullin3 E3 Ligase as a Dishevelled Interacting
Complex." Nature.com. Nature Publishing Group, 19 Mar. 2006. Web. 14 Apr. 2015.
How do you express the TAP tag protein?
How do you transfect a TAP tags into a cell?
TAP-tag fusion protein
"Transfection." Genebank Biosciences Inc. GBI, n.d
1. TAP tag expression
via cloning:
"BIOL2060: Sexual Reproduction, Meiosis and
Genetic Recombination (a)." Pearson
Education, 2012. Web. 11 Apr. 2015.
2. TAP tag expression via homologous recombination
"Millennium Science." Yeast. ABN, 2013. Web. 14 Apr. 2015.
TAP: Advantages
ProtA
14
Angers, Stephanie. "Identification of the KLHL12–Cullin3 E3 Ligase as a Dishevelled Interacting
Complex." Nature.com. Nature Publishing Group, 19 Mar. 2006. Web. 14 Apr. 2015.
TAP: Advantages
ProtA
in vivo approach
High yield
Identifies proteins that are weakly interacting
Highly specific
Only need to know bait protein sequence
Can manipulate TAP tag components to alter specificity
Angers, Stephanie. "Identification of the KLHL12–Cullin3 E3 Ligase as a Dishevelled Interacting
Complex." Nature.com. Nature Publishing Group, 19 Mar. 2006. Web. 14 Apr. 2015.
Disadvantages
"Study: Online Self-Injury Information Often Inaccurate." Mom
Psych. University of Geulph, 31 Mar. 2014. Web. 12 Apr. 2015.
Functional organization of the
yeast proteome by systematic
analysis of protein complexes.
Gavin et al, 2002
What were the goals of the research?
Identify and analyze the components of multiprotein complexes
Determine how proteins are organized into functional unit
Compare yeast and human complexes
What organism did they use and why?
http://www.microbiologyonline.org.uk/about-microbiology/introducing-microbes/fungi
http://www.microbiologyonline.org.uk/themed/sgm/img/slideshows/3.1.4_fungi_2.png
Fig1: How did they setup their TAP tag screen?
Fig 1c: Overview of steps to purify and identify TAP complexes
*596 of 1,739 genes were nonorthologous
Fig 2a & 2b: Where did the proteins localize?
*Only 14% of membrane proteins were analyzed
numbers = % of total proteins
Fig 2c & 2d: How many novel proteins were identified?
Left side of Fig. d is what makes this technique special!
Fig 2e & 2f: What was the functional distribution of the proteins identified?
Organization of the protein assemblies
589 different proteins
245 known
purifications
242 new
purifications
98 known non redundant
complexes in yeast database
134 new
complexes
*102 remaining proteins- no detectable protein interactions
Fig.3 How did they validate the TAP tag method?
Proposed Model of the “Machinery”
Polyadenylation Complex
underline = new
*Similar band is observed when different components are used as entry points
Fig 4: How did they determine relationships between networks?
Red-cell cycle
Dark green-signaling
Dark blue-transcription, DNA maintenance,
chromatin structure
Pink-protein and RNA transport
Orange-RNA metabolism
Light green-protein synthesis and turnover
Brown-cell polarity and structure
Violet-intermediate and energy metabolism
Light blue-membrane biogenesis and traffic
Used a “Relaxed” algorithm to create these networks
How do these networks look today?
Fig 4: How did they determine relationships between a few complexes?
All the small proteins make up the larger circles that form the complex
Fig.5a/b: Do complexes share similar composition in yeast and humans?
Yeast genes have equivalent genes found in humans
Fig. 5c: Do complexes share similar composition in yeast and humans?
Simplifies gene study in humans
Conclusions
Steps towards identifying the “core eukaryotic proteome”
Helped explain the assembly and disassembly of protein
complexes
Proteins that are metazoan orthologues preferentially bind
to the same set of proteins
Grouped cellular proteins into 200 complexes
Future Implications
Creation of Higher-Order maps
Provide insight for drug discovery programs
Provide an efficient way to identify gene and gene function
http://www.celiaccentral.org/drugdevelopment/
Why is TAP tag better than Y2H?
TAP vs Y2H
Easy
Relies on binary interactions
Allows for creation protein network
maps
Transient protein interactions
Works in membrane proteins
High Yield
Low false negatives
Works in a variety of organisms
Relies on previous information such
as YPD
Viewed as complimentary to
TAP
Questions?