Characterizing protein:protein interactions

Many proteins function with partners or as components of a large multiprotein complex. Understanding these interactions is critical to our understanding of biological pathways and cellular function. Discovery of protein:protein interactions is accomplished via several different methods including yeast two hybrid, co-immunoprecipitation and tandem affinity purification (TAP). Using these methods to characterize protein  interactions can be a time consuming and difficult process.

 

Glutathione-S Transferase (GST) pull-down is becoming an increasingly important tool for validation of suspected protein:protein interactions and also for identification of new interacting partners. GST pull-down uses a GST-fusion protein (bait) expressed typically in E.coli and then bound to glutathione (GSH)-coupled particles to affinity purify any proteins (prey) that interact with the bait from a pool of proteins in solution.

Prey proteins can be obtained from multiple sources including cell lysates, purified proteins and cell-free expression systems. Using cell free systems enables the researcher to easily express a variety of prey proteins and map the domain necessary for a successful interaction to occur. 

Schematic of GST Pull-Down.

Schematic of GST Pull-Down.

 

 

 

A short animation illustrating this technique can be viewed at: http://www.promega.com/paguide/animation/selector.htm?coreName=tnt01

 

 For examples of using this technique to define required protein domains or for the analysis of the effects of mutations refer to these publications:

Kruse J-P and Gu,W (2009) J. Biol. Chem, 284, 3250-3263

Galan, R-O. et al. (2009) J. Biol. Chem. 284, 4404-4412

Rokudai, S. et al. (2009) J. Biol. Chem 284, 237-244

Wang, Y. et al. (2009) J. Biochem. 145, 331-343

Schneider, J, et al. (2009) J. Virology, 83. 701-712

Purbey, J-K. et al. (2009) Mol.Cell. Biol. 29, 1321-1337

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Gary Kobs

Strategic Marketing Manager at Promega Corporation
Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

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