Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique

Bacterially expressed glutathione S-transferase (GST)-fused

proteins are used as probes to perform direct measure of protei

n-protein interactions and for affinity purification.

The GST Fusion protein pull-down technique (Kaelin et al.1

991) uses the affinity of GST for glutathione-coupled beads to p

urify interacting proteins from a solution of non interacting protei

ns.

The GST fusion protein probe is expressed and purified from bacte

ria;

In parallel, a cell lysate (which can be 35S-labled or unlabled) is pre

pared;

The GST fusion protein probe and the cell lysate are mixed, in the

presence of glutathione-agarose beads and incubate the mixture to

allow protein associations to occure;

By centrifugation, collect the GST fusion probe protein and any as

sociated molecules;

The complexes are washed and can be eluted from the beads with

excess free glutathione or boiled directly in SDS-PAGE buffer;

The proteins are resolved by SDS-PAGE, and processed by Weste

rn blotting, autoradiography or protein staining.

GST Protein X GST

35S-labled cell lysate

(glutathione-sepharose beads) (glutathione-sepharose beads)

35S-labled cell lysate

GST Protein XGST

GST

Interact at 40C

Microfuge to collect complexes

1 2 3

autoradiograph

Analyze by SDS-PAGE

Lane1. Marker

Lane2. GST-protein X

Lane3. GST

The schema of a GST pull-down experiment

GSTGST Protein X

Two general uses :

To identify novel interactions between a fusion (or probe) protein and unknown (or target) proteins;

1) The protein concentrations,

2) Is the probe protein normally expressed in that particular cell or tissue?

3) Is the goal to compare different types of cell populations?

To confirm suspected interactions between the probe protein and a known proteins.

antibodies to the target protein, or:

1) the 35S-labeled in vitro translated protein, or the target protein can be tagged with an epitope;

2) Cell in culture can be transfected with a plasmid encoding the target protein to increase the abundance;

3) To control the specificity of binding, the best is the inclusion of a GST fusion protein with a mutated interaction domain,

4) To test for binding between the putative protein and GST.

Method Preclearing the cell lysate---cell lysate + glutathione agarose beads + GST

Probing the cell lysate---

Detecting interacting proteins

Incubation, 40C, 2h

End-over-end mixing

Centrifugation,40C

supernatant

Precleared cell lysate + glutathione agarose beads + GST

Precleared cell lysate + glutathione agarose beads + GST fusion probe protein

Incubation, 40C, 2h

End-over-end mixing

Centrifugation,40C

Wash the beads 3 times Elute the GST fusion protein, any proteins

optional

Mix the beads or the eluted protein with SDS-PAGE gel loading buffer

the samplesboiling SDS-PAGE analysis

Detecting (Western blotting, autoradiography or protein staining) .

To optimize the GST pull-down technique for each protein complex :The buffer (such as RIPA )in which the interactions take place;

The amount of target protein mixed with the fusion(probe) protein;

The condictions used to wash the beads to eliminate nonspecific interactions.

Troubleshooting GST pull-down experiment

The GST pulldown is a related technique in which either 1) a

single defined in-vitro-expressed protein, 2) an unknown protein

present in a pool of proteins in a cell lysate, or 3) an unknown p

rotein expressed from a pool of in-vitro-translated cDNAs is coll

ected by its interaction with a fusion protein composed of the tar

get protein linked to a GST moiety. This technique can also be

used to derive semiquantitative estimates of the affinity of protei

n-protein interactions.