Affinity purification of GST fusion proteins

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Affinity chromatography is one of the most selective types of chromatography, and it can be a very useful technique for protein purification. It employs a specific interaction that takes place between one kind of molecule in the solute and a second molecule that is immobilized to the stationary phase. The high affinity binding that occurs between protein molecules and their specific ligands can be exploited by this technique.Examples are histidine binding to metal ions, and glutathione-S-transferase binding to glutathione, as will be further discussed in this protocol.

A convenient method of protein expression and subsequent purification is to fuse a protein with a glutathione-S-transferase (GST) domain. The DNA encoding for this 25 kDa protein domain is ligated in-frame with the gene for the desired protein so that, upon expression, your desired protein is fused to the GST domain. This is an incredible help in protein purification, since GST binds glutathione extremely strongly. The general purification strategy is thus to bind the GST fusion protein on a column of immobilized glutathione, wash away all the other stuff, and then elute the protein.

The protein can then be used directly in experiments, with the GST domain still attached, although in many cases one must then control the experiments with GST to rule out interactions between GST and other molecules. Alternatively, the GST fusion protein is often constructed with a protease cleavage site between the GST domain and the protein, so that digestion with a protease such as thrombin or blood coagulation

Factor Xa and subsequent separation will remove the GST domain altogether.

II. Purification of a GST Fusion Protein

Starting with the supernatant of the cell lysis, there are two steps to GST fusion protein purification. First, the GST fusion protein is separated from all other proteins by running the supernatant over a glutathione column; the GST fusion protein binds to the glutathione column and all other proteins are washed away. The GST protein is then eluted from the column with glutathione. Second, the eluted GST protein is run over a Nap10 column to remove the glutathione, resulting in a very pure sample containing only the GST fusion protein.

III. Thrombin Cleavage

If the protein is desired without GST attached to it, and there is a cleavage site built in to the fusion between GST and your protein, you can use a protease to remove the GST. The following protocol describes cleavage with thrombin using the Novagen Thrombin Kit.

Thrombin is an endoprotease that cleaves at the sequence Leu-Val-Pro-Arg--Gly-Ser. There are two ways to accomplish cleavage. The first (and most common)method involves carrying out cleavage while the GST fusion protein is still bound to the glutathione column. This method is excellent if you are only interested in recovering your protein, because after cleavage the GST is still bound to the glutathione and the

protein elutes by itself.

If you need to recover pure GST as well, purify the sample as described above,then carry out the thrombin reaction to completion in a tube. Run the completed reaction back through a glutathione column as described above using 1x Thrombin Buffer as Buffer A. Flow-through will contain your protein plus thrombin, and then you can remove thrombin as described below. Finally, you can elute GST from column as

described above. Time and amount of thrombin required for cleavage reaction is dependent on the protein. You may want to optimize the reaction conditions on a small scale first, starting with a general estimate of 1 unit of thrombin per mg of target protein.