This chapter describes the use of N-terminal glutathione S-transferase (GST)-tagged Recombinant gene fusion proteins as a method for high-level protein expression and inducible purification from bacterial cell lysates. The protein is expressed in a pGEX vector, with the GST residue located at the N-terminus followed by the target protein. The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and the fusion protein can often be expressed as a soluble protein rather than in inclusion bodies. In addition, the GST fusion protein can be readily affinity-purified without denaturation or the use of mild detergents.
The fusion protein is captured by the immobilized glutathione and the impurities are removed. The fusion protein is then eluted under mild, non-denaturing conditions using reduced glutathione. If desired, removal of the GST affinity tag is achieved through the use of a site-specific protease recognition sequence located between the GST moiety and the target protein. The purified proteins have been used successfully in immunological studies, structure determinations, vaccine production, protein-protein and protein-DNA interaction studies, and other biochemical analyses.
glutathione S-transferase (GST), pGEX, protein expression, protein purification, thrombin, factor Xa, fusion tags
1. GST fusion protein expression
- LB medium per litre: 10 g tryptone, 5 g yeast, 5 g NaCl. Adjust to pH 7.2 with NaOH. Autoclave.
- Ampicillin, 5 mg/mL stock solution, filter sterilization
- Glycerol stock of transformed E. coli cells expressing the GST fusion protein in the pGEX vector
- Isopropyl-1-thio-β-D-galactopyranoside (IPTG), stock 100 mM
2. Affinity purification of the GST fusion protein
- Glutathione Sepharose 4B Bulk Matrix (GE Healthcare or Sigma)
- Column (BioRad Econo-Column 2.5 cm × 10 cm)
- 10 mM glutathione buffer: 50 mM Tris, 10 mM reduced glutathione, pH 8.0 (make fresh daily)
- PBS/EDTA/PMSF: 1X PBS, 5mM EDTA, 0.15mM PMSF, pH 7.4
- E. coli pellets containing expressed target fusion protein
- PBS/EDTA: 1X PBS, 5mM EDTA, pH 7.4
- Lysis buffer: 50 mM Tris, 50 mM NaCl, 5 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 0.15 mM PMSF, 1 mM DFP, 1 mM 2-ME, pH 8.0. Caution: DFP is a very dangerous neurotoxin; and the precautions provided.
3. Enzymatic cleavage to remove the GST affinity tag
- Affinity purified fusion protein
- The vector-specific enzyme (thrombin, factor Xa, or PreScission protease)
- PMSF 0.15 M in isopropanol
- PBS/EDTA/PMSF: 1X PBS, 5mM EDTA, 0.15mM PMSF, pH 7.4
4. GST affinity tag removal after protease cleavage
- Cleaved fusion protein dialyzed in PBS/EDTA/PMSF
- Glutathione Sepharose Column
5. Further purification of cleaved recombinant protein
- cleaved fusion protein
- Concentrator (Amicon Ultra)
- HPLC system with the online detector and fraction collector
- Gel filtration column compatible with the molecular weight range of the sample to be purified
GST Label Definition
Glutathione-S-transferase (GST), a 26 kDa sequence of 211 amino acids, is another widely used affinity tag that increases the solubility of the desired protein. The GST tag has an affinity for immobilized glutathione and is most often used for prokaryotic expression. It can be fused to the N-terminus or the C-terminus of a protein.
Glutathione Affinity is an efficient method for the one-step purification of proteins fused with a GST (glutathione S-transferase) tag. GST can be expressed as a soluble protein in the cytoplasm of E. coli in large quantities and with full enzymatic activity. Furthermore, many eukaryotic proteins that are insoluble when expressed in E. coli have been shown to be at least partially soluble when expressed as a GST fusion protein.
How to purify GST fusion proteins?
To generate constructs that express GST fusion proteins, the sequences encoding the protein of interest can be inserted into commercially available vectors. In addition to its use for affinity purification, the GST tag is frequently used in so-called pull-down experiments to investigate protein-protein interactions.
The scale of purification of GST-tagged proteins depends on the amount of protein in the preparation. A column size and total binding capacity should be chosen to roughly match the amount of protein to be purified. Very few unlabeled proteins are generally retained on the resin if the target protein occupies nearly all of the available glutathione binding sites (GST fusion sites). If too much matrix is used, other proteins can non-specifically bind to the unoccupied sites.
In GST fusion protein purification, GST-tagged proteins contained in a clarified lysate bind to immobilized glutathione (GSH, Bind). And unbound proteins are washed from the matrix (Wash) and bound GST fusion proteins are eluted from the support by the addition of excess reduced glutathione (Elute).
Purification of GST fusion proteins with affinity chromatography
Affinity chromatography is one of the most selective types of chromatography and can be a very useful technique for protein purification. A convenient method of protein expression and subsequent purification is to fuse a protein with a glutathione-S-transferase (GST) domain. The general purification strategy is therefore to bind the GST fusion protein on an immobilized glutathione column, wash all the rest, and then elute the protein.
There are four main steps to purify a GST fusion protein:
1. Solutions to prepare buffer A for the glutathione column
2. Column purification of glutathione
3. Evaluation of purification
4. Glutathione Removal on a Nap-10 Column
Besides affinity purification, other applications for GST-tagged fusion proteins are possible with the help of glutathione ligand chemistry or GST-tag specific antibodies, such as microplate coating, protein interaction extraction and ELISA or Western blot.