GST-Agarose

The effective way for purification of recombinant Glutathione-S-transferase fusion proteins

Biontex offers with its Glutathione Agarose an effective way for purification of recombinant Glutathione-S-transferase fusion proteins from cellular lysates.

The product is prepared by coupling Glutathione through its central sulphydryl group by a linker, which minimizes steric hindrance, to activated agarose.

Glutathione-S-transferase-Technology

The Glutathione-S-transferases (GST) are a family of enzymes that catalyze the addition of the tripeptide glutathione (see figure) to endogenous and xenobiotic substrates which have electrophilic functional groups:

They play an important role in the detoxification and metabolism of many xenobiotic and endobiotic compounds, because their adducts have increased solubility in water and are subsequently enzymatically degraded to mercapturates and excreted. Glutathione-S-transferases are dimeric with subunits of 25kDa molecular weight.

In general the GST gene fusion system is an integrated system for the expression, purification and detection of fusion proteins expressed in bacterial, yeast, mammalian and insect cells. The sequence encoding the GST protein is incorporated into an expression vector, generally upstream of the multi-cloning site. The sequence encoding the protein of interest is then cloned into this vector. Induction of the vector results in expression of the fusion protein- the protein of interest fused to the GST protein. The fusion protein can then be released from the cells and purified.

Purification of the fusion protein is facilitated by the affinity of the GST protein for glutathione residues, which are coupled to Biontex GST Agarose resin. The expressed fusion protein product is brought in contact with the resin and will bind to the glutathione-agarose complex. Now, all other non-specific proteins can be washed off. The fusion protein can then be released from the resin using a mild elution buffer including reduced glutathione or by cleavage from the fusion protein by using a specific protease (e.g. thrombin, factor X), which cleave specific sites between GST and the protein of interest.

Fusion proteins can also be detected easily with a number of GST antbodies available on the market.

Highlights of GST-Agarose

Widely used system
Suitable for use in all expression systems: bacterial, yeast, mammalian, and baculovirus cleavage sites to remove GST from protein of resin.
Direct purification from crude bacterial lysates.
High purity of protein (more than 90%).
pH stability of 3–13 (short term 2-14).
Binding capacity of 5 mg fusion protein per ml agarose.
Resin can be regenerated for multible uses and no time wasted in regenerating resin.
Extremely cost effective.
Generic tool for protein expression, purification and detection: no need to produce individual antibodies to each protein of interest.
GST antibodies are available for detection of fusion protein.
No risk of cross-contamination of one prep by another protein.
High flexibility: Use of resin volumes from 20ml preparative columns to 20 µliters for quick verification of expression, molecular weight, integrity of taq is possible.

Applications*

Production of antigens for in vitro diagnosis
Production of vaccines and adjuvants
Production of chemical intermediates
Antigen production for generation of antibodies
Generation of research reagents

* Important Information

Biontex GST-Agarose is developed and sold for research purposes and in vitro use only. It is not intended for human therapeutic or diagnostic purposes. Appropriate care should be exercised when handling many of the reagents described in this publication.

General Principle of Protein Purification

Sample Preparation
GST Agarose preparation
Sample Application
Washing
Elution
Cleavage of GST fusion proteins from the eluate (or directly from the agarose)

For more details and specifications of resin see our GST-Agarose Solutions Guidebook.

Application Notes

Protein	Language	Download
GST-HCK (Kinase domain of HCK ca. 30kDa)	english
GST-GFP-NLS (ca. 60kDa)	german

References

J.V. Frangioni, B.G. Neel: "Solubilization and purification of enzymatically active Glutathione-S-transferase (pGEX) fusion proteins", Anal. Biochem. 210, 179-187 (1993)
P.C. Simons, D.L. Vanderjagt: "Purification of Glutathione-S-transferases for human liver by glutathione affinity chromatography", Anal. Biochem. 82, 334-341 (1977)
R. Janknecht et al.: "Rapid and efficient purification of native histidine-tagged protein expressed recombinant vaccinia virus", Proc. Natl. Acad. Sci. USA 88, 8972-8976 (1991)
P. Riggs et al.: Current Protocols in Molecular Biology, 16.4.1-16.6.14 (1990)
D.B. Smith, K.S. Johnson: "Single-step purification of polypeptides expressed in Eschericia coli as fusions with Glutathione-S-transferase", Gene 7, 31-40 (1988)

Prices

Reagent	Order No.	Price
GST-Agarose 5 x 1 ml For purification of Glutathione-S-transferase fusion proteins (GST)	R030-02.5	167 €
GST-Agarose 10 ml For purification of Glutathione-S-transferase fusion proteins (GST)	R030-10.1	209 €
GST-Agarose 50 ml For purification of Glutathione-S-transferase fusion proteins (GST)	R030-50.1	807 €
GST-Agarose 2 x 50 ml For purification of Glutathione-S-transferase fusion proteins (GST)	R030-50.2	1226 €