Purification using Glutathione Sepharose^® High Performance, Glutathione Sepharose^® 4 Fast Flow, and Glutathione Sepharose^® 4B

These three chromatography media are all used for the purification of GST-tagged recombinant proteins and other S-transferases or glutathione-dependent proteins. They allow mild elution conditions that preserve protein structure and function. All are supplied preswollen in 20% ethanol and are also available in various prepacked formats, such as GSTrap, as described later in this chapter. Appendix 2 (Characteristics of Glutathione Sepharose products) for the main characteristics of all Glutathione Sepharose media.

In Glutathione Sepharose High Performance, the glutathione ligand is coupled to highly cross-linked 6% agarose. The medium has an average bead size of 34 µM and can be used for high-resolution purification and elution of a more concentrated sample.

In Glutathione Sepharose 4 Fast Flow, the glutathione ligand is coupled to highly cross-linked 4% agarose. The medium has an average bead size of 90 µM. It is a good choice for scale-up due to its good binding capacity and flow properties. This medium is also suitable for batch and gravity-flow purifications.

In Glutathione Sepharose 4B, the glutathione ligand is coupled to 4% agarose. The medium has an average bead size of 90 µM. It provides very high binding capacity and is recommended for small-scale purification as well as batch and gravity-flow operations.

Glutathione Sepharose 4 Fast Flow and Glutathione Sepharose 4B are also available prepacked in 96-well filter plates.

Procedures for both batch and column purification of GST-tagged proteins follow.

Figure 5.3. Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B for purification of GST-tagged proteins.

Sample Preparation

Refer to General considerations for purification of GST-tagged proteins for general considerations before beginning this procedure.

Adjust the sample to the composition and pH of the binding buffer by additions from concentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange.

Pass the sample through a 0.22 µM or a 0.45 µM filter and/or centrifuge it immediately before sample application. If the sample is too viscous, dilute it with binding buffer to prevent it from clogging; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.

Buffer Preparation

Use high-purity water and chemicals, and filter all buffers through a 0.45 µM filter before use.

Binding buffer:	PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.3
Elution buffer:	50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

1 to 20 mM DTT may be included in the binding and elution buffers to reduce the risk of oxidation of free -SH groups on GST, which may cause aggregation of the tagged target protein, resulting in lower yield of GST-tagged protein.

Batch purification of GST-tagged proteins using Glutathione Sepharose HP,  Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B

Glutathione Sepharose chromatography media are supplied preswollen in 20% ethanol. The media are used at a final slurry concentration of 50%.

1. Determine the bed volume of Glutathione Sepharose required for your purification.
2. Gently shake the bottle to resuspend the slurry.
3. Use a pipette or measuring cylinder to remove sufficient slurry for use and transfer to an appropriate container/tube.
4. Sediment the chromatography medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant.
5. Wash the Glutathione Sepharose HP, FF, or 4B by adding 5 mL of PBS per 1 mL of slurry (= 50% slurry).

Glutathione Sepharose media must be thoroughly washed with PBS to remove the ethanol storage solution because residual ethanol may interfere with subsequent procedures.

6. Sediment the chromatography medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant.
7. Repeat steps 5 and 6 once for a total of two washes.

For cleaning, storage, and handling information, refer to Appendix 2 (Characteristics of Glutathione Sepharose products).

Batch Purification

1. Add the cell lysate to the prepared Glutathione Sepharose medium and incubate for at least 30 min at room temperature, using gentle agitation such as end-over- end rotation.
2. Use a pipette or cylinder to transfer the mixture to an appropriate container/tube.
3. Sediment the medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant
   (= flowthrough) and save it for SDS-PAGE analysis to check for any loss of unbound target protein.
4. Wash the Glutathione Sepharose medium by adding 5 mL of PBS per 1 mL of slurry (= 50% slurry). Invert to mix.
5. Sediment the medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant (= wash) and save it for SDS-PAGE analysis.
6. Repeat steps 4 and 5 twice for a total of three washes.
7. Elute the bound protein by adding 0.5 mL of elution buffer per 1 mL slurry of Glutathione Sepharose medium. Incubate at room temperature for 5 to 10 min, using gentle agitation such as end-over-end rotation.
8. Sediment the medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant (= eluted protein) and transfer to a clean tube.
9. Repeat steps 7 and 8 twice for a total of three elutions. Check the three eluates separately for purified protein and pool those eluates containing protein.

Column purification of GST-tagged proteins using Glutathione Sepharose HP,  Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B

Column Packing

See instructions supplied with the products or refer to Appendix 6 (Column packing and preparation) for general guidelines for column packing.

Purification

For recommended flow rates, Appendix 6 (Column packing and preparation), Table A6.6.

1. Equilibrate the column with approximately 5 column volumes of binding buffer.
2. Apply the pretreated sample at low flow rate (approximately one-third of the flow rate used during wash and elution).
3. Wash the column with 5 to 10 column volumes of binding buffer or until no material appears in the flowthrough. Save the flowthrough for SDS-PAGE analysis to check for any loss of unbound target protein.
4. Elute the bound protein with 5 to 10 column volumes of elution buffer. Collect the fractions and check separately for purified protein. Pool those fractions containing the GST-tagged target protein.