Glutathione Sepharose

When the solubility assay gave an 5 value larger than 0.125, the GST-fiised protein extracted in a soluble fraction was purified on glutathione-sepharose beads. 

The glutathione sepharose supplied by GE Healthcare is approximately 75% slurry, and the storage solution contains 20% ethanol. Therefore, the glutathione sepharose should be washed with TBS and replaced by TBS. 

Following induction with IPTG and an incubation period to allow for the expression of GST, the cells are harvested and lysed by sonication in phosphate buffer containing 150 mM NaCl. Triton X-100 (a non-ionic or nondenaturing detergent) is added to help solubilize the membranes and release proteins that may be peripherally associated with them. The clarified cell extract is then incubated with the glutathione Sepharose 4B resin to allow GST to bind the immobilized ligand. The Triton... 

Obtain 0.1 ml of a 50% slurry of Glutathione sepharose 4B resin in PBS buffer. Centrifuge the slurry for 30 sec in a 1.5 ml plastic microcentrifuge tube and discard the supernatant. 

Figure 10-3 Purification of glutathione-S-transferase with glutathione Sepharose 4B resin.

Is there another method that could be used to elute GST from the glutathione sepharose 4B resin besides the addition of reduced glutathione Are there any advantages to eluting the enzyme with the addition of free substrate as opposed to these other methods ... 

You have isolated a protein of interest tagged with the GST domain. At this point, the fusion protein is bound to the glutathione Sepharose 4B resin and the unbound proteins have been washed away. Describe, in detail, two different techniques that you could use to isolate the protein without the affinity tag using only thrombin, glutathione Sepharose 4B, and reduced glutathione. 

In this experiment, you will identify the C-terminal domain of glutathione-S-transferase (GST) from Schistosomajaponicum (purified from an Escherichia coli cell extract in Experiment 10) through the use of a Western blot. After the proteins present in the crude cell extract, unbound fraction, and the fraction eluted from the glutathione Sepharose resin are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins will be electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Fig. 18-1). Although membranes composed of nitrocellulose or nylon can also be used for Western blotting, PVDF membranes are particularly effec-... 

Glutathione Sepharose 4B—We obtain this from Pharmacia (catalog 17-0756-01). Prepare a 50% slurry of this resin in PBS buffer according to the manufacturer s instructions. NOTE The resin can be regenerated and used in following semesters by washing the resin in 3 M NaCl. The Sepharose 4B matrix is chemically stable in the pH range of 4 to 9. 

Purified GST standard (prepared by instructor)—Prepare a 1-liter culture of the GST expression strain, harvest the cells, and purify the protein according to the protocol. Use 1 ml of the immobilized glutathione Sepharose 4B per 1 liter of culture. Adjust the concentration of the purified protein to 1 mg/ml, and store at —20°C in 1-ml aliquots. One preparation of GST will give enough protein to act as a standard for many semesters. 

Add 1 pi of a 50% Glutathione Sepharose equilibrated in sonication buffer per ml original culture volume to the supernatant and incubate for 30 min at 4°C with gentle agitation. 

Fig. 4. Identification of GST subunits in rat liver cytosol. SDS-PAGE was performed in a 12% polyacrylamide resolving gel. Samples were run from the cathode (top) to the anode (bottom) and the gel was stained with Coomassie brilliant blue. The fractions are as follows (1) liver cytosol from normal rats, (2) liver cytosol from nodule bearing rats that had been fed aflatoxin Bj (2 ppm) for approx 16 weeks, (3) hepatic cytosol from normal rats that did not bind the glutathione-Sepharose affinity matrbt, (4) hepatic cytosol from aflatoxin Bj-treated rats that was not retained by the glutathione-Sepharose affinity gel, (5) affinity-purified GST from normal rat liver, and (6) affinity-purified GST from rat livers that contained aflatoxin-induced preneoplastic nodules. The positions of the Ya, Yb, Yc, and Yf subunits are shown. The horizontal arrow adjacent to track 1 shows the position where the Yf (or Yp) would migrate if present. These data are taken from Hayes et al. (H23).

H24. Hayes, J. D., Kerr, L. A., Peacock, S. D., Cronshaw, A. D., and McLellan, L. I., Hepatic glutathione S-transferases in mice fed on a diet containing the anticarcinogenic antioxidant butylated hydroxyanisole. Isolation of mouse glutathione S-transferase heterodimers by gradient elution of the glutathione-Sepharose affinity matrix. Biochem. J. 277, 501-512 (1991). 

Glutathione S-transferase Glutathione Sepharose CL-4B with aliphatic diamines 51... 

Glutathione sepharose 4B (GE Healthcare) or Glutathione agarose (Sigma). 

While the lysate is centrifuging, add 10 mL of glutathione sepharose slurry to a Biorad Econo column (or similar) and equilibrate the column with at least two column volumes of lysis buffer. 

Pack 1-2 mL bed volumes ofthe appropriate resin (glutathione-Sepharose for GST, amylose for MBP) in a disposable column... 

Takatsu et al, 2002) is expressed in E. coli BL21(DE3) cells and purified using glutathione-Sepharose 4B beads (Amersham Biosdences, Tokyo, Japan) as described in the manufacturer s instructions. The composition of GST-fusion protein binding buffer and washing buffer are described above. 

The supernatant (cell lysate containing 500 /rg protein) is then precleared with glutathione-Sepharose 4B beads for 30 min at 4° with gentle shaking. 

The supernatant is incubated with the GST-GGAl-GAT domain ( 40 pg) prebound to glutathione-Sepharose 4B beads for 1 h at 4° with gentle shaking and centrifuged at 2,000 xg for 5 min at 4°. 

---