Recombinant Glutathione S-transferase (gst)
Description
Vector Systems
Commonly used pGEX vectors and their restriction sites:
Affinity Chromatography
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Binding: GST binds to glutathione-Sepharose matrices with K~d~ ≈ 10^−7^ M .
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Elution: Achieved using 10–20 mM reduced glutathione in Tris-HCl buffer (pH 8.0) .
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Yield: Typical E. coli expression yields 10–50 mg/L culture .
Biochemical Characteristics
| Parameter | Specification |
|---|---|
| Molecular Weight | 25.4 kDa (monomer) |
| Purity | ≥90% (SDS-PAGE) |
| Specific Activity | >30 units/mg (CDNB assay) |
| Storage Stability | 12 months at −80°C with 0.1% HSA/BSA |
CDNB assay conditions: 1 mM GSH, 1 mM CDNB, 25°C, pH 6.5 .
Drug Metabolism Studies
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AZD1979: GSTA2-2 catalyzes glutathionylation of this obesity drug candidate .
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Exemestane: GSTA1 and GSTM3 isoforms metabolize this breast cancer therapeutic .
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Cisplatin Resistance: GSTYb1 overexpression confers 1.5-fold resistance in mammalian cells .
Proteomics
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Mitochondrial GST isoforms (alpha3, kappa1) show decreased abundance in diabetic mouse models (P < 0.05) .
Technical Limitations
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Protease Cleavage Efficiency: Thrombin digestion requires optimization (0.01–0.06 U/mg, 2–16 hrs) .
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Solubility Issues: 15–30% of GST fusions form inclusion bodies under standard IPTG induction .
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Batch Variability: Activity fluctuates ±12% across production lots due to oxidation state .
Anticancer Drug Resistance
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GSTπ overexpression increases doxorubicin resistance 1.3-fold in COS-7 cells (P = 0.0001) .
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GSTYa reduces chlorambucil cytotoxicity by 2.9-fold through enhanced detoxification .
