Expression and purification of human gamma-glutamylcysteine synthetase.
Article Details
- CitationCopy to clipboard
Misra I, Griffith OW
Expression and purification of human gamma-glutamylcysteine synthetase.
Protein Expr Purif. 1998 Jul;13(2):268-76.
- PubMed ID
- 9675072 [ View in PubMed]
- Abstract
gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-glutamate and L-cysteine to form L-gamma-glutamyl-L-cysteine; this is the first and rate-limiting step in glutathione biosynthesis. Inhibitors of gamma-GCS such as buthionine sulfoximine are widely used as tools for elucidating glutathione metabolism in vivo and as pharmacological agents for reversing glutathione-based resistance to chemotherapy and radiation therapy in certain cancers. Although gamma-GCS is readily isolated from rat kidneys, future drug design efforts are better based on structure-activity relationships established with the human enzyme. We report here the coexpression in Escherichia coli BL21(DE3) of the human gamma-GCS catalytic (heavy) subunit and regulatory (light) subunit using pET-3d and pET-9d vectors, respectively. Intracellular assembly of the holoenzyme occurred without difficulty, and levels of expression were acceptable (approximately 32 mg holoenzyme/100 g cells). Recombinant human gamma-GCS was purified to homogeneity in an overall yield of 45% by ammonium sulfate fractionation followed by sequential chromatography on Q-Sepharose ion-exchange, Superdex 200 gel filtration and ATP-affinity resins. Trace amounts of E. coli gamma-GCS were removed by immunoaffinity chromatography. The specific activity of the isolated enzyme was >1500 units/mg, comparable to the best preparations from rat kidney. The Km values for L-glutamate, L-cysteine, L-gamma-aminobutyrate (an L-cysteine surrogate), and ATP are 1.8, 0.1, 1.3, and 0.4 mM, respectively. Recombinant human gamma-GCS, like native rat gamma-GCS, is feedback inhibited by glutathione and is potently inhibited by buthionine sulfoximine and cystamine.