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| Protocol |
Glutaredoxin (GRX) expression in host bacteria Escherichia coli BL21 |
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| Glutaredoxin (GRX) expression in host bacteria Escherichia coli BL21 (LyS). |
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| Bacteria containing the expression plasmid (human glutaredoxin± pET) were cultured overnight at 37 °C in Luria±Bertani medium supplemented with ampicillin (100 lg}ml), transferred to fresh medium, and further cultured with vigorous shaking. When culture D'!! was approx. 0.7, isopropyl-b-d-thiogalactoside was added to a final concentration of 0.5 mM. After 4 h of induction, bacteria were collected by centrifugation and the pellet was frozen at -70 °C. |
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| Purifiication of the expressed protein was performed at 4 °C unless otherwise indicated. Approx. 2 g of frozen bacteria were resuspended in 20 ml of 50 mM Tris}HCl, pH 7.8, containing 1 mM PMSF, 5 lg}ml aprotinin and 1 lg}ml leupeptin. Bacteria were disrupted by sonication (Kontes) and cell debris was sedimented by centrifugation at 23000 g (4 °C) for 20 min. The resulting supernatant was incubated for 10 min in a water bath at 75 °C. Denatured proteins were removed by centrifugation at 23000 g (4 °C) for 20 min, and the supernatant was dialysed overnight in 10 mM phosphate buffer, pH 6.5. The dialysate was applied to carboxymethyl-Sepharose (1.0 cm¬10 cm) equilibrated with 10 mM phosphate buffer, pH 6.5. The column was washed with 50 ml of 10 mM phosphate buffer, pH 7.5. Bound proteins were eluted with a linear NaCl gradient of |
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