GST融合蛋白纯化方法

GST 融合蛋白纯化方法 Purification of GST Fused Proteins Abstract: Many people have vented out frustration over insoluble GST-fused proteins. This is a protocol for enzymatically active soluble GST-fused proteins. All GST-fused proteins are rendered soluble with this technique though enzyme activitiy can range from 30-90%. Materials and Reagents 1. STE Buffer 10 mM Tris-HCl, pH 8.0 1 mM EDTA 150 mM NaCl 2. Lysozyme solution 10 mg/ml in water (make fresh) 3. PBS 4. Elution Buffer 50 mM Tris.Cl, pH 9.0 20 mM GSH 5. 10% Sarkosyl in STE Buffer 6. 10% Triton X-100 in STE Buffer 7. 1 M DTT 8. 100 mM IPTG Procedure Day 1 1. Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin. Day 2 1. Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicillin. 2. Grow at 37oC to an A600 of 0.6 to 0.8. 3. Induce with 0.1 mM to 2 mM of IPTG. Grow for 3 hr at 37oC or grow overnight at room temperature. Lower IPTG concentrations and lower growing temperatures tend to produce greater solubility at the expense of yield. 4. Pellet cells by centrifuging at 3000 g, 4oC for 10 min. Decant media and resuspend cells in 30 ml ice-cold PBS to wash. Transfer to a 40-ml Oak Ridge tube and centrifuge at 3000 g, 4oC for 10 min. Decant PBS. 5. This is a convenient point to stop and to store pellets at -80oC. Else continue to lyse cells. 6. Thaw pellet on ice if cells are frozen else proceed to the next step. 7. Resuspend pellet in 10 ml of ice cold STE Buffer. 8. Add 100 ml of freshly prepared lyozyme solution, incubate on ice for 15 min. Just before sonication, add 100 ml of 1 M DTT and 1.4 ml of 10% Sarkosyl. Mix thoroughly by inversion and sonicate for a tota...