GST融合蛋白纯化方法
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
STE Buffer
10 mM Tris-HCl, pH 8.0
1 mM EDTA
150 mM NaClLysozyme solution
10 mg/ml in water (make fresh)PBS
Elution Buffer
50 mM Tris.Cl, pH 9.0
20 mM GSH10% Sarkosyl in STE Buffer
10% Triton X-100 in STE Buffer
1 M DTT
100 mM IPTG
Procedure
Day 1
Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin.
Day 2
Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicillin.
Grow at 37oC to an A600 of 0.6 to 0.8.
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.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.
This is a convenient point to stop and to store pellets at -80oC. Else continue to lyse cells.
Thaw pellet on ice if cells are frozen else proceed to the next step.
Resuspend pellet in 10 ml of ice cold STE Buffer.
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 total time of 1 min.
Centrifuge 16,000 rpm for 20 min on the SS34 rotor to pellet debris. Transfer supernatant to a 50-ml conical tube and discard the pellet. Add 4 ml of 10% Triton X-100 and top up with STE Buffer to 20 ml. The effective concentration of Sarkosyl and Triton X-100 will be 0.7% and 2% respectively. Incubate at room temperature for 30 min.
Pour the lysate to 1 ml bed of prepared Glutathione Sepharose in PBS. Incubate at room temperature for 30 min to 1 hr with agitation.
To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml tube. Fill to 50 ml with PBS, invert tube a few times. Centrifuge to 2000 rpm on a swing bucket centrifuge then switch off. Carefully suck off PBS and resuspend beads with 1 ml of PBS.Wash the beads with 3 X 50 ml of PBS. Finally resuspend in 5 ml of PBS. Pour to a dispo-column. Wash the 50-ml conical tube with an additional 5 ml of PBS. Pool with the first 5 ml in the dispo-column.
To wash, use the same centrifugation technique for preparing the beads. When transferring beads to column, do not pipette but pour. The beads tend to stick to pipette tips.If desired, elute with 10 x 1 ml fractions of Elution Buffer. Determine desired fractions with SDS PAGE.
Reference
Frangioni and Neel.Anal. Biochem. 210, 179-187 (1993)
