Protein Purification
HD exon 1, 16, 38, 56, and 82 CAGs fused with GST.
Making constructs.
Exon I of HD gene was amplified from partial cDNA constructs obtained from normal (16 CAGs) and patient (48 CAGs). The primers were engineered with restriction enzyme BamH I site in the sense primer and EcoR I in the antisense primer. the primer sequences are: sense, 5' CGG CCC CAG GGA TCC GGG GAC TGC 3'; and antisense, 5' TGC GGC TGA GGC AGA ATT CGG GTG TCG 3'. digested PCR products were ligated directionally to corresponding digested bacterial expession vector, pGEX2T (4.9 kb) after phosphatase treatment of the vector with calf intestine alkaline phosphatase. Transformation of ligated products was performed by using Top 10 (Invitrogen, Inc) competent cells, HD exon I insert both for normal (16 CAG), expanded (48 CAG) and other repeat sizes were checked by digesting with BamH I and EcoR I restriction enzymes and electrophoresis as well as by sequence analysis.
Expression of GST fusion protein.
Grow 5ml of overnight culture at 37°C using LB broth (with 100mg/liter Amicillin). Next day add overnight culture to fresh LB (amp in it) at 1:20 ratio and till OD reaches 0.6-1.0 at fixed wavelength A 600.
Induce expression of fusion proteins by adding isopropyl-B-D-thiogalactoside (IPTG) to a final concentration of 1.0mM. Allow the cells to grow for an additional 3 hours at 37°C. Set up two tubes for each colony/construct, one with IPTG and the other one without IPTG.
Take 1 ml of cells and spin down from all samples both for induced and uninduced. To the cell pellet add 100µl reducing solution. Boil cells with reducing sol. for 5 minutes and sonicate and load 10µl of both induced (plus IPTG) and uninduced (minus IPTG).
Note: The constructs p16, p38, p56 and p82 are checked for expression and purified protein: for additional purification like analytical gel filtration and other purposes these constructs are ready to use. Expression is good at 3 hours IPTG induction.
Purification of GST fusion protein.
Grow a 5ml culture of cells HD gene in LB (containing 100mg/litre ampicillin) at 37°C . Use this culture inoculate and expand the culture. Inoculate 1 litre of LB broth (containing 100mg/1 liter) with 100ml of cell culture (1:10 culture and LB dilution). Grow the cells till OD reaches 0.6-1.0 at 600nm fixed wave length. Induce cells with IPTG final concentration of 1mM for 3 hours (at 3 hours you get good expression).
Spin down cells for 15 minutes at 2000 RPM and wash cells three times with 1X PBS. Keep cells on ice at all times. Add 10ml lysis sol. (containing protease inhibitors Aprotonin 1% from 1000x and AEBSF 1mM). Sonicate cells for 45 seconds for three times. At this stage you can see light brown transilluminant sol. Add triton X (1% final concentration). Place tubes in rotary shaker at 4°C for 15 minutes. Spin cells for 15 minutes at 7000 RPM, collect supernatant into Beckman centrifuge tube. Spin again for 30 minutes at 45 K. Separate supernatant (our protein is soluble).
Add 2ml of 50% gluthione sepharose beads (from Pharmacia) to the lysed cells, incubate at 4°C for 5 hours or overnight on a rotator.
Spin beads and separate supernatant (at this stage target protein bind to sepharose beads). Wash beads 3 times with 50 volumes of 1X PBS (containing 1% triton). Wash once with 50 volumes of 50 mM Tris (pH 7.5) and 150 mM NaCl.
Elute protein from beads using 3-4 mls of 10mM reduced gluthione in 50 mM Tris (pH 8.0). Elute again 1-2 mls of the 10mM gluthione. Dialyse protein in dialysis buffer for 5-8 preferably overnight. Run the dialysed protein on 10% SDS PAGE gel to verify size and purification procedure.
Reagents
Reducing solution.
- pinch bromophenol blue
- 2.5ml 0.5M Tris pH 6.8
- 2ml glycerol
- 2ml 10% SDS
- 2.5ml ddH2O
- 1ml beta-mercaptoethanol
Lysis buffer.
- 1X PBS
- 100mM EDTA
- 1% from 1000x apropotin
- 1mM AEBSF
- 0.5mM DTT
Dialysis buffer.
- 20 mM Hepes
- 150mM KCL
- 0.2mM EDTA
- 1mM AEBSF
- 20% glycerol
