Purification of the helicase domain of NS3h HCV protein
Cell Transformation, Growth, and Protein Expression
Start this procedure “early” in the morning. It takes two days.
1. Thaw a 20 uL aliquote of BL21(DE3) competent cells (Novagen) on ice for 10 min. in 1.7 mL
tube. Add a small amount (0.2 L is enough) of 1 mg/mL solution of pET21b-NS3HCV plasmid,
originally obtained from Dr. Joonho Choe (Kim, D.W., Gwack, Y., Han, J.H., and Choe, J. (1995)
Biochem.Biophys.Res.Commun. 215, 160-166).
2. Incubate cells on ice for 30 min..
3. Heat shock at 42C for 40 s and put cells back on ice.
4. Add 80 L of SOC medium to the cells and shake the tube at 37 C for 60 min..
5. Spread the cells on agar-LB-Amp Petri dish forming a gradient of cell density. This will make
possible to isolate a single colony even if the number of transformants is large.
6. Incubate the Petri dish at 37 C until the colonies become visible. This will take at least 6 hours.
7. Prepare one 150 mL and twelve 1 L cultures of LB.
8. To prepare 15 mL of 1000xAmp, dissolve 1.5 g of ampicillin and filter the solution through 0.8/0.2
m syringe filter.
9. In the evening inoculate a 150 mL LB-Amp culture with a single colony. Shake the culture at
room temperature overnight.
10. Early in the morning, day two, make sure the OD600 of the 150 mL culture does not exceed 1.
Inoculate 12 one-liter LB-Amp cultures with 10 mL of overnight culture each. Shake the cultures
at 200 rpm, 37 C.
11. To prepare 13 mL of 0.5 M IPTG solution dissolve 3.12 g of IPTG in water.
12. When the OD600 of one liter cultures reaches 0.6, collect 1 mL sample of the culture and induce
cultures by adding 1 ml of 0.5 M IPTG solution to each flask. Continue shaking flasks for two
more hours.
13. Prepare 400 mL of Buffer A: 100 mM NaCl, 50 mM Tris-HCl, 10% sucrose, pH 7.5.
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14. Harvest cells by centrifuging cultures at 15 k g (10 k rpm for SLA-3000 rotor) for 10 min..
Resuspend cells in the Buffer A to the total volume of 400 mL. Make sure that the cell suspension
is homogenous. Add 0.1 g of chicken egg lysozyme. Dissolve 0.1 g of PMSF in 3 mL of
isopropanol and add the solution to the cell suspension.
15. Dispense the cells into 50 mL polypropylene tubes and freeze them in liquid nitrogen. The cells
can be stored at -80 C.
Crude Extract Preparation
1. Lyse cells by thawing the 50 mL tubes at 37 C and freezing them in liquid nitrogen three times.
2. Centrifuge cell lysate at 4 C, 180 k g (40 krpm for T-647.5 rotor) for 1 hour.
3. The viscosity of the supernatant can be reduced by passing it through French press.
4. Precipitate proteins from the crude extract with 80% saturated ammonium sulfate by gradually
adding 608 g of solid Am2SO4 per liter of crude extract and stirring the solution on ice for 30 min..
Ni-Chelating Chromatography Purification
High purity imidazole (eg. Fluka, Microselect >99.5% GC) should be used.
The chelating column loaded with Ni++ SHOULD NOT BE stored in imidazole buffer for more that a
few hours. Prolonged exposure of charged resin to imidasole causes redistribution of nickel.
Protin sample and running buffers SHOULD NOT contain any reducing or chelating agents.
1. Pack a 1.6x10 cm column with His-Bind metal chelating resin (Novagen)
2. Wash, charge, and equilibrate the column by passing the following solutions:
- 100 mL 1 M NaCl, 50 mM EDTA
- 20 mL water
- 100 mL 2% SDS
- 50 mL water
- 100 mL 50 mM NiSO4
– 50 mL water
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- 50 mL 0.5 M NaCl, 300 mM imidazole, 20 mM Tris-HCl, pH 7.2 (His Elution buffer)
- 100 mL 0.5 M NaCl, 5 mM imidazole, 20 mM Tris-HCl, pH 7.2 (His Loading buffer)
3. Dissolve the protein precipitate in about 60 mL of His Loading buffer
4. Centrifuge at 4 C, 30 k g (15 k rpm for SA-600 rotor) for 10 min..
5. Load the supernatant on the column with a flow rate of 0.5 mL/min.. Collect the eluent.
6. Wash the column with His Loading buffer until the base line stabilizes. Collect the eluent.
7. Elute the bound proteins with a gradient of His Elution buffer from 0 to 100% in 400 mL. Collect
eluent in 12 mL fractions. The second peak usually contains the NS3h protein.
8. Run SDS 10%PAGE to determine which fractions contain NS3h. To prepare samples for SDSPAGE take 50 L aliquots from elution fraction, add 150 L of acetone, incubate the mixture at 20 C for 30 min, centrifuge at 4 C, 16 k g (14 k rpm for a table top centrifuge) for 10 min..
Discard the supernatant and dry the pellet of the protein for 10 min. at room temperature. Dissolve
the pellet in at least 10 L 20 mM NaOH, 2% SDS.
9. Combine NS3h containing fractions and precipitate proteins with 85% saturated ammonium sulfate
Anion Exchange Chromatography Purification
1. Dissolve the precipitated protein in about 10 mL of 50 mM Tris-HCl, 1 mM EDTA, 5 mM DTT,
pH 7.0 (Q Loading buffer).
2. Dialyse the sample once against 1 L of Q Loading buffer overnight. Some salt should remain in
the sample to keep NS3h protein soluble. Therefore, the amount of dialysis buffer SHOULD NOT
BE increased.
3. Pack 1.6x15 cm Q Sepharose Fast Flow column. Wash the column with 1 M NaCl and equilibrate
with Q Loading buffer.
4. Centrifuge the dialized sample at 4 C, 30 k g for 10 min..
5. Load the sample on the column at flow rate of 0.5 mL/min..
6. Wash the column with 20 mL of Q Loading buffer while collecting fractions.
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7. Elute the protein from the column with a gradient of 0 to 0.5 M NaCl in 400 mL of Q Loading
buffer.
8. A single peak should appear at about 0.15 M NaCl. Determine which fractions contain pure
protein by running SDS PAGE as described previously.
9. Combine the fractions together and precipitate the protein with 85% saturated ammonium sulfate
10. Dissolve the precipitate in about 3 mL of 50 mM MOPS-Na, 10 mM NaCl, 10 mM DTT, 1 mM
EDTA, pH 7.0.
11. Dialize the solution against 1 L of the same buffer plus 15% glycerol overnight.
12. Clear the solution by centrifugation (30 k g, 10 min.).
13. Dispense the protein solution in 0.7 mL eppendorf tubes in 100, 50, and 20 uL aliquotes. Freeze in
liquid nitrogen.
14. Determine the concentration of the protein in the stock by its absorbance at 280 nm in 8 M urea
using extinction coefficient of 280nm= 47,600 M-1 cm-1 . This concentration should be the same as
the one determined by BioRad/Bradford method with BSA as a standard.
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