HRV 3C Expression and Purification

Protocol 1, Autoinduce

1. transform pET28b-Cold-Native 3C construct into E. coli BL21 Star (DE3) pRARE2. Plate out on LB-agar containing kanamycin (30 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at 37°C.

The BL21 Star (DE3) pRARE2  strain is derived from BL21 Star (DE3), transformed with  pRARE2 plasmid from Rosetta2 (DE3).

2.  Pick 2 colonies from the plate to inoculate 2 x 4 ml LB medium containing kanamycin (30 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at 37°C in a shaker/incubator.

3. Use the overnight cultures to inoculate 2 x 1 L ZYM 5052 autoinduction medium containing kanamycin (100 μg/ml) and chloramphenicol (33 μg/ml) in 2-L flasks.

4. Grow the cultures at 37°C until the OD600 is approx. 1. Then lower the temperature of the shaker/incubator to 20°C and incubate the cultures overnight.

5. Harvest the cells by spinning for 5 min at 6,000 rpm using a S3 rotor.

6. Resuspend the pellet in 40 ml lysis buffer.

7. Sonicate (2 x 3 min on ice-water) to lyse the cells.

8. Spin the cell lysate for 15 min at 12,000 rpm. Spin again at 25000 rpm at 20 min.

9. Load the supernatant to a 5-ml Ni column.

10. Wash the column with washing buffer until no more protein elutes (monitored by the absorbance at 280 nm).

11. Elute the protein with elution buffer (150 mM imidazole).

For GST-3C

9. Load the supernatant to a 5-ml GSTrap column equilibrated with wash buffer.
10. Wash the column with wash buffer until no more protein elutes (monitored by the absorbance at 280 nm).
11. Elute the protein with elution buffer. Collect the eluate in 2-ml fractions.

12. Pool the protein containing fractions and dialyze overnight against 50 ml storage buffer at 4°C.

13. For product: dilute the final solution with storage buffer to a final concentration of 1 mg/ml and store in 0.5-ml aliquots at -80°C.

The amount of His6-3C protease can be determined by measuring the absorbance at 280 nm of the protein solution against the dialysis buffer. The concentration can be calculated using a specific extinction coefficient of 0.253 (a His6-3C protease solution of 1 mg/ml gives an A280 of 0.253). The molecular weight of His6-3C protease is 21.7 kDa.

The amount of GST-3C protease can be determined by measuring the absorbance at 280 nm of the protein solution against the dialysis buffer. The concentration can be calculated using a specific extinction coefficient of 1.005 (a His6-3C protease solution of 1 mg/ml gives an A280 of 1.005). The molecular weight of His6-3C protease is 46.4 kDa.

ZYM-5052 medium for auto-induction

958 ml ZY
20 ml 50 x M
20 ml 50 x 5052
2 ml 1 M MgSO4
0.2 ml 1000 x trace elements

ZY

tryptone  1%
yeast extract  0.5%

50 x M

Na2HPO4  25 mM
KH2PO4  25 mM
NH4Cl  50 mM
Na2SO4  5 mM

50 x 5052

glycerol  0.5%
glucose  0.05%
alpha-lactose  0.2%

1000 X Trace elements

50 mM FeCl3
20 mM CaCl2
10 mM MnCl2
10 mM ZnSO4
2 mM CoCl2
2 mM CuCl2
2 mM NiCl2
2 mM Na2MoO4
2 mM Na2SeO3
2 mM H3BO3

500 x MgSO4

1 M MgSO4-7H2O
24.65 g/100 ml


Preparation of 100 ml 1000x trace elements stock solution (in ~60 mM HCl).
Prepare stock solutions of the salts (except FeCl3) as mentioned in the table below.
Dissolve 0.1 M FeCl3 in 50 ml of 100x diluted concentrated HCl.
Add to 36 ml sterile water:
50 ml  0.1 M FeCl3-6H2O  2.70 g/100 ml
2 ml  1.0 M CaCl2-2H2O  15.8 g/100 ml
1 ml  1.0 M MnCl2-4H2O  19.8 g/100 ml
1 ml  1.0 M ZnSO4-7H2O  28.8 g/100 ml
1 ml  0.2 M CoCl2-6H2O  4.76 g/100 ml
2 ml  0.1 M CuCl2-2H2O  1.70 g/100 ml
1 ml  0.2 M NiCl2-6H2O  4.76 g/100 m
2 ml  0.1 M Na2MoO4-2H2O  2.42 g/100 ml
2 ml  0.1 M Na2SeO3  1.73 g/100 ml
2 ml  0.1 M H3BO3  0.62 g/100 ml

Lysis Buffer

50 mM Tris-HCl, pH 8.0
300 mM NaCl
10 mM imidazole
10% (v/v) glycerol
0.2% (v/v) NP-40
0.01% (v/v) 1-thioglycerol or 1 mM beta-ME

Washing Buffer

50 mM Tris-HCl pH 8.0
300 mM NaCl
20 mM imidazole
10% (v/v) glycerol
0.01% (v/v) 1-thioglycerol or 1 mM beta-ME

Elution Buffer

50 mM Tris-HCl pH 8.0
300 mM NaCl
150 mM imidazole
10% (v/v) glycerol
0.01% (v/v) 1-thioglycerol or 1 mM beta-ME

GST-3C Washing Buffer

50 mM Tris-HCl pH 8.0
300 mM NaCl
10% (v/v) glycerol
0.01% (v/v) 1-thioglycerol

GST-3C Elution Buffer

50 mM Tris-HCl pH 8.0
300 mM NaCl
10 mM reduced glutathione
10% (v/v) glycerol
0.01% (v/v) 1-thioglycerol or 1 mM beta-ME

Storage buffer

50 mM Tris-HCl pH 8.0
150 mM NaCl
10 mM EDTA
50% (v/v) glycerol
1 mM DTT

Protocol 2

Expression

Vector: 6XHis rhinovirus 3C protease  in a pDB.His.GST expression vector

Strain: E. coli Rosetta BL21(DE3)plyS, or Nico(21)-pLysN

Grow in 2YT (5 g NaCl, 16 g tryptone, 10 g yeast extract) or LB containing 50 μg mL−1 kanamycin and 34 μg mL−1 chloramphenincol.

Inoculate  7 liters of culture with 20 ml/L of an overnight starter culture grown from a freshly transformed single colony

Grow at 37 °C to OD600 =0.8.

Lower temperature to 30 °C.

After approximately 30 min at 30 °C,  add IPTG to 0.2 mM. Grow o/n at 30 °C.

Sample Prep

Spin down cells.

Resuspend in 50 mL lysis Buffer per liter culture (Buffer A: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM beta-ME, 10% glyerol, 0.1 mM EDTA,  10 mM imidazole).

Freeze cells with dry ice.

Lyse cells by thawing  in water bucket.

Discrupt sticky DNA using a homogenizer.

Spin at  10,000 rpm for 20 min (high speed centrifuge),  then 25000 rpm for 20 min (ultracentrifuge), at 4 oC, collect supernatant

Ni Column

Load supernatant into 10 to 20 ml Ni column equilibrated with buffer A. Do not discard Flow through.

Wash with >5 CVs of Buffer A supplemented with 10 mM imidazole.

Elute protein with Buffer B (Buffer A + 300 mM imidazole), 2~5 CVs. Collect 3 ml per fraction.

Check protein  in each fraction (10 ul) using Braford reagent (75 ul)

Combine fractions, set aside 20 ul for SDS-PAGE analysis

Size-exclusion TSK Gel 3000 SW

50 mM Sodium Phosphate Buffer,  pH 7, or Tris-HCl, pH 8.0,  200 mM NaCl,  5 mM beta-ME, 1 mM EDTA and 5% (by vol.) glycerol,

Collect 3ml per fraction.

Run SDS gel to see if protein is pure

Anion/Cation Exchange Chromatography

If purity is not good:

• Q Sepharose 50 mM Tris-HCl pH 8.8 - 9,  5 mM beta-ME. Usea linear NaCl gradient (0 - 1 M NaCl) to elute

• or S-Sepharose fast-flow column. Load crude extract in 50 mM sodium acetate, pH 6.5, 5 mM beta-ME. Usea linear NaCl gradient (0 - 0.5 M NaCl) to elute

Guideline:

Buffer pH >PI: protein will bind anion exchange column.

Buffer pH <PI: protein will bind cation exchange column.

Concentration of enzyme by dialysis

Dialyse purified 3C protein over night at 4 °C with Glycerol Concentration Buffer (50 mM TRIS-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 5 m DTT, 50% glycerol) to 10 mg∕mL

Aliquot and store at −80 °C.