Production of GST-3C (PreScission®) protease (1-liter preparation)
Construct
N-terminal Glutathione (GST)-tagged 3C protease gene in a pGEX vector (ampicillin
resistance).
Construct name: pGEX/3C
Protocol
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Freshly transform the construct into E. coli BL21 Star (DE3) pRARE2. Plate out on
LB-agar containing ampicillin (100 µg/ml) and chloramphenicol (33 µg/ml). Incubate
overnight at 37°C.
The used strain is similar to the commercial E. coli Rosetta2 (DE3).
Pick 2 colonies from the plate to inoculate 2 x 4 ml LB medium containing
carbenicillin (100 µg/ml) and chloramphenicol (33 µg/ml). Incubate overnight at
37°C in a shaker/incubator.
Use the overnight cultures to inoculate 2 x 0.5 L ZYM 5052 autoinduction medium
containing carbenicillin (100 µg/ml) and chloramphenicol (33 µg/ml) in 2-L flasks.
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.
Harvest the cells by spinning for 20 min at 6,000 rpm using a SLC 6000 rotor in an
Evolution RC centrifuge (Thermo Scientific).
When not immediately used store the pellet at -20°C.
Resuspend the pellet in 40 ml lysis buffer.
Sonicate (2 x 3 min on ice-water) to lyse the cells.
We use a large probe (VS 70 T) in a Bandelin Sonopuls HD 2200 sonicator with the
following settings: cycle 30% and power 60%.
Spin the cell lysate for 60 min at 20,000 rpm using a SS-34 rotor in an Evolution RC
centrifuge (Thermo Scientific). Filter the supernatant over a 0.22-µm filter.
Apply the supernatant to a 5-ml GSTrap column on an Äkta Purifier (GE Healthcare),
equilibrated with wash buffer.
Wash the column with wash buffer until no more protein elutes (monitored by the
absorbance at 280 nm).
Elute the protein with elution buffer. Collect the eluate in 1.8-ml fractions.
Pool the protein containing fractions and dialyze overnight against 1 L storage buffer
at 4°C.
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 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.
Lysis buffer
Wash buffer
50 mM Tris-HCl pH 8.0
300 mM NaCl
10% (v/v) glycerol
0.2% (v/v) NP-40
0.02% (v/v) 1-thioglycerol
1 mg/ml lysosyme
2 µg/ml DNase I
50 mM Tris-HCl pH 8.0
300 mM NaCl
10% (v/v) glycerol
0.01% (v/v) 1-thioglycerol
Elution buffer
Storage 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
50 mM Tris-HCl pH 8.0
150 mM NaCl
10 mM EDTA
20% (v/v) glycerol
0.01% (v/v) 1-thioglycerol
Arie Geerlof - Helmholtz Zentrum München
3 November 2010