Production of N-His6-TEV protease (1-liter preparation)
Construct
N-terminal His6-tagged TEV protease gene in a pET-24d (+) vector (kanamycin resistance).
Construct name: pET/TEV
Protocol
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Freshly transform the 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 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
kanamycin (30 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 kanamycin (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: duty 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.
Add imidazole (1M) to the supernatant to a final concentration of 20 mM.
Apply the supernatant to a 5-ml Chelating Sepharose column on an Äkta Purifier (GE
Healthcare), charged with NiCl2 and equilibrated with chelating buffer.
Wash the column with chelating buffer.
Wash the column with chelating buffer containing 50 mM imidazole (90% chelating
buffer + 10% elution 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.
TEV protease is not stable in buffers containing a high concentration of imidazole.
Therefore, you need to exchange the buffer of the protein solution immediately after
elution from the Chelating Sepharose column. Keep the protein at 4°C.
Pool the protein containing fractions and apply this to a 50-ml HiTrap 26/10
Desalting column (GE Healthcare), equilibrated with desalting buffer. Collect the
eluate in 1.8-ml fraction.
Keep the protein at 4°C as much as possible.
Pool the protein containing fractions and determine the protein concentration.
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Dilute the preparation to a final concentration of 1 mg/ml by the addition of pure
glycerol to a final concentration 50% (v/v) and desalting buffer. Store the final
solution in 0.5-ml aliquots at -80°C.
Alternatively, the Chelating Sepharose eluate can be dialyzed overnight against 1 L of
storage buffer. This has the additional advantage that the protein is concentrated by a
factor of 2-3. Store the final solution in 0.5-ml aliquots at -80°C.
The amount of TEV protease can be determined by measuring the absorbance at 280 nm of
the protein solution against the desalting buffer. The concentration can be calculated using a
specific extinction coefficient of 1.14 (a His6-TEV protease solution of 1 mg/ml gives an A280
of 1.14). The molecular weight of His6-TEV protease is 27.7 kDa.
Lysis buffer
Chelating buffer
50 mM Tris-HCl pH 8.0
300 mM NaCl
20 mM imidazole
20% (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
20 mM imidazole
20% (v/v) glycerol
0.01% (v/v) 1-thioglycerol
Elution buffer
Desalting buffer
50 mM Tris-HCl pH 8.0
300 mM NaCl
300 mM imidazole
20% (v/v) glycerol
0.01% (v/v) 1-thioglycerol
50 mM Tris-HCl pH 8.0
150 mM NaCl
20% (v/v) glycerol
0.01% (v/v) 1-thioglycerol
Storage buffer
50 mM Tris-HCl pH 8.0
150 mM NaCl
50% (v/v) glycerol
0.01% (v/v) 1-thioglycerol
Arie Geerlof - Helmholtz Zentrum München
3 November 2010