Expression and purification of TEV-HIS protease
30 litre culture
Jeuken lab adaptation from V. Postis
Materials
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Agar (Oxoid, LP0011)
Escherichia coli strain BL21 STAR DE3 harbouring the plasmid pRARE2
(Novagen) (home-made)
Tris (FW 121.14) (Melford, B2005)
Carbenicillin (Melford, C0109)
Chloramphenicol (Alfa Aesar, B20841.22)
Glycerol (Melford, G1345)
Imidazole (Acros, 122025000)
IPTG (isopropyl-β-D-thiogalactoside, FW 238.30) (Generon, C9H1805S)
KH2PO4 (FW 136.10) (Melford, P0574)
K2HPO4 (FW 174.18) (VWR, 26931.263)
MgSO4◦7H2O (FW 246.48)(BDH, 101514Y)
NaCl (FW 58.44) (Fisher, S3160/63)
NaH2PO4 (FW 119.98) (Melford, S2318)
Na2HPO4 (FW 141.96) (Melford, S2002)
TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, FW 286.65) (Sigma,
C4706)
EDTA (FW 372.25) (Melford, E0511)
Tryptone (Melford, T1332)
Yeast extract (Melford, Y1333)
Media and solutions
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Lysogeny Broth (LB)-medium. Per litre:
Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in H 2O to give a final
volume of 1 litre then sterilise by autoclaving.
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Terrific Broth (TB)-medium. Per litre:
Buffer1. TB medium 900 ml. Dissolve 12 g tryptone, 24 g yeast extract, 4 mL
glycerol in H2O to give a final volume of 900 mL then sterilise by autoclaving.
Buffer2. Potassium salt 100 mL (0.17M KH2PO4 and 0.72M K2HPO4). Dissolve
2.31 g KH2PO4 and 12.54 g K2HPO4 in 80 ml H2O. After the salt has completely
dissolved, adjust volume to 100 mL with H2O then sterilise by autoclaving.
Do not add to TB broth until needed.
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LB-agar: LB supplemented with 1.5 % agar.
1
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100 mg/mL Carbenicillin.
Dissolve 1g in 10 mL of H2O. Filter sterilise through 0.22 µm filter. Dispense into
aliquots and store at -20°C.
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30 mg/mL Chloramphenicol.
Dissolve 300 mg in 10 mL EtOH. Dispense into aliquots and store at -20°C.
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0.2M Sodium Phosphate Buffer (NaPi) pH 7.4.
Adjust pH with 77.4 ml Na2HPO4 and 22.6 ml NaH2PO4.
0.2M Na2HPO4: Dissolve 14.196 g in H2O to give a final volume of 500 mL.
0.2M NaH2PO4: Dissolve 11.98 g in H2O to give a final volume of 500 mL.
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1 M Tris-HCl, pH 8.0
Dissolve 121.14 g Tris in 800 mL H2O. Adjust the pH to 7.0 with HCl then use a
volumetric flask to make up to a final volume of 1 litre.
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0.5 M EDTA, pH 8.0
Dissolve 9.307g EDTA in about 40 mL H2O. Warming will probably be necessary.
Cool to room temperature, adjust pH to 8.0 by addition of 1 M NaOH, then make
up volume to 50 mL. Store at 4 °C, or frozen for extended periods.
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IPTG (1 M):
Dissolve 1.906 g IPTG to a final volume of 8 mL in H 2O then filter sterilise
through a 0.22 µm filter. Dispense into aliquots and store at -20°C.
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4M Imidazole, pH 8.0:
Dissolve 136.16 g Imidazole in ~400 mL H2O. Adjust pH to 8.0 with HCl then
make up the volume to 500 mL.
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4 M NaCl
Dissolve 116.88 g NaCl in ~400 mL H2O then make up the volume to 500 mL.
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Lysis buffer (B) for disrupting cells (50 mM TRIS pH 8, 400mM NaCl, 0.5 mM
EDTA). Per 750 ml:
Mix 37.5 mL 1M TRIS pH 8.0, 75 mL 4M NaCl, 0.75 mL 0.5M EDTA then dilute to
give a final volume of 750 mL in H2O.
2
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Loading buffer with imidazole (50 mM TRIS pH 8, 400mM NaCl, 30 mM
Imidazole pH8.0). Per 750 mL:
Mix 37.5 mL 1M TRIS pH 8.0, 75 mL 4M NaCl, 5.6 mL 4M Imidazole pH 8.0 then
dilute to give a final volume of 750 mL in H2O. Sterilize by filtration.
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Binding-Equilibration buffer (50 mM TRIS pH 8, 400mM NaCl, 40 mM
Imidazole pH8.0, 0.32 mM TCEP). Per 2L:
Mix 100 mL 1M TRIS pH 8.0, 200 mL 4M NaCl, 20 mL 4M Imidazole pH 8.0,
0.172g TCEP then dilute to give a final volume of 2L in H2O. Sterilize by filtration.
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Elution buffer (50 mM TRIS pH 8, 400mM NaCl, 500 mM Imidazole pH8.0, 0.32
mM TCEP). Per 1L:
Mix 50 mL 1M TRIS pH 8.0, 100 mL 4M NaCl, 125 mL 4M Imidazole pH 8.0,
0.086g TCEP then dilute to give a final volume of 1L in H2O. Sterilize by filtration.
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Dialysis buffer (50 mM TRIS pH 8, 200mM NaCl, 2 mM EDTA, 0.125 mM
TCEP). Per 1L:
Mix 50 mL 1M TRIS pH 8.0, 50 mL 4M NaCl, 2 mL 0.5M EDTA, 0.035g TCEP
then dilute to give a final volume of 1L in H2O.
Expression and isolation of membranes:
Day 1: Transform E. coli strain BL21 STARTM (DE3) Invitrogen harbouring the
plasmid pRARE2 (Novagen), with pTH24 TEV SH, plate on LB-agar supplemented
with carbenicillin (100 µg/mL) and chloramphenicol (30 µg/mL) and incubate
overnight at 37°C.
Day 2: In the morning inoculate 2x20 mL LB medium in 50 mL Falcons,
supplemented with same antibiotics, with a single colonies from the plate and
incubate overnight at 37°C with orbital shaking at 200 rpm. In the afternoon to 2x2L
flasks containing 300 mL LB, supplemented with same antibiotics add 6 mL of pre
seed prepared above.
Day 3: In the morning to the 30L fermenter containing media components TB
(Tryptone 360g, Yeast Extract 720 g, KH2PO4 69.3g, K2HPO4 376.2g and antifoam –
9 mL) sterilized in situ add:
Carbenicillin, 3g (in 150ml autoclaved MilliQ H2O which and filter sterilised)
Chloramphenicol, 0.9g (in 50ml EtOH)
MgSO4◦7H20, 12.198g (in 100 mL autoclaved MilliQ H20 and filter sterilised)
Glycerol, 360 ml (1.2%) (in 1 litre MilliQ H20 and autoclaved)
Inoculum 300 mL (OD600nm 6.966)
3
Initial OD600nm of the 30L fermenter containing inoculum is around 0.05 AU. Incubate
at 20°C with agitation and monitor D600nm value. When it reaches 0.6AU (about 3
hours) induce with 0.1mM (0.7149g IPTG). Leave the culture to grow for
approximately 20 hours at 20°C.
Day 4: Final OD600nm is around 33 AU. Harvest the cells for 20 min. In total 890g of
wet cells paste could be obtained from harvesting and frozen at -80° in batches of
around 80g.
Affinity purification of protein
All purification steps should be done at 4°C
1. Re-suspend the cell paste at a ratio of 2 mL of buffer per gram of wet cell
paste in Lysis Buffer B.
2. Homogenize the cells twice for 1 min using Ultra Turrax then disrupt the cells
twice at 30 kPSi. Wash out the cells left in the disruption chamber 2 x 10 ml of
Lysis Buffer B.
3. Centrifuge suspension for 60 min at 40000rpm in the Beckman Coulter
Optima L-80XP Ultracentrifuge using Ti45 rotor to pellet insoluble material.
4. Filter suspension with 0.45 µm filter.
5. Dilute the supernatant to a final volume which corresponds to that of a gram
of wet cell paste per 10 ml with Loading buffer with Imidazole.
6. The sample is loaded onto the column (HiTrap) at 4 mL/min.
7. Wash columns 50CV of Binding buffer (flow rate 4 mL/min).
8. The protein is eluted with 15CV of Elution buffer at 4 mL/min.
9. 5 mL fractions are collected for the peak in 50 mL Falcon tubes following
AKTA program for HiTrap 5ml column and using a gradient approach.
10. Measure the A280nm of the eluted fraction to provide an indication of protein
concentration, using elution buffer as the blank.
11. Transfer fractions to the dialysing tube (Snake Skin, Thermo, 88243) and
dialyse for two hours three times in 300 mL Dialysis buffer.
12. If precipitation occurs spin at 100 000g in Ultracentrifuge using Ti50.2 rotor
(33 000 rpm) for 20 min.
13. Make 100 µL aliquots by snap freezing in the liquid nitrogen to prevent protein
degradation. Store at -80°C.
4
Absorbance
Volume
Fig. 1. Elution profile of TEV-His protease from Ni-column.
kDa
170
130
100
70
55
40
35
25
15
10
M
S
F
W1
W2
W3
E1
E2
E3
D
Fig. 2. Characterization of the purification results by SDS-Page. Lane M, marker. Lane S, supernatant in loading
buffer. Lane F, flowthrough. Lanes W1-W3, washes. Lanes E1-E3, fractions of TEV protease (respectively F3, G2,
G4 from Fig.1). Lane D, dialysed TEV.
5
References
1
van den Berg, S., Löfdahl, P.A., Härd, T. & Berglund, H. (2006) Improved solubility of TEV
protease by directed evolution. J. Biotechnol. 121, 291-298.
The sequence of the encoded protein is as follows:
MRSSTSLYKKAGSGESLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNN
GTLLVQSLHGVFKVKDTTTLQQHLVDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTN
FQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNY
FTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMNEL
VYSQDPAFLYKVVDLEGPRFEGKPIPNPLLGLDSTRTGHHHHHH
Calculated molecular weight 33442.9 Da (that given in paper is 33.5 kDa).
The blue text in the above shows residues 2038-2279 of P04517, Genome polyprotein, Tobacco etch
virus, with the positions mutated (T17S, N68D, I77V, S219N) shown in yellow highlight. The V5
epitope sequence is in cyan highlight. The positions encoded by the att sites are in green highlight, as
indicated below
attB1 sequence, from Gateway manual at http://wolfson.huji.ac.il/expression/gatewayman.pdf:
T S L Y K K A G S
ACAAGTTTGTACAAAAAAGCAGGCT
attB2 sequence:
D P A F L Y K V V
GACCCAGCTTTCTTGTACAAAGTGGT
Analysis
Length
Molecular Weight
1 microgram
Molar Extinction coefficient
1 A280nm unit
A280nm of 1 mg/ml solution
Isoelectric Point
Charge at pH 7
Entire protein
296 aa
33552.07
29.804 pmol
36610
0.92 mg/ml
1.09 AU
8.82
4.97
6
We use a C-terminally His-tagged version from van den Berg et al. [1]. The following shows the map
of the vector
T7 Promoter
XbaI (48)
NdeI (88)
ATG
BglII (6518)
HindIII (134)
SphI (6329)
EcoRV (169)
SacI (177)
lacI
SphI (389)
NdeI (453)
TEV SH
EcoRV (5350)
SpeI (510)
C-terminally His-tagged TEV
pTH24 TEV SH
SacII (897)
6536 bp
V5 epitope
6XHis
T7 term
EcoRV (1117)
HindIII (1271)
ClaI (1278)
ROP
ScaI (1823)
amp(R)
PstI (2062)
pBR322 ori
7