Expressing and Purifying Tagged (His6, GST, sugar-binding) Proteins
Growth and expression will have to be optimized for your individual protein.
Cell Growth and Expression (4 L prep of BL21(DE3) or JM109(DE3) cells with your
transformed DNA). For smaller cultures, protocols of growth and induction still
apply, but lysis should be performed by either sonication with lysozyme or B-PER
(Pierce) extraction.
Day 1
- Autoclave 4.05 L TB media (includes 10 ml for spectrophotometer blank)
- Inoculate 1 x 45 ml TB with a single colony (starter culture) (e.g. 41 ml TB + 4 ml phosphate salts + 45 µl antibiotics + single colony)
- Grow o/n at 250 rpm and 37 °C
Day 2
- Transfer 5-10 ml of starter culture into 4 x 1 L TB with salts and antibiotics, grow at 37ûC
- Monitor cell OD at 600 nm and induce just before end of log growth.
Usually this induction point is between 1.2 and 1.8, but
this growth curve should be plotted for any untested
spectrophotometer. Induction after this point results in
poor expression.
- Once induction point is reached, stop shaking cultures and bring them to induction temp.
- Place flasks in ice in 4 °C room, to cool to 17 °C (~15 min)
- Induce with IPTG
- Grow cells at induction temperature as follows:
Temp (C) Time (hr) IPTG (mM)
17 12-18 0.15
30 4-7 0.50
35 3-5 1.00
Day 3
- Harvest cells in 1 L centrifuge tubes for 20 min at 5000 rpm (HA6000 rotor)
- Measure cell mass from cultures (Dry 1L tubes with caps, Nalgene = 95.4g)
- Either freeze cells with liquid N2 now or resuspend in lysis buffer
- Keep cells at 4 °C from this point on
- Resuspend cells in cold lysis buffer that is stirring with a large stir-bar:
100-150g cells
Protease inhibitors (i.e. 4.5 ml PMSF, 0.5 ml Leupeptins, 0.5 ml Pepstatins)
4 mg DNase
500 ml Binding Buffer for affinity column
- Pass resuspended cells through large volume French Press (EmulsiFlex C-50)
- Collect lysis material in beaker on ice
- Ultracentrifuge at 18,000 rpm and 4 °C for 45 min in SS-34 rotor
- Filter lysis supernatant through 0.45 µm syringe filter
- Incubate filtered lysis supernatant in batch with Ni or GST resin on nutator at 4 C for 1 hr
- Pour lysis slurry with resin into small 30 ml column fitted with large funnel
- Drain column as quickly as possible and remove funnel
- Wash resin with 200 ml Binding Buffer
- Elute successively with a total of 15 ml elution buffer
- Removal of His6 / GST tag with thrombin
Removal of tag (His6, GST, sugar-binding) with thrombin
- Measure protein concentration by either A280 (Current Protocols) or dye binding assay
- Place eluted protein in dialysis tubing with appropriate [thrombin]
- Prepare 2 L thrombin cleavage buffer for dialysis, and dialyze 15 hrs at 4 C
- Stop cleavage reaction with 100 l PMSF stock
- Pass protein over 250 l Benzamidine Sepharose beads in plastic column
- Collect flow through and discard beads
- Pass protein over small Ni-NTA/Glutathione column
- Collect and keep Ni-NTA/Glutathione flow through (your protein without tag)
- Wash and Elute Ni-NTA/Glutathione beads to asses % cleavage
Additional Purification
Additional chromatography steps may be required at this point. Consult
your FPLC manual for specific procedures. Use the following columns as
appropriate:
Column When to use:
MonoQ Anion exchange (i.e. your protein has a low pI)
MonoS Cation exchange (i.e. your protein has a high pI)
Phenyl-
Superose Hydrophobic separation, used near end of purification for persistent impurities
Superdex 200 Size separation (e.g. 200 kD from 400 kD)/buffer exchange
Superdex 75 Size separation (e.g. 60 kD from 30 kD)/buffer exchange
Gels
- Resuspend cells from 250 µl culture, before and after induction in 1x SDS sample buffer, 100 and 150 µl respectively
- Run SDS-PAGE lanes to asses proteins:
Before/after IPTG
Fractions from each column
Thrombin cleavage rxn (including final column bump
and Ni-NTA/Glutathione beads if necessary
Media TB 48 g Bacto-tryptone TB salts 23.1 g KH2PO4 (MonoBasic)
96 g Yeast extract 125.4 g K2HPO4 (DiBasic)
16 ml Glycerol Add dH2O to 1 L and autoclave
Add dH2O to 3.6 L and autoclave
Buffers Ni-NTA: Binding Elution
0.2 M Buffer of choice same
0.2 M NaCl same
10 mM Imidizole 100 mM Imidizole
Precautions [EDTA]=0, [DTT]<1 mM, pH > 7.0
Glutathione: Binding Elution
0.2 M Buffer of choice same
0.2 M NaCl same
20 mM Glutathione (reduced)
Precautions [DTT]<1 mM
Thrombin Cleavage: 50 mM Tris pH 8.0
150 mM NaCl
3 mM CaCl2
5 mM MgCl2
10 % Glycerol
1 mM DTT or 5 mM BME
General Points
- Antibiotic and protease inhibitor solutions not described are from Molecular Cloning, Sambrook, Fritsch and Maniatis
- Do not throw away Ni-NTA beads. This is VERY EXPENSIVE material. Ni-NTA can be used 3 or 4 times for the same step in a protein prep.
- Detergents can be added to any or all buffers starting with lysis buffer.