CDPK-pET28b
This page includes experimental information on uniform and selective isotopic labeling as well as deuteration of CDPK constructs.
- All culture media to contain 50 mg/ml Kanamycin Sulphate
- Grow all cells at 37°C and 270-300 rpm.
1) Grow an overnight (5-10 ml) culture of pet-JC (from frozen glycerol stock) in 2xYT or LB.
2) Use 10 ml of overnight culture per liter of main culture.
3) Grow cells until OD600 = 0.5 -0.7 then induce protein expression by adding 0.5 mM IPTG (500 ml of 1M IPTG stock per liter of culture).
4) After 3-6 hours, harvest cells:
5) Centrifuge culture at 6000 rpm for 20 min. Discard supernatant.
- Keep proteins and buffers at ca. 4 °C as far as possible during the purification.
- Assessments of purity can be made via SDS-PAGE. (10 ml aliquots of each stage of the procedure should be kept for SDS-PAGE analysis)
- PMSF should be freshly prepared as an ethanolic solution.
A: 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.2 mg/ml lysosyme, 1mM PMSF.
B: 20 mM Tris-HCl pH 8.0, 100 mM NaCl.
C: 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 300 mM Imidazole.
1) Resuspend pellet in 10-20 ml (per liter of culture) of buffer A.
2) Transfer pellet to a plastic mini-beaker and stir for 5 min.
3) Sonicate in a plastic beaker on ice, e.g. using 50% duty cycle at intermediate power in 1 minute bursts, with 2 minute intervals. Repeat 3 times.
4) Transfer to 50 ml Oakridge centrifuge tubes and centrifuge at 14,000 rpm for 30 min.
5) Ni-NTA agarose column (gravity method)
a) Make a small Ni-Agarose column (3-5 ml suspension per liter of culture)
b) Equilibrate column with 25 ml buffer B
c) Load lysate onto the column
d) Wash column with 20 ml buffer B
e) Elute protein with 15 ml buffer C
6) Thrombin Cleavage: The protein contains a thrombin cleavage site to remove the purification tag
a) Prepare Ni-affinity eluate for thrombin cleavage by dialyzing against 4 L buffer B.
(Prepare regular dialysis tubing by stirring in boiling water (500 ml) to which has been added a spatula of EDTA and ammonium bicarbonate for 5 min. Alternatively use a Slide-A-Lyzer dialysis cassette (10 Kda MWCO) from Pierce).
I have written a CDPK-specific procedure for the reaction with thrombin, which appears on our web pages: http://structbio.vanderbilt.edu/chazin/wisdom/labpro/thrombin.html.
React protein with thrombin at room temperature for ca. 1 h. Inactivate the thrombin with 1 mM PMSF
7) Ni-NTA agarose column 2.
a) Make another small Ni-Agarose column (2 ml suspension per liter of culture)
b) Equilibrate column with 25 ml buffer B
c) Load protein/thrombin mixture onto the column. Collect eluate
d) Elute column further with 6 ml buffer B
Mix eluate fractions from steps 7 c) and d)
8) Benzamidine-sepharose column (suspension available from Pharmacia)
In a manner similar to the Ni-NTA step above:
a) Make a Benzamidine-sepharose column (1 ml suspension per liter of culture)
b) Equilibrate column with 25 ml buffer B
c) Load mixed eluate fractions from the 'Ni-NTA agarose column 2' step onto the column. Collect eluate
d) Elute column further with 6 ml buffer B
Mix eluate fractions from steps 8 c) and d).
Further Purification (if required)
1) Superdex- 75 gel-filtration FPLC step on the AKTA system.
The protein should be concentrated (via ultracentrifugation with a 10 Kda MWCO filter) to ca 5 mg / ml before loading onto the column. Limit load to 1-5 ml. Flow should be at 1 ml/min with detection at 280 nm.
Suitable buffers for this step include:
50 mM Tris-HCl pH 8.0, 100 mM NaCl , 1mM DTT.
or 100 mM Ammonium Bicarbonate.
Ammonium bicarbonate solution is volatile and evaporates upon lyophilization. Both JC and the CaM-LD proteins are stable to lyophilization and/or can be stored as frozen solutions. JC-3G should be stored in frozen solution.
General
1) Use M9 minimal media prepared exactly according to the recipe on the Chazin wisdom page: http://structbio.vanderbilt.edu/chazin/wisdom/labpro/M9.html
2) Prior to using the initial overnight 2xYT culture for inoculation of the main culture, the cells should be pelleted (via gentle centrifugation at 2000 rpm) and resuspended (gently!) in an equivalent volume of the M9 media. Again, use 10 ml of these resuspended cells per liter of main culture.
3) When 13C-labeling is required use only 2 g/L of 13C-glucose.
For deuteration it is important to prepare an inoculum that is suitably adjusted to growth in D2O. This can be done in several ways with the overall aim being to gradually adjust the cells to growing on deuterium rich media. Adapt cells to 35%, 68%, then 99% D2O. It is important make test runs (small scale, no 13C,15N) before trying large-scale overexpression. Cells will grow at a significantly and often unpredictably attenuated rate in deuterated media and it is necessary to be patient and develop a stable system.
1) M9 minimal media preparation for deuteration
-Prepare M9 using D2O instead of H2O. Do not autoclave, sterile filtration is sufficient.
-Add the MgSO4, CaCl2, NH4Cl and kanamycin sulphate in solid form.
-Use only 5 ml (per liter of media) of Basal Vitamins solution.
-If protonated glucose is used, then the level of deuteration will be ca. 80 %
2) Grow an overnight (10 ml) culture of pet-JC (from frozen glycerol stock) in 2xYT. Prepare the 2xYT in D2O.
3) Pellet cells according to Labeled Preparations:General:2 above and resuspend cells in 50 ml of deuterated M9. Transfer cells to a beveled 50 ml culture flask and grow cells to OD600 > 0.7. This may take anywhere from 6-36 h, so be patient.
If cells do NOT grow during this period then go to step 9)
4) Use 25 ml of this culture per liter of main deuterated culture.
5) Grow cells until OD600 = 0.5 -0.7 then induce protein expression by adding 0.5 mM IPTG (500 ml of 1M IPTG stock per liter of culture).
6) Length of induction will vary according to cell growth rate. For CDPK constructs John used 10 hours and achieved reasonable expression (10 mg/L)
7) Centrifuge culture at 6000 rpm for 20 min.
8) Purify protein as for unlabeled cultures.
If cells have not grown during step 3) then do step 9)
9) It may be necessary to acclimatize cells to growth in M9-D2O in several stages.
e.g. via 50:50 D2O:H2O M9 mix initially, then a 75:25 mix, before finally using only D2O. Employ small (5-10 ml) cultures to achieve this using the gently pelleted cells from each stage as the inoculum for the next stage.
For selective labeling only: add 25-40 mg of amino acid to be incorporated at point of induction. Grow for 90 min only. With BL21(DE3) cells, John found Lys a-15N labeling to result in no scrambling of the label while attempted a-15N labeling of Leu and Phe residues resulted in extensive scrambling.
If the periplasmic target protein content after lysis (estimated via SDS-PAGE band intensity) differs significantly from that when the whole cell is disrupted (via boiling in SDS) then it is likely that significant quantities of the target protein are in inclusion bodies.
The protein in inclusion bodies can be extracted using urea denaturation:
1) As far as possible dissolve pellet in 8M urea, 50 mM Tris-HCl pH 8.0, 10 mM DTT.
This will require extensive vortexing and heating to 50°C (OK to do this since CDPK constructs are stable at this temperature).
2) Transfer to 50 ml Oakridge centrifuge tubes and centrifuge at 14,000 rpm for 30 min.
3) Transfer the supernatant to prepared dialysis tubing and refold the protein by dialyzing against 4 L of buffer B. Exchange the dialysis buffer 3 times over the period of 1 day.
4) Subject dialyzate to the purification beginning from stage Purification:B:5 (Ni-NTA column)
1) The same general conditions apply for CaM-LD and JC-3G preparations. For JC-3G preparation, grow protein for 90 min. post induction. The protein will predominantly be in the inclusion body even after such a short induction, but will most likely be refoldable to a stable protein.
2) John recommends avoiding the MonoQ cation exchange column for CDPK constructs since he has observed a tendency for on-column irreversible oligomerization.
3) The benzamidine-sepharose column step should NOT be used for preparation of the CaM-LD. The protein interacts irreversibly with this matrix.
4) The adventurous may attach the Ni column to a peristaltic pump and adjust flow to 4-5 ml/min.
1) Protein masses should be confirmed by MALDI mass spectrometry.
Further assessments of purity can be via SDS-PAGE and analytical gel-filtration HPLC (via Chazin lab TSK2000SW gel filtration column, Tosohaas, use 20 mM Tris-HCl pH 8.0, 100 mM NaCl, flow rate 1 ml/min, 280 nm detection).
2) Proteins concentrations should be determined via the Bradford and/or the BCA protein assay methods (Pierce), relative to BSA. John found that similar results were obtained via both methods.
If you have any questions, comments - email: johnchrs@structbio.vanderbilt.edu
JC/Aug01