Reference: Ikura, M., Marion, D., Kay, L.E., Shih, H., Krinks, M., Klee, C.B. and Bax, A. (1990) Heteronuclear 3D NMR and Isotopic Labelling of Calmodulin: Toward the Complete Assignment of the 1H-NMR Spectrum. Biochem. Pharm. 40:153-160. ============================================================================== Preparation of Uniformly 13C and/or 15N-Labelled Calmodulin from AR58[pXCaM71] Cells by Thermal Induction Recipes for solutions are described after the protocol. ------------------------------------------------------------------------------ A. Preparation of a 2 L Cell Suspension: (1) Initial Culture (three methods: in M9 or LB medium) 25 mL Culture in M9 (with control) ------------------- - Prepare 50 mL of 1X M9 medium in a 125 mL erlenmeyer flask as follows: 5 mL 10X M9 salts (including 15N-NH4Cl for 15N-CaM) 45 mL H2O - Autoclave the 1X salt solution above, or prepare using sterile H2O, then add: 1.25 mL 20% (w/v) glucose (or 0.5 mL 13C-glucose for 13C-CaM) 5 uL 1 M CaCl2 50 uL 1M MgSO4 10 uL 1% (w/v) thiamine 10 uL 1% (w/v) d-biotin 250 uL 10 mg/mL ampicillin - Transfer 25 mL of the medium to a second 125 mL flask (to be used as a control). - Inoculate 25 mL of the labelled medium with 1 colony of AR58[pXCaM71] cells (grown on an LB plate containing 50 ug/mL ampicillin and 12.5 ug/mL tetracyclin). - Incubate the inoculated and control flasks at 32C in a shaking bath. - Monitor the cell growth by measuring optical desity at 650 nm, and incubate until the cells have reached saturation (OD650 > 1.5, typically requires 24-36 h). 25 mL Culture in LB ------------------- - Grow the overnight culture in 25 mL of LB medium containing 0.1 mg/mL (250 uL of 10 mg/mL stock solution) ampicillin. (1 L of LB medium contains 10 g bacto-tryptone, 5 g bacto-yeast extract and 10 g NaCl, pH 7.5). - Cells will reach saturation more quickly, and the large volume culture which is innoculated with this initial culture (see below) will also grow more quickly. 25 mL Culture in LB/M9 alternative ---------------------------------- - Inoculate 5 mL of LB with 1 colony. Shake in a bath at 32C overnight. If cloudy the next day, add 0.25 mL of this culture to 25 mL M9 (prepared as described above). Shake again at 32C overnight. (2) Large Volume Culture: - Prepare 2 L of labelled 1X M9 medium in a 6 L erlenmeyer flask: 200 mL 10X M9 salts (including 15N-NH4Cl) 1800 mL H20 - Autoclave the above 1X salt solution and, when cool, add: 50 mL 20% (w/v) glucose (or 20 mL 13C-glucose) 200 uL 1 M CaCl2 2 mL 1 M MgSO4 400 uL 1% (w/v) thiamine 400 uL 1% (w/v) d-biotin 10 mL 10mg/mL ampicillin - Inoculate the 2 L of M9 medium with 20 mL of the small volume culture from (1). - Incubate at 32C in a shaking bath until OD650 reaches 0.8 to 1.0 (no greater), at which point the cells are ready for induction. - Plot a growth curve in order to predict when the OD will reach this target in order to prepare for the thermal induction (typically 18-32 h required; average time to 0.8 AU is 20-22 h. NOTE: If LB medium is used in the overnight culture (1) which is used to innoculate this large volume, the time required to reach 0.8 AU is typically 16-18 h). (3) Preparation for Thermal Induction: - Approximately 1 h before the culture in (2) has reached the appropriate OD, have ready 600 mL of labelled M9 medium, prepared as follows: 60 mL 10X M9 salts (including 15N-NH4Cl) 540 mL H2O - Autoclave the 1X salt solution unless prepared with sterile H2O, and add: 15 mL 20% (w/v) glucose (or 6 mL 13C-glucose) 60 uL 1 M CaCl2 600 uL 1 M MgSO4 120 uL 1% (w/v) thiamine 120 uL 1 % (w/v) d-biotin 3.0 mL 10 mg/mL ampicillin - Heat the 600 mL of M9 medium to 65C in a water bath so that it has reached this temperature by the time the 2 L cell culture in (2) has reached the target OD650. - Approximately 1 h before the cells are to be induced, warm a shaking water bath to 42C. (4) Thermal Induction: - Remove a small aliquot (around 1 mL) of the cell suspension in (2) for SDS-PAGE before proceeding with the thermal induction (centrifuge and discard the supernatant; store the pellet at -70C). - When the cell suspension in (2) is ready for thermal induction, remove the M9 medium from the 65C bath in (3) and quickly pour the hot medium into the 2 L culture in (2). Swirl the flask and quickly place it in the 42C shaking bath (the objective is to quickly heat the cells to 42C). - Incubate the culture at 42C for 3-4 h. - At the end of the induction, measure the OD650 to complete the growth curve, and remove a small aliquot of the suspension for SDS-PAGE (as above). (5) Final Cell Pellet: - Centrifuge the suspension in 500 mL plastic bottles for 10 minutes at 6500 rpm, 4C. - Set aside the supernatant (store at 4C) until isolation and purification of the labelled CaM is complete. - The cell pellet (expected yield about 25 g wet cells) can be stored at -70C. - Check the success of the thermal induction by SDS-PAGE analysis of the cell pellets before and after induction. Continue if a band at 16700 Da is present in the post-induction cell suspension. ------------------------------------------------------------------------------ B. Isolation and Purification of Calmodulin: (6) Cell Lysis: - Combine the cells pellets from (5) and suspend in a total volume of 100 mL Lysis Buffer. - Stir on an ice bath. - Dissolve 24 mg of lysozyme in 20 mL of Lysis Buffer, and add to the cell suspension. - Stir on ice for 40 min. (7) DNase Treatment: - Add 1 M MgCl2 to the supernatant from (6) so that the final [MgCl2] = 3 mM (3/1000 dilution of MgCl2, approximately 360 uL required). - Add 10 mg/mL DNase so that the final [DNase] = 10 ug/mL (1/1000 dilution of DNase, approximately 120 uL required). - Stir on ice for 40 min. - Centrifuge for 20 min at 15000 rpm (27000 x g), 4C. Set aside the pellet until purification is complete (store at -70C). (8) TCA Precipitation of Unwanted Protein: - Chill an aliquot (at least 20 mL) of 50% (w/v) TCA on an ice bath. - While stirring the solution from (7) on ice, add 50% TCA so that the final [TCA] = 3% (3/50 dilution of TCA, approximately 7.2 mL required). - Stir on ice for 20 min, and continue stirring while adding the following: 6 M NaOH (add gradually until the pH reaches 5.3) 0.5 M EDTA (1/1000 dilution, approximately 130 uL required) 0.1 M PMSF for a final [PMSF] = 0.1mM (1/1000 dilution, approximately 130 uL required). - Stir on ice for 30 min. - Centrifuge for 10 min at 6500 rpm, 4C (be careful to keep the suspension cold at all times). (9) TCA Precipitation of CaM: - Decant and measure the volume of the supernatant (approximately 135 mL). Transfer to a clean centrifuge bottle and stir on ice. - Add cold 50% (w/v) TCA for a final [TCA] = 6% (supernatant for (8) already contains 3% TCA; this step involves addition of another 3%) (3/50 dilution of this aliquot of TCA, approximately 8.1 mL required in this step). - Stir on ice for 10 min. - Centrifuge for 20 min at 6500 rpm, 4C. - Decant the supernatant and set aside until the purification is complete (store at 4C). (10) Phenyl Sepharose Purification: - Dissolve the pellet from (9) in 400 uL 1 M Tris-base, 6 mL 50 mM Tris.HCl, pH 7.5 (1X buffer a) and 2 mL H2O. - Adjust to pH 7.5 by the addition of more 1 M Tris-base if necessary. - Stir until pellet is dissolved (may require up to 1 h). The pI of CaM is low (between pH 4-5), so dissolution is slow if the pH is too low. - If necessary, the dissolved pellet may be stored at -20C. - Prepare a phenyl sepharose (CL-4B, Pharmacia) column as follows (to be run at room temperature): - Fill a 2.5 x 15cm glass column with phenyl sepharose to a height of 10 cm (bed volume of approximately 50 ml). The void volume will be approximately 27 mL. - Equilibrate the column with 100 mL Buffer A, using a flow rate of 1 mL/min. - Connect a detector to the column outlet and set to 280 nm and 0.5-1.0 AU full scale (or, be prepared to take samples from the flow-through to a spectrophotometer) - NOTE - take care not to disturb the column beads by washing slowly. Do not let the column run dry. Siphoning the buffer solutions may be necessary. - To the crude solution of CaM, add 1 M CaCl2 for a final [CaCl2] = 5 mM (1/200 dilution of CaCl2, approximately 45 uL required). - Just before Buffer A has run through completely, load the entire sample onto the column and maintain the flow rate at 1 mL/min. - Wash the column with Buffer A until the OD280 (use a glass cuvette; reference is Buffer A) is less than 0.05 (about 250 mL required). - Wash the column with Buffer B until the OD280 is less than 0.01 (about 150 mL required). NOTE: OD may increase as high as 1.0 before decreasing, due to presence of salt, so a larger volume of Buffer B wash will likely be required. - Wash the column with 30 mL Buffer C. - Connect the detector outlet to a fraction collector and adjust for the collection of 1 mL fractions. - Elute CaM with Buffer D (typically less than 100 mL required). - Pool the fractions containing CaM and measure the final volume of the pooled solution. (11) Decalcification (two methods: by EGTA or TCA Precipitation) Decalcification with EGTA ------------------------- - Add 0.5 M EGTA to the pooled fractions collected from the phenyl sepharose column in (10), so that the final [EGTA] = 50 mM (1/10 dilution of EGTA; approximately 3 mL required). - Lyophilize the CaM/EGTA solution and redissolve in 2 mL H2O. - Prepare a calcium-free PD-10 column as follows: - Wash the column with 25 mL decalcified H2O (prepared by elution through a Chelex (BioRad) column) and 25 mL 50 mM NH4HCO3, pH 8.0, prepared using decalcified H2O. - Apply 5ml 0.5 M EDTA, pH 8.0 to the PD-10 column and elute with 25 mL of the calcium-free NH4CO3 solution. - Apply the CaM/EGTA sample to the column and elute with calcium-free NH4HCO3. - Collect 8 x 1 mL fractions, measure the OD280 values of each, and pool fractions 1-4 (fraction a) and fractions 5-8 (fraction b). Fraction a will contain the desalted CaM, and fraction b will contain residual CaM along with some salt. (The CaM in fraction b can be recovered by passage through a second PD-10 column as above.) - Lyophilize fraction a. - Go to step (12a). Decalcification by TCA Precipitation ------------------------------------ - Stir the pooled phenyl sepharose fractions on ice. - Add cold 50% (w/v TCA) until the final [TCA] = 5% (1/10 dilution of TCA, approximately 3 mL required). - Stir on ice for 10 min. - Centrifuge for 20 min at 8000 rpm, 4C. - Go to step (12b). (12a) Desalting by PD-10 Chromatography (from EGTA): - Dissolve fraction a in 1 mL 50 mM NH4HCO3, (pH 8) and apply to another PD-10 column, prepared and eluted as above, in order to completely desalt the protein. - Pool fractions 1-4 from this PD-10 column in preweighed plastic tube and lyophilize to obtain the final calcium-free CaM. - Weigh the plastic tube containing the dry product to determine the final yield of CaM (typically 20-30 mg). (12b) Desalting by PD-10 Chromatography (from TCA): - Dissolve the pellet from (11) in 50 uL Tris-base and 2 mL 50 mM NH4HCO3, pH 8. Adjust to pH 8 if necessary by addition of more Tris-base. - Prepare 2 Pharmacia PD-10 (G-25) columns by washing each with 25 mL H2O and 25 mL 50 mM NH4HCO3, pH 8. - Weigh a 50 mL Falcon tube, with cap. - Apply 2 mL of the protein solution to each of the PD-10 columns (discard flow-through) and elute with 3.5 mL 50 mM NH4HCO3, collecting in the Falcon tube. - To be as thorough as possible, wash out residual protein solution with water. - Cover the tube with parafilm (keep the cap) and poke holes using a needle point. Freeze using liquid nitrogen (be careful not to crack the parafilm). - Lyophilize - 1.5 days - Total dry product is typically 20-30 mg. ------------------------------------------------------------------------------ Solutions Required for the Preparation of Labelled Calmodulin A. Solutions Required for Cell Culture: 1. 10X M9 Salts (270 mL) 35.6 g Na2HPO47H2O (MW 268.1) 8.10g KH2PO4 (MW 136.1) 1.35g NaCl (MW 58.4) 2.70g NH4Cl (or 15N-NH4Cl) (MW 53.5 or 54.5) Dissolve in 200 mL H2O. Adjust to pH 7.40 with 6 M NaOH. Make up to 270 mL with H2O. Autoclave (store at room temperature or 4C). 2. 20% (w/v) Glucose (70 mL) 14.0 g glucose (or 13C-glucose) Dissolve and make up to 70 mL with H2O. Sterilize by filtration (store at room temperature or 4C). 3. 1 M CaCl2 (10 mL) 1.47 g CaCl2.2H2O (MW 147.0) Dissolve and make up to 10 mL with H2O. Sterilize by filtration (store at room temperature). 4. 1 M MgSO4 (10 mL) 1.20 g MgSO4 (MW 120.4) Dissolve and make up to 10 mL with H2O. Sterilize by filtration (store at room temperature). 5. 1% (w/v) Thiamine (10 mL) 0.10 g thiamine.HCl Dissolve and make up to 10 mL with H2O. Sterilize by filtration (store at 4C). 6. 1% (w/v) d-Biotin (10 mL) 0.10 g d-biotin Dissolve in 5 mL H2O. Add 1M NaOH until solid dissolves with shaking (a few drops required). Make up to 10 mL with H2O. Sterilize by filtration (store at 4C). 7. 10 mg/mL Ampicillin (15mL) 15 mg ampicillin sodium salt Dissolve and make up to 15 mL with H2O. Sterilize by filtration (store at 4C). B. Solutions Required for Isolation and Purification 8. 10X Buffer a: 0.5M Tris.HCl, pH 7.5 (500 mL) 39.4g Tris.HCl (MW 157.6) Dissolve in 400 mL H2O. Adjust to pH 7.50 with 6 M HCl. Make up to 500 mL with H2O (store at 4C). 9. 1X Buffer a: 50 mM Tris.HCl, pH 7.5 (500 mL) 50ml 10X Buffer a Make up to 500ml with H2O (store at 4C). 10. 0.5 M EDTA (10 mL) 1.86 g EDTA disodium salt.2H2O (MW 372.2) Suspend in 7 mL H2O. Adjust to pH 8.0 with 6 M NaOH while stirring to dissolve. Maintain at pH 8.0 by addition of 6 M NaOH as necessary while solid dissolves. Make up to 10 mL with H2O (store at room temperature). 11. Lysis Buffer: 50 mM Tris, 2 mM EDTA, 1 mM DTT, pH 7.5 (150 mL) 15 mL 10X Buffer a 0.6 mL 0.5 M EDTA 23 mg DTT (MW 154.2) Dissolve in 100 mL H2O. Adjust to pH 7.50 if necessary. Make up to 150 mL with H2O (store at 4C). 12. 1 M MgCl2 (10 mL) 2.03 g MgCl2.6H2O (MW 203.3) Dissolve and make up to 10 mL with H2O (store at room temperature). 13. 10 mg/mL DNase (1.0 mL) 10 mg DNase Dissolve in 10 mL with H2O. Aliquot in 5 x 200 uL portions (store at -20C). 14. 0.1 M PMSF (200 uL) 3.5 mg PMSF (MW 174.2) Dissolve in 200 uL isopropanol (store at -20C). Destroy unused solution by addition of an equal volume of 6 M NaOH. 15. 50% (w/v) TCA (50 mL) 25.0 g TCA Dissolve and make up to 50 mL with H2O (store at 4C). 16. 1 M Tris-base (10 mL) 1.21 g Tris-base (MW 121.1) Dissolve and make up to 10 mL with H2O (store at 4C). 17. Buffer b: 50 mM Tris.HCl, 0.5 M NaCl, pH 7.5 (250 mL) 25 mL 10X Buffer a (0.5 M Tris.HCl) 7.3 g NaCl (MW 58.4) Dissolve and make up to 250 mL with H2O (store at 4C). 18. Buffer A: 50 mM Tris.HCl, 1 mM MgCl2, 1 mM CaCl2 (500 mL) 50mL 10X Buffer a 0.5 mL 1 M MgCl2 0.5 mL 1 M CaCl2 Make up to 500 mL with H2O (store at 4C). 19. Buffer B: 50 mM Tris.HCl, 0.5 M NaCl, 1 mM MgCl2 1 mM CaCl2 (200 mL) 200 mL Buffer b 0.2 mL 1 M MgCl2 0.2 mL 1 M CaCl2 Store at 4C. 20. Buffer C: 50 mM Tris.HCl, 50 mM NaCl (50 mL) 4.5 mL 10X Buffer a 5 mL Buffer b Make up to 50 mL with H2O (store at 4C). 21. Buffer D: 50 mM Tris.HCl, 50 mM NaCl, 1 mM EDTA (200 mL) 18 mL 10X Buffer a (0.5 M Tris.HCl) 20 mL Buffer b 0.4 mL 0.5 M EDTA Make up to 200 mL with H2O (store at 4C). 21. 50 mM Ammonium Bicarbonate, pH 8.0 (250 mL) 0.99 g NH4HCO3 (MW 79.1) Dissolve in 200 mL H2O. Adjust to pH 8.00 with 1 M NaOH. Make up to 250 mL with H2O (store at 4C). 22. O.5 M EGTA, pH 8.0 (10 mL) 1.90 g EGTA (MW 380.4) Dissolve and makeup to 10 mL with H2O, pH 8.0 as described for EDTA.