Recombinant Catalytic Subunit Preparation
(cAMP-dependent Protein Kinase A)
Susan Taylor (University of California at San Diego) January 3, 1997
Clone Culture, Expression & Harvest
1. Prepare 12 liters of sterilized YT Media by placing 18 YT capsules/Liter of MQ H20 and Autoclaving. Let the media cool to at least 37°C and add to each flask 1 ml of a 100 mg/ml MQ H20 filtered ampicillin solution (1.2 g Ampicillin/12 ml H2O). 2. Add 1.1 ml of sterile YT to the frozen bacterial cell stock. Vortex well. Using sterile techniques, add 100 ml of this solution to each flask. If you inoculate the media that is at room temperature, let the cells grow for 8-8.5 hours at 37°C until OD600 is greater than 0.55 and less than 0.80. If you inoculate media that is warm, the cells will grow to the appropriate OD in less time. **Alternate Method: Inoculate cold media flasks (with Ampicillin) at night. Place flasks in the shaker and set the temperature to 37°C and RPM to 225. Set timer to start in the early morning, ex: 1:00 or 2:00 am. Let grow 8-8.5 hours at 37oC until OD600 is approximately between 0.55-0.80. Record the OD in your notes. 3. When cultures reach an OD600 of 0.55 to 0.80, induce them with 0.5 M IPTG (1.62 g/12 ml). Add 1 ml to each liter observing sterile technique. Final concentration of IPTG ==> 0.5 mM. Adjust the temperature control of the shaker to 24°C. Leave lid open or the temperature will continue to rise past 24°C. Grow for an additional 6 hours. 4. Harvest the cells by spinning in 1 liter bottles for 11 minutes at 5,000 RPM. Use Sorvall centrifuge. 5. Consolidate the pellets in a 40 ml centrifuge tube. Label completely and store the cells in the -70°C freezer.
A Day Before Your Prep Starts or Early the First Day of Your Prep:
6. Prepare P-11 phosphocellulose resin A. Stir 250 mls of 0.5 M NaOH into 12-13 g of P-11 and let sit for 5 minutes B. Filter off the supernatant and wash with MQ water (about 2 liters) until the pH is below 11 (Use pH paper to measure approximate pH's) C. In filter funnel mix resin with 250 mls 0.5 M HCl and let sit for 5 minutes D. Filter off the supernatant and wash with MQ water (about 2 liters) until the pH is above 3.0 E. Transfer the P-11 into approximately 3 volumes (300 mls) of 10X P-11 running buffer F. Titrate the slurry with NaOH until the pH is 6.5. Let the solution sit in the cold room G. Decant the supernatant and add 250 ml of 1X running buffer. Stir the slurry and pH again to 6.5 H. Leave in cold room overnight or until you add the total cell supernatant.
Day 1 of Cat Protein Prep
1. Remove frozen pellets from -70°C freezer and let thaw at room temperature on bench or in a beaker of water. Resuspend the thawed pellets in 240 mls 1X P-11 lysis buffer [Dilute 10X P-11 (cold room) and add 43 ml b-me to 5mM] 2. Lyse the cells in the French Press 2X. Save 20 ml of the total cell lysate and record the total volume 3. Spin the lysate for 40 minutes at 15,000 RPM (JA-20 rotor) in 50 ml centrifuge tubes. Collect and combine the supernatants. Take a 20 ml aliquot and record total volume. Save the pellet from one of the centrifuge tubes and resuspend with water in the same volume as total cell lysate. Take a 20 ml aliquot and save. 4. Measure and record the initial conductivity of the supernatant. Dilute the supernatant with cold water (4°C) until the conductivity reaches 1.2 mS/cm. Check the pH and make sure it is at 6.5. Save a 50 ml aliquot of the diluted supernatant and record volume. 5. Check pH of the resin (6.5). Decant off supernatant of resin. Add resin to the diluted cell supernatant. Mix Slowly (Batch bind) for 3 hours minimum (or overnight) in an appropriately sized beaker.
End of Day 1 or Day 2 of Cat Protein Prep
6. Decant resin through funnel (plastic). Resuspend in 1X Running buffer. 7. Pour Slurry into a 100 ml column. Pack and wash with 200 mls of 1X running buffer (or batch wash the resin in a beaker for 10 minutes at 4oC.). 8. Column wash with 200 mls of: 1X Running buffer + 50 mM KPO4. Collect elution in bottle or beaker and save. (or batch wash in beaker for 10 minutes). 9. Wash with 200 mls of: 1X Running buffer + 90 mM KPO4. Collect elution in bottle or beaker and save. (or batch wash in beaker for 10 minutes and pour column). 10. Elute with 250 mls of 1X Running buffer + 250 mM KPO4. Collect 8-10 ml fractions. 11. Run 12.5% SDS PAGE gels. Include standard, total cell lysate (2 ml), lysate pellet (2 ml), lysate supernatant (2 ml), diluted lysate supernatant (5 ml), flow through (5 ml), 1X Running Buffer Wash, (20 ml), 50 mM Wash (20 ml), 90mM Wash (20 ml), and selected fractions (20 ml). 12. Pool wanted protein (proteins that show little or no contaminating bands) in a small beaker. Dialyze the protein in 2L of Buffer A (20 mM KPO4), pH 6.5 overnight.
Day 2 or 3 of Cat Protein Prep
1. Check pH of Dialysis buffer, make sure it is at pH 6.5. 2. Measure the concentration of your dialyzed protein. Record concentration and volume of your pooled protein. Take a 20 ml aliquot. 3. Load up to 50-60 mgs of protein onto Mono S 10/10 HR of FPLC. 4. Watch for peaks. Protein usually starts eluting between 15-25% of Buffer B. 5. Run a 12.5% SDS PAGE Gel. Include standard, load (10 ml), and all necessary fractions 6. Run IEF Gel to determine peaks of individual fractions (if necessary). 7. Check Cat activity and record. 8. Dialyze protein for crystallographers in : 50 mM Bicine 150 mM Ammonium Acetate 10 mM b-me pH 8.0 9. Dialyze protein to be frozen at -20°C in: 100 mM MOPS 150 mM KCl 0.1 mM DTT pH 7.1 10. Record total amount of protein recovered, separating them by peaks. 11. Record distribution of protein and amount distributed.
Buffers for Recombinant Cat Preparation
Lysis buffer: 1X 10X 30 mM MES 300 mM MES 1 mM EDTA 10 mM EDTA 50 mM KCl 500 mM KCl 5 mM b-me pH 6.5 pH 6.5 P-11 Running buffer: 1X 10X 30 mM MES 300 mM MES 1 mM EDTA 10 mM EDTA 5 mM b-me pH 6.5 pH 6.5 Mono S: Buffer A: Buffer B: 20 mM KPO4 20 mM KPO4 1M KCl 5 mM b-me or 5 mM b-me or 2 mM DTT 2 mM DTT pH 6.5 pH 6.5 S-100: 1X: 10X: 200 mM KCl 2 M KCl 25 mM KPO4 250 mM KPO4 5 mM b-ME pH 7.0 pH 7.0 For all solutions: pH when cold, add b-me to final solutions only (350 ul/1L).