Recombinant His-6 RII Purification
(cAMP-dependent Protein Kinase A)
Susan Taylor (University of California at San Diego) January 3, 1997
---Revised 7/95 ---Typical yield approximately 15 mg per liter of cells
Growing Cells and Expressing Protein
1. Prepare 6 liters of YT Media by placing 25 LB capsules/1 Liter MQ H2O and autoclaving. Let the media cool to at least 37°C and add to each flask 1 ml of filtered 100 mg/ml Ampicillin stock. (0.6g Ampicillin/6ml H2O) 2. Add 0.6 ml of sterile YT to the frozen bacterial stock. Vortex well. Using sterile technique, add 100 ml of this solution to each flask. If you inoculate the media that is at room temperature, let the cells grow 5-7 hours in the shaker 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 than predicted. ***Alternate Method: Inoculate media that is at room temperature after addition of sterile Ampicillin in the evening. Place flasks in the shaker and set the temperature to 37°C and RPM to 225.Set the timer to start the shaker early in the morning, ex: 3:00 or 4:00 a.m. Let the cells grow 5-7 hours at 37°C until OD600 is between 0.55-0.80. Record the OD in your notes. 3. When cultures reach an OD600 of 0.55-0.80, induce them with 1.0 M IPTG (1.62g/6ml). Add 1 ml to each liter observing sterile technique. Final concentration of IPTG in the media ==> 1.0 mM. Grow for an additional 6 hours at 37°C. 4. Harvest the cells by spinning in 1 liter bottles for 11 minutes at 5,000 RPM. Use Sorvall centrifuge. Measure and record the average OD's before centrifuging 5. Consolidate the pellets in a 40 ml centrifuge tube. Label completely and store the cells in the -70°C freezer.
Isolation of Protein from Bacteria (Day 1)
1. Make 150 mls His6 Lysis Buffer. Resuspend cell pellet in 60 mls/6L of His6 buffer [Dilute 10X His6 buffer, add Benzamidine-HCL, leupeptin, TLCK, TPCK, PMSF add 54 ml b-me, pH 8.0] ***If the resuspended cells are viscous, sonicate several times with large probe then french press 2. Lyse the cells with two passes through a french press. Save 20 ml aliquot of the total cell lysate and record the total volume. 3. Spin the lysate for 40 minutes at 15,000 RPM in 50 ml centrifuge tubes. 4. While the lysate is spinning, equilibrate the Ni2+ resin. Use 15 mls of resin for a 6 L WT His6-RII prep, less for mutants. Equilibrate by putting the resin in a 50 ml conical tube and spinning down the slurry. Decant the supernatantand resuspend the packed resin in His6 Lysis Buffer, pH 8.0. Repeat several times. Load the resin onto a column or keep in conical tube for batch method purification. 5. When lysate spin is complete, collect and combine the supernatants. Take a 20 ml aliquot of the supernatant and record the total volume of proteinsupernatant pooled. Save the pellet from one of the centrifuge tubes and resuspend with water in the same volume as the total cell lysate. Take a 20 ml aliquot of the resuspended pellet and save. 6. Check the pH of the supernatant protein solution. Re-pH, if necessary to 8.0. Load protein supernatant onto the column slowly (1 ml/min). Collect the flow through and pass over column again. Wash with 5 column volumes His6 lysis buffer. ***Alternatively, batch bind the supernatant with the Ni2+ Resin for at least 30 minutes (longer if necessary...but not more than 2 hours). Then, spin the resin down into one 50 ml conical tube. Wash the resin in the conical tube 2 times with 30-40 mls of His6-Lysis Buffer. 7. Step elute His6-RII from Resin (batch method or on column) with: 1) 2 column volumes (Ex:30 + 30mls) His6 buffer + 20mM imidazole, pH 7.0, 2) 2 column volumes His6 buffer + 50 mM imidazole, pH 7.0 8. Elute the protein off the resin with 10 column volumes (150 mls) His6 buffer + 100 mM imidazole, pH 7.0. Collect small fraction sizes, ex: 5 ml fractions. (can collect overnight)
Day 2 of His6-RII Protein Prep
9. Run 10.0% SDS PAGE gels. Include standard, total cell lysate (2ml), lysate pellet (2ml), lysate supernatant (2ml), flow through (5ml), Wash #1 (20ml), Wash #2 (20ml), 20 mM Wash (20ml), 50 mM Wash (20ml), and selected fractions (20ml). 10. Pool wanted protein (proteins that show little or no contaminating bands) in a small beaker or conical tube. Step Dialyze the protein in 1 L of: 20 mM Tris and 150 mM NaCl, pH 7.3 for six hours. Then, dialyze the pooled protein in 2 L of: 20 mM Tris and 50 mM NaCl, pH 7.3, overnight.
Day 3 of His6-RII Protein prep: Cleaning up Protein
1. Check the pH of your Dialysis buffer, make sure it is still at pH 7.3, if it isn't, record the pH and re-pH to 7.3. 2. Measure and record the concentration and volume of your dialyzed pooled protein. Take a 20 ml aliquot. 3. Load up to 50-60 mgs of protein that has been filtered, onto Mono Q 5/5 HR of FPLC. 4. Watch for peaks. Protein usually starts eluting between 35-45% of Buffer B (1 M KPO4, pH 7.2). Make sure to hold the gradient when peaks show and protein starts to elute. 5. Run a 10.0% SDS PAGE Gel. Include standard, load (20ml), flow through (20ml), and all necessary fractions from FPLC Run (2- 30ml) 6. Dialyze protein for crystallographers in R Crystallography Buffer and any excess in RII Storage Buffer: 7. Record total amount of protein recovered 8. Record distribution of protein and amount distributed.
Buffers for His6 Recombinant RII Purification:
His-6 Lysis and Running Buffer: 1X 10X For Qiagen Resin 50 mM KPO4 500 mM KPO4 300 mM NaCl 3 M NaCl 5 mM b-me pH 7.0 or 8.0 pH 7.0 His 6 Lysis and Running Buffer: 1X 10X For Invitrogen Resin 20 mM KPO4 200 mM KPO4 500 mM NaCl 5 M NaCl 5 mM b-me pH 7.0 or 8.0 pH 7.0 **Add to all Lysis Buffers, for 150 mls (Not necessary for running buffer): 10 mM Benzamidine (0.234 g/150 mls) 1 mM or 0.5 mg/ml Leupeptin (75 ml/150 mls) 70 mg/ml TPCK (150 ml of 7.0 mg/ml stock/150 mls) 37 mg/ml TLCK (150 ml of 3.7 mg/ml stock/150 mls) 0.5 mM PMSF (750 ml of 100mM stock/150mls) Mono Q Buffer: Buffer A Buffer B 20 mM Tris 1 M KPO4 5 mM b-me 5 mM b-me pH 7.3 pH 7.2 R Crystallography Buffer: 1X 10X 10 mM MES 100 mM MES 1 mM EDTA 10 mM EDTA 1 mM EGTA 10 mM EGTA 5 mM b-me, pH 6.0 pH 6.0 RII Storage Buffer: 1X 10X 50 mM MES 500 mM MES 150 mM NaCl 1.5 M NaCl 1 mM EGTA 10 mM EGTA ~20% Glycerol 5 mM b-me pH 6.8 pH 6.8 KMOPS 130: 1X 10X 10 mM MOPS 100 mM MOPS 130 mM KCl 1.3 M KCl 2 mM DTT or 5 mM b-me pH 6.5 pH 6.5 Optional: add 1 mM EDTA, 1 mM EGTA to 1X to stabilize RII For all solutions, pH when cold and b-me to final solutions only