Recombinant His-6-Cat Purification
(cAMP-dependent Protein Kinase A)
Susan Taylor (University of California at San Diego) January 3, 1997
---Typical yield approximately 15 mg per liter of cells ---Use this protocol for most His-6-Proteins including GFP
Growing Cells and Expressing Protein
1. Prepare 6 liters of YT Media by placing 18 YT capsules/1 Liter MQ H2O and autoclaving. Let the media cool to at least 37&deq;C and add to each flask 1 ml of filtered 100 mg/ml Ampicillin stock. (0.6g Ampicillin/6ml H2O) 2. Add 0.5 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 5.5-7 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. Adjust the temperature 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.
Isolation of Protein from Bacteria (Day 1)
1. Resuspend cell pellet in 60 mls of His6-Cat buffer [Dilute 10X His6-Cat buffer and add 22 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-Cat prep, less for mutants. Equilibrate by putting the resin in a 50 ml conical tube and spinning down the slurry. Decant the supernatant and resuspend the packed resin in His6-Cat 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 supernatant 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-Cat 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 H6-Cat Lysis Buffer. 7. Step elute His-6-Cat from Ni2+ (either batch method or on column) with: 1) 2 column volumes (Ex: 30 + 30 mls) His6-Cat lysis buffer + 20 mM imidazole, pH 7.0 2) 2 column volumes Cat lysis buffer + 50 mM imidazole, pH 7.0 8. Elute the protein off the resin with 10 column volumes (150 mls) Cat lysis buffer + 100 mM imidazole, pH 7.0. Collect small fraction sizes, ex: 5 ml fractions. 9. Run 12.5% 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).
Cleaning up the Protein (Day 2/3)
1. Pool wanted proteins in a small beaker or conical tube. Dialyze the protein in 2 L of Buffer A (20 mM KPO4).