Sosnick:Purifying ctAcP
ctAcP is a 98-residue protein with a relatively high pI so it is well purified using ion-exchange chromatography (FPLC) followed by reverse-phase HPLC purification.
Growth
1. Transform BL21 DE3 e. Coli or appropriate expression strain and plate on Amp-resistant plates (200mg/l).
2. Cut several colonies out of the agar plate and place in 250-mL flask of LB with 200mg/l amp. Shake for 4-8 hrs until OD at 600nm is >0.3. Place in fridge overnight with 100mg/l additional amp.
3. Next day, innoculate four 1.5L Fernbach flasks with 1:100 dilutions of cell culture (15mL/flask) with 200mg/l amp. Grow to OD600 > 0.5 at 37C then induce with 100mg/flask of IPTG. It should take 3-4 hrs to get to this point.
4. Harvest after three hours and freeze pellets overnight.
Purification:
5. Thaw cells on benchtop for 15 minutes while preparing lysis buffer. Prepare 30ml buffer to 1L of growth media before being spun, so a four flask, 1.5L growth should be six liters and 180mL of lysis buffer (10mM HEPES pH 7.5 and 1mM EDTA)
6. Resuspend cells one bottle at a time and place resuspended liquid in one bottle sitting in an ice bucket. When cells are resuspended completely, add a small amount of lysozyme (spatula-tip or so) and swirl to mix. Lyse 30-45 mins on ice.
7. When cell mixture is viscous, sonicate for 5 minutes. Then sonicate the surface foam to break up any floating DNA. Spin in SS-34 tubes for 20 mins at 18k RPM.
8.