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.

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.