Sosnick:Purifying ctAcP

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m (Purifying ctAcP moved to Sosnick:Purifying ctAcP: Lab Specific protocol)
 

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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. Pour supernatant into a beaker and keep on ice.

8. FPLC will be conducted using cation exchange (SP) resin at pH 7.5 at 10ml/min. Make 1L each high and low salt buffer: 10mM HEPES pH 7.5 plus 1M NaCl in high-salt only. Equilibrate column with 2 column volumes of Buffer B (high salt) followed by 4 CV of Buffer A.

Note: I usually clean the column beforehand with a slow wash of high-concentration of Guanidine HCL and/or isopropanol but this is not required every time.

9. When column is equilibrated, load supernatant onto column at 5ml/minute and save the flowthrough. If the column gets clogged then you have too much DNA on it and you may have reduced yield. In future, sonicate more and dilute the cell solution more as well.

10. Once the supernatant is loaded, wash with Buffer A until the OD goes to zero. Then run a 1200ml gradient from 0-100%B and collect 20ml fractions. You should see two peaks emerge, the first of which is DNA and the second is ctAcP. Confirm this by spectrophotometry. The protein should elute at roughly 15-30%B.

11. Run the peak on a C8 HPLC column with 0.1%TFA vs 0.1%TFA in Acetonitrile. Elute over 1000ml and protein will elute at about 40%B. Freeze in the -80C and lyophilize to powder. Confirm mass by MS.

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