Luke Wheeler: S100 purification protocol

S100s* expression and Purification Protocol

Luke Wheeler

*This protocol works for most members of the S100 family, but does not work for the calgranulin clade.

Expression of the proteins in Rosetta DE3 pLysS:

• Transform/streak out a glycerol stock of the relevant clone onto an LB ampicillin/chloramphenicol plate.
• Inoculate a 10-20mL (LB + amp+cam) culture from a single colony.  Place at 37 °C, shaking @ 220-250 rpm.  Grow overnight. (Alternatively, can inoculate directly from glycerol stock)
• Inoculate a 1.5 L (LB + amp + cam) culture with the overnight culture.  Place at 37 °C, shaking @ 220-250 rpm.
• Follow growth by OD600.  Aim for logarithmic growth and an OD600 of ~0.8-1. as measured using a plastic cuvette in the Eppendorf BioSpec.  (Note: measure the OD600 using the “OD600” option, not the absorbance protocol.  Because of how the instrument processes the raw data, the cell culture and absorbance values are radically different). An OD600 of 0.8 is usually achieved within ~3.5 hr.
• Induce growth by adding 1.5 mL 1M IPTG (1 mM) and 7.5 mL 50% glucose (0.2%).  Drop the growth temperature to 16 °C.
• Allow cultures to grow overnight with shaking (>= 200 rpm) @ 16C.
• Spin cultures down for 20 min @ 3,000 rpm (J-6B centrifuge in IMB shared centrifuge room) and place centrifuge bottles in -20 °C freezer. Leave them in the freezer for ~1 hr to overnight.  Tip: leave the bottles at an angle with the pellet pointing downward makes the pellets much easier to pop out after freezing.
• Use a stiff metal spatula to pop the pellets out of the bottles into a 50 mL conical labeled with the protein name, the pellet mass, your name, and the date. Tip: as the pellets thaw they are harder to remove from the bottle.  Pull the bottles out two at a time, rather than all at once, to make it easier to pop the pellets.
• Move onto the lysis step. Otherwise, place the conical with the pellets into the –20 °C freezer.  The pellets will keep for >1 month.

Buffers: (filter all FPLC buffers, 0.22uM vacuum filter)

All buffers should be at pH 7.4

His A (1L)

His B (1L)

HIC A (1L)

HIC B (1L)

1. Lyse cell pellets in His A buffer (3.5mL/g cell pellet), 2uLlysozyme/g, and 2uL Dnase/g. Resuspend pellet in buffer by vortexing repeatedly. Sonicate for 5min @ 50% duty cycle with power setting applicable to sample volume. Bring the sample up to ~50mL in His A.

2. Centrifuge cell lysate at 15,000 rpm (large centrifuge in centrifuge room, JA-20 rotor) for ~60 minutes. Collect lysate in clean conical and filter (0.45um syringe) if necessary.

3. Meanwhile, wash HisTrap affinity column with filtered ddH2O. Then wash the system with appropriate buffers and equilibrate His column with His buffer A.

4. Load spun-down cell lysate into the 50mL superloop and run over His column, washing with 25mL His A and then elute with a 25mL gradient to 100% His B. Collect fractions throughout the run.

5. Wash the column with 25mL ddH2O, then with 25mL filtered 20% EtOH and store.

6. Run SDS PAGE on the fractions (Only recommended to do this the first time the protein is purified. Skip this step in subsequent purifications and save a gel).

7. Pool fractions that have the His6-S100 fusion (should be the large, sharp peak that comes off in the His B elution gradient).

8. Add 300-500uL TEV protease (from frozen stock) to the pooled fractions, spike with ~5mM DTT, and incubate at room temperature for ~3hr. Then incubate overnight @ 4 degrees Celsius (Note: be sure to save a small ~15uL sample of before/after TEV for comparison on SDS PAGE).

9. Add calcium to saturation in sample before loading on column (2mM should be sufficient). This step is necessary for the protein to stick to the HIC column. 

    

10. Wash Hydrophobic Interaction Chromatography (HIC) column with 25mL ddH2O. Then equilibrate with 25mL HIC A (5 column volumes).

11. If there is any precipitate in sample, centrifuge or filter with a 0.45um syringe filter. Load sample into 50mL superloop and run over HIC column. Wash with 30mL HIC A, then do a 30mL gradient to 100% HIC B. Collect fractions throughout this step.

12. After the run, wash the column with 25mL ddH2O and then with ~25mL 0.5M NaOH, then again with 25mL ddH2O. Finally equilibrate with 20% EtOH and store.

13. Run SDS PAGE to check fractions. (For a first time/previously untested protein it’s best to check fractions from throughout the purification pipeline). Pool good fractions and dialyze overnight into 5L 25mM Tris, 100mM NaCl, 1mM DTT, pH 7.4.

14. Wash a His column with 25mL ddH2O and then equilibrate with 25mL His A.

15. Remove pooled fractions from dialysis and load into 50mL superloop. Run over the His column, washing with 25mL His A. Then step to 25mL 100% His B. Collect fractions throughout the run (purified S100 should be in the flow-through fractions from the His A wash). The step elution will remove the residual uncleaved protein from the column.

16. After run, wash His column with 25mL ddH2O, then with 25mL 20% EtOH and store.

17. Run SDS PAGE to check fractions. Pool good fractions and dialyze overnight into 2L of 25mM TES, 100mM NaCl, pH 7.4 with ~2g chelex resin suspended in dialysis vessel. Overnight @ 4 degrees Celsius.

18. Measure A280 of protein and calculate concentration. Filter sterilize the dialyzed protein (sterile 0.2um syringe) dropwise into liquid N2 to flash freeze. Store at -80°C. (Save a sample of unfrozen protein for step 19 -> for first time purification only).

19. Measure far UV CD spectrum of before and after (flash freezing) samples to make sure that freezing does not alter protein secondary structure.

20. Measure molecular mass of purified protein via MALDI-TOF mass spectrometry to confirm size.