Purification of recombinant profilin from E. coli

RDM 4/30/03

Lysis buffer
10 mM Tris-HCl pH 8.0
1 mM EDTA
1 mM PMSF
2 mM DTT

Wash buffer
10 mM Tris-HCl pH 8.0
1 mM EDTA
100 mM NaCl
2 mM DTT

Storage buffer
10 mM Tris-HCl pH 8.0
1 mM EDTA
1 mM DTT (or 0.5 mM TCEP)

N.B. Reducing agents may be omitted when purifying w.t. Acanthamoeba profilin, which has no cysteines.

Day 1:

Transform BL-21 cells with DNA. Plate onto LB Amp and place in 37 C incubator to grow overnight. IMPORTANT: Always start with a fresh transformation. Never use colonies more than 1 day old. The only acceptable shortcut is to transform and plate cells onto LB Amp and then put the plates directly into the 4 C refrigerator. The night before you need cells, transfer a plate to the 37 C incubator. Transformed cells may be stored this way for up to 2 weeks.
Pre warm 1-4 liters of TPM medium by incubating at 37 C in shaker overnight.

Day 2:

Pick about 200 individual colonies to inoculate each 1 liter flask of medium. Resuspend them in about 10 ml of warm TPM by vigorously rotating the inoculating loop. Vortex the tube to break up clumps and then transfer to 1 liter Fernbach flask. Add Amp to TPM to final concentration of 50 µg/ml.

Grow cells at 37 C, and 250 rpms orbital shaking. When cells reach a density (OD at 600 nm) of 0.7 to 0.9, induce expression by adding 0.5 mM IPTG. Grow for 2-3 more hours (actually you can get away with up to 6 hours but 2 hours is optimal).

Harvest cells by spinning for 15 minutes at 4000 RPM in RC3B centrifuge.
Resuspend each liter-worth of cells in 50 ml ice-cold, distilled, deionized water. Wash 1x by centrifugation and discard excess water. Freeze pellet overnight at –20C.

Day 3:

Thaw cell pellet and resuspend cells in lysis buffer. Lightly vortex if necessary to break up clumps.

Lyse cells by passing 2X through microfluidizer.

Spin lysate for 30 minutes at 20,000 rpms, 4 C in SS34 rotor (RC5B centrifuge) and take supernatant.

Pass cleared lysate over poly-L-proline column (see protocol for coupling poly-L-proline to Sepharose 4CL) and wash with 100 ml wash buffer.

Wash pLp column with 100 mls of 4 M urea in wash buffer.

Elute pLp column with 8 M urea in wash buffer.

Dialyze to equilibrium 2-3 X vs. storage buffer.