REAGENTS

• Lysis Buffer: 20 mM Tris-Cl (pH 7.4), 300 mM KCl, 10% glycerin, 1 mM EDTA, 1mM PMSF, 5 ug/mL leupeptin, 2 mM DTT.
• Wash Buffer (A): 20 mM Tris-Cl (pH 7.4), 2 mM CaCl2, 20 mM BME.
• Elution Buffer (B): 20 mM Tris-Cl (pH 7.4), 5 mM EGTA, 20 mM BME.
• Dialysis Buffer (C): 20 mM Tris-Cl (pH 7.4), 20 mM BME.

TRANSFORMATION

pGEMEX vector encoding the human S100A2 gene is transformed into E. coli BL21(DE3)-pLysS competent cells (Stratagene) and grown overnight on LB/(amp+chloramphenicol) plates at 37 °C.

PROTEIN EXPRESSION

Luria Broth (100 mg/ml Amp., 34 mg/ml chloramphenicol)

• Single colony is used to start a 10 ml overnight culture of LB (supplemented with 0.2% glucose to stabilize the plasmid) and grown for 12-14 hrs at 37°C.
• Use the overnight culture to inoculate 500 ml of LB media.
• Allow cells to grow at 37°C till OD600~0.5 before inducing with IPTG to a final concentration of 0.5 mM.
• Harvest the cells after 3-4 hrs by spinning at 8000g for 30 mins.

Minimal Media (100 mg/ml Amp., 34 mg/ml chloramphenicol)

• 50 ml overnight culture of LB (supplemented with 0.2% glucose) from a single colony and grown for 12-14 hrs at 37°C.
• Spin the cells down at 200g and discard the supernatant. To remove the residual LB resuspend cells in 1 ml of M9 media, spin the cells and discard the supernatant. Resuspend the cells in 1 ml M9 media and use the same to inoculate 500 ml of M9 media supplemented with 15 ml 100x basal vitamin solution (Gibco). Use all the cells from the 50mL O/N to inoculate 500mL M9.
• It normally takes 4-5 hrs for the OD to reach a value of 0.7 before inducing with IPTG to a final concentration of 1 mM. Harvest the cells after 3-4 hrs by spinning at 8000g for 30 mins.

PROTEIN PURIFICATION

• Thaw the cells and suspend in 30 ml of lysis buffer (cells from a 2L growth). Cell lysis is initiated by two freeze (-20°C) thaw cycles followed by sonication on ice with 30 sec bursts/15 sec pause for 2-3 mins.
• The lysed cells are spun down at 36000g.
• The supernatant is brought slowly to an ammonium sulfate concentration of 30 % (0.194g/ml) on ice and left overnight at 4°C. Add a few crystals of sodium azide.
• The precipitated proteins are spun down at 36000g and the supernatant brought to 5 mM CaCl2 concentration before applying it to a phenyl sepharose column.

The column was poured and run using a peristaltic pump hooked to a fraction collector.

• The 15 ml column was packed with phenyl sepharose HP media from amersham pharmacia. Before loading the supernatant from precious step it is pre-equilibriated with wash buffer. After loading the sample wash the column thoroughly with buffer A (5 CV). Elute protein with buffer B (5 CV). At this point the protein is fairly pure except for some very high molecular weight impurities. Dialyze (MWCO of 3500) the eluant extensively against buffer C at 4°C.
• Bring the protein up to 30% ammonium sulfate concentration. Spin the solution at 36000g for 40 mins. Discard any precipitate and bring the supernatant up to 5 mM CaCl2 and repeat previous step. This second step helps to get rid of any remaining impurities.

Average yield of purified protein is 20-30mgs/L.