REAGENTS

• Lysis Buffer: 50 mM Tris, 5 mM CaCl2, 1 mM PMSF, pH 7.5.
• Wash Buffer (A): 50 mM Tris, 5 mM CaCl2, pH 7.5.
• Wash Buffer (B): 600 mM NaCl, 50 mM Tris, 5 mM CaCl2, pH 7.5.
• Elution Buffer (B): 20 mM Tris-Cl (pH 7.4), 5 mM EGTA

(In case of mouse and human calcyclin there are free cysteines that require the use of DTT/BME in all the buffers)

TRANSFORMATION

pET-R1120 vector encoding the S100A6 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 chloramophenicol)

• Single colony is used to start a 5 ml overnight culture of LB (supplemented with 0.2% glucose to stabilize the plasmid) and grown for 12-14 hrs at 37°C.
• Use 5ml overnight culture to inoculate 500 ml of LB media.
• Since the expression is leaky the cells are allowed to grow at 37°C for 24 hrs without inducing with IPTG.
• Harvest the cells by spinning at 8000g for 30 mins.

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

• 5 ml overnight culture from single colony of M9 minimal media was grown for 15 hrs at 37°C. Makesure the solution is saturated.
• This culture is used to inoculate 500 ml of M9 media and allowed to grow till OD600 reaches a value of 0.7 (3-5 hrs) before inducing with IPTG to a final concentration of 0.01 mM.
• Harvest the cells after 24 hrs by spinning at 8000g for 30 mins.

PROTEIN PURIFICATION

• Thaw the cells (1L growth) and suspend in 30 ml of lysis buffer. 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 80 % (0.516g/ml) on ice and left with stirring for 2-3 hrs.
• Spin the solution at 36000g for 1 hr.

The column was packed and run using a peristaltic pump hooked to a fraction collector. However it can be improvised for runs on the ÄKTA.

• The ammonium sulfate pellet was resuspended in about 10-20 ml of buffer A. Spin down any precipitate before loading the supernatant onto a phenyl sepharose column. (15 ml column poured with Pharmacia HP phenyl sepharose).
• Wash the column with a low salt buffer B (5 CV).
• Elute the protein with EGTA gradient of buffer C (5 CV).
• Dialyze the protein extensively against buffer A (MWCO 3500) at 4°C. Load onto a phenyl sepharose column and repeat steps (vi) and (vii).

Average yield of purified protein is 10-20 mgs/L.

Please note comparable yields of S100A6 are obtained if a procedure identical to S100A2 is followed for protein expression and purification in minimal media.