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): 25 mM Tris-Cl (pH 7.4), 300 mM NaCl, 10 mM BME, 1mM PMSF, 5 mM Imidazole.
• Elution Buffer (B): 25 mM Tris-Cl (pH 7.4), 300 mM NaCl, 10 mM BME, 1mM PMSF, 150 mM Imidazole.

TRANSFORMATION

pH6 TEV vector encoding the bovine S100B gene is transformed into E. coli BL21 Codon Plus (DE3) RIL competent cells (Stratagene) and grown overnight on LB/(Kanamycin) plates at 37°C (The his-TEV tag has the following sequence MGSSHHHHHHENLYFQGA).

PROTEIN EXPRESSION

Luria Broth (15 mg/ml Kanamycin)

• 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 (15 mg/ml Kanamycin)

• 50 ml overnight culture of LB (supplemented with 0.2% glucose) induced 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 5 ml 100x basal vitamin solution (Gibco). Use all the cells from the 50mL O/N to inoculate 500mL M9.
• It normally takes 3-4 hrs for the OD600 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.

All the following steps are done on the ÄKTA.

• The supernatant is brought upto 5 mM imidazole and loaded onto a Ni-NTA column (20 ml of resin) pre-equilibriated with buffer A and washed extensively with the same (5 CV). Check pH (~7.5) of supernatant before loading on column.
• Elute protein with 5 to 300 mM imidazole gradient of Buffer B over 10 CV. The protein is almost 95 % pure after first Ni column. Dialyze the eluant extensively against buffer A.
• Repeat last two steps.
• The his-tag can be cleaved off by the TEV protease but requires unreasonable amounts of the protease and so the tag is left on.

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