Expression and preparation of proteins

Expression and purification of Abl Kinase 

The coding sequence of Abl^248-534  and Abl^83-534  were cloned into the pET16b vector for expression as maltose binding protein (MBP)-His₆ fusion protein including a tobacco etch virus (TEV) protease cleavage site. Equimolar amounts of the Abl^248-534 (or Abl^83-534), GroEL(pREP4), and YopH phosphatase(164-468) (pCDFDuet-1) were co-transformed into Escherichia coli BL21(DE3) cells (NEB) and were grown at 37 °C in Luria-Bertani (LB) medium. Protein expression was induced by adding 0.2 mM IPTG at 16 °C when absorbance at 600 nm(A₆₀₀) reached 0.8. Cells were harvested ~24-48 h thereafter at A₆₀₀~1.7-1.9, and re-suspended in lysis buffer (50mM Tris-Hcl, 500mM NaCl, 1mM PMSF, 5 mM BME, pH 8.0). Cells were disrupted using a sonicator or a high-pressure homogenizer and centrifuged at > 18,000 g. The supernatant was further purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare). Preparing the following buffers for Ni Sepharose purification, TEV cleavage and gel filtration:

1. Equilibration buffer (50mM Tris-Hcl, 500mM NaCl, 5 mM BME, pH 8.0)
2. Wash buffer 1 (50mM Tris-Hcl, 1mM NaCl, 5 mM BME, 25 mM imidazole, pH 8.0)
3. Wash buffer 2 (50mM Tris-Hcl, 0.15mM NaCl, 5 mM BME, 25 mM imidazole, pH 8.0)
4. Elution buffer (50mM Tris-Hcl, 0.15mM NaCl, 5 mM BME, 300 mM imidazole, pH 8.0)
5. Dialysis buffer (50mM Tris-Hcl, 0.15mM NaCl, 5 mM BME, pH 8.0)
6. Gel filtration buffer (25 mM sodium phosphate buffer, 75 mM NaCl, 3.0 mM BME, pH 7.1)

First, equilibrate the resin with equilibration buffer;

Second, load the supernatant onto the resin;

Third, wash the resin with wash buffer 1 and wash buffer 2 sequentially;

Fourth, elute the protein with the elution buffer;

Fifth, add TEV to the eluted protein solution, and dialyze against the dialysis buffer 

          for 12-16 hours at 4 °C. 

Sixth, pass the solution through the Ni Sepharose resin again, collect the 

            flow-through.

Seventh, concentrate the flow-through to 4-5 ml, and run gel-filtration.

Eighth, run gel and mass spec.

Note: please keep the protein on ice during the purification!!!

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