Stefan B. Schmidbauer and Oliver K. Strobel

Boehringer Mannheim GmbH, Penzberg, Germany

3. MATERIALS AND METHODS

Reagents

Water used in buffer preparation was deionized and free of organic impurities. All buffers and salts were analytical grade or higher, and solutions were degassed and filtered (0.45 µm) prior to use.

Determination of protein concentration

Protein concentration of the cell lysate and the purified fusion protein was determined spectrophotometrically using the Protein Assay ESL (Boehringer Mannheim) as described in the accompanying pack insert.

Preparation of bacterial lysate

Genetically engineered E. coli cells which express the fusion protein GST(His)₆, a kind gift from Dr. Thomas Emrich (Boehringer Mannheim), were suspended in 150 mM NaCl, 50 mM sodium phosphate, pH 6.8 buffer, and lysed with a French press. The lysate was then centrifuged to remove cell debris prior to the chromatographic separation. Total protein content in the starting sample was 9.0 mg/ml.

Column preparation

The column size, bed volume, and flow rate are given in Figure 1. The POROS MC/M column (Boehringer Mannheim) was prepared as described in the pack insert. Briefly, the column was cleaned with 30 column bed volumes (CV) of 50 mM EDTA in 1 M NaCl, washed with 10 CV of H₂0, and finally charged with 30 CV of 50 mM ZnCI₂. Excess metal ions were washed out with 10 CV H₂0, followed by 10 CV 0.2 M NaCl. The column was then equilibrated with 10 CV of starting buffer (see Figure 1).

Figure 1.Purification of GST-(His)₆ on POROS MC/M, 4.6 mm x 100 mm (Column volume = 1.8 ml). Sample: 200 µl centrifuged bacterial lysate containing rec. GST-(His)₆; Buffer A: 50 mM potassium phosphate, 500 mM NaCl, pH 7.8; Buffer B: buffer A + 500 mM imidazole, pH 7.8; Flow rate: 2500 cm/h (7 ml/min); Gradient: 0% B for 5 CV, 0-10% B in 1 CV, 10% B for 7 CV, 10-100% B in 2 CV, 100% B for 10 CV.

If Ni²⁺ is used for charging the column, caution must be exercised since the metal is a known allergen and several nickel salts are carcinogenic.