Stefan B. Schmidbauer and Oliver K. Strobel

Boehringer Mannheim GmbH, Penzberg, Germany

4. RESULTS AND DISCUSSION

The column was charged with Zn²⁺ as described earlier and 1.8 mg (200 µl) of crude protein extract applied to it. As shown in the chromatogram in Figure 1, a large majority of the injected sample's components was not retained and eluted in 100% buffer A. Such conditions are very desirable since the bulk of the column binding capacity is reserved for binding the molecules of interest. When the stringency of the eluting buffer was increased (50 mM imidazole), several components were eluted. These probably represent proteins that naturally contain a few histidines or other amino acids on their surfaces that interact weakly with the separation media. These proteins are readily separated from the recombinant protein since the hexa-histidine tag binds much more tightly to the chromatographic media. Finally, upon increasing the imidazole concentration to 500 mM, a single chromatographic peak was observed. The whole separation process was complete in less than 5 min.

The peaks were collected and analyzed using denaturing polyacrylamide gel electrophoresis. The results of this analysis are shown in Figure 2. The starting material contained numerous bands. Peaks that eluted before the application of 50 mM imidazole did not show the characteristic band for recombinant GST fusion protein (31 kD, results not shown). The peak that eluted following application of 500 mM imidazole produced only a single band (Figure 2, lane 1) with the expected molecular weight. The total pooled protein content of this peak (0.025 mg) indicates that a 72-fold purification was achieved in the 5-minute separation.

Figure 2. SDS electrophoresis of POROS MC-purified fusion protein and crude bacterial lysate. Lane 1: POROS MC-purified fraction; Lane 2: crude bacterial cell lysate; Lane 3: molecular weight marker.