(LI-COR, Lincoln, NE). BLAST software was used for sequence similarity searching (Altschul et al. 1990).

                                     Results

Purification of Recombinant His6-PduA Protein

     E. coli strain BE230 was constructed to produce high levels of recombinant His6PduA protein. Samples of induced and uninduced boiled cells of this strain were analyzed by SDS-PAGE to determine if the His6-PduA protein was being expressed. When induced by IPTG, strain BE230 produced high levels of an 11.8 kDa protein (Figure 3-1, lane 3), which was not seen in the uninduced sample (Figure 3-1, lane 2). This observed mass correlates well with the predicted mass of 11.4 kDa for the recombinant His6-PduA protein. Electron microscopy revealed that strain BE230 produced rod-like structures in the cytosol when induced with IPTG (Figure 3-2A and B) and SDS-PAGE showed that these inclusion bodies (Figure 3-2C) were composed mainly of the His6-PduA protein (Figure 3-1, lane 5). Significant amounts of the His6-PduA protein were not found in the soluble fraction (Figure 3-1, lane 4).

     The His6-PduA protein was purified from inclusion bodies by Ni2+-chromatography under denaturing conditions. The His6-PduA protein eluted from the column at approximately 160 to 200 mM imidazole. The fractions containing the His6-PduA protein were analyzed by SDS-PAGE. A single band at the predicted mass for the His6PduA protein was observed as well as three faint bands at higher molecular masses (Figure 3-1, lane 6). About 600  tg of the partially purified His6-PduA protein was isolated on a preparatory SDS-PAGE gel, and the band of interest was excised and used as a source of antigen for polyclonal antiserum production.