based on the Periodic Acid-Schiff (PAS) staining method (Zacharius et al. 1969). No glycosylated proteins were detected in the polyhedral organelle preparation (20 [Lg). Two-Dimensional Electrophoretic Analysis of Purified Polyhedral Organelles

      Samples of purified polyhedral organelles were separated by 2D-IEF-SDS-PAGE. Seventeen major (1-12, 16, 18, and 20-22) and five minor protein spots (13-15, 17 and 19) were observed (Figure 4-5). Each protein present in Figure 4-5 was reproducibly observed following 2D-electrophoresis of three different organelle preparations. N-Terminal Sequencing

      Subsequent to 2D electrophoresis, the identities of the most prevalent protein spots were assigned by Edman sequential N-terminal degradation followed by sequence similarity searching against the S. enterica genome (Table 4-3) and/or peptide mass fingerprinting via MALDI-TOF MS (Table 4-4) with subsequent searching of the Genpept, SwissProt, and NCBI-nr databases using MS-Fit and the NCBI-nr database using Profound. N-terminal sequencing was carried out on the protein spots 1-8 (Figure 4-4). The results are summarized in Table 4-3. Spots 1, 6, 7, and 8 were identified as the PduC, PduE, PduA and PduJ proteins, respectively, confirming the results of the western blotting experiments described above. The N-terminal sequence of protein spots 2 and 5 identified these proteins as PduG and PduB, respectively. Surprisingly, spots 4 and 5 corresponded to the PduB protein and a shorter version of this same protein (PduB') that lacked 37 N-terminal amino acids. Examination of the DNA sequence of thepduB gene revealed potential start sites for each protein with an appropriate ATG and ShineDalgarno sequence; thus it appears that pduB and pduB' represent overlapping genes although at this time post-translational proteolytic processing remains a possibility. In addition, both of these proteins appear to have the methionine cleaved after translation.