José Godinho da Silva Junior

Complete amino acid sequence and location of Omp-28, an important immunogenic protein from Salmonella enterica serovar typhi

Omp-28 isolated from Salmonella enterica serovar typhi presented a subunit molecular mass of 9,632 Da by MALDI-TOF MS. It was denatured, S-alkylated, and 1) directly submitted to Edman sequencing, 2) cleaved with CNBr, and 3) hydrolyzed either with endoproteinase Glu-C or Asp-N. The major CNBr peptide containing the C-terminal portion of Omp-28 was isolated by tricine-SDS-PAGE and electroblotted whereas Omp-28 enzymatic peptides were isolated by C18-RP-HPLC. All peptides were sequenced. This approach allowed the elucidation of the complete primary structure of Omp-28. Its amino acid sequence is identical to that deduced from part of the DNA of the “putative periplasmic transport protein” of either S. enterica serovar typhimurium and a multiple drug resistant S. enterica serovar typhi. Omp-28 homologous protein sequences were also deduced from Escherichia coli and Yersinia pestis genomic DNA. All proteins had their secondary structures predicted. Immunogold cytochemistry indicated that Omp-28 is found on the bacterium outer membrane.

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp‐28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp‐28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp‐28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N‐terminal amino acid sequence of Omp‐28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c‐DNA. ELISA and Western blotting identified Omp‐28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp‐28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp‐28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp‐28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.