For several years investigators have examined and characterized some of the surface antigens of the bacterium Neisseria gonorrhoeae, including pili, lipopolysaccharide (LPS), and various proteins of the outer membrane (for review see references 1 and 2). The particular interest in our laboratory has been to understand how all of the components within the gonococcal outer membrane function to the advantage of the organism. In this effort, we have isolated and purified members of the protein I family and found them to be trimeric complexes that form voltage-dependent, anion-specific transmembrane porins (3-5). Furthermore, we have purified several members of the protein II family, one of which confers the opaque phenotype to gonococcal colonies (6) whereas another seems to be implicated in the association of gonococci with polymorphonuclear leukocytes (7). In order to complete our information about the outer membrane proteins and help us understand how these protein components of the gonococcal outer membrane might work in concert, we turned our attention to the last remaining major outer membrane protein family, the reductionmodifiable proteins, or proteins III .Protein III is present in all strains of gonococci examined to date and differs from the other surface-exposed gonococcal outer membrane proteins by its high degree of intrastrain and interstrain homology in molecular weight, structure, and immunology (8, 9). These characteristics make protein III unique among the major outer membrane antigens of the gonococcus, as proteins I and II, as well as LPS and pili, all appear to display a considerable degree of variability either within a strain or between strains (1, 2). Because it has been hypothesized that this variability was in part due to the immunologic pressure of the human host, it was puzzling that gonococci would rigidly conserve such a surface antigen. For this reason it seemed that we should isolate and purify this protein in order to study its biochemical and immunochemical nature in more detail . Thus, this article will present a newly devised method of isolating and purifying protein III and a partial characterization of the structure and immunochemistry of the molecule based on our investigations that use the purified protein . The method used for isolating protein III in large quantities is a simple refinement of the
We have purified protein III (PIII) from several strains of gonococcus by extractions with Zwittergent 3,14 followed by cation exchange chromatography and gel filtration. The pI of 8.6 determined by isoelectric focusing was in keeping with the high content of basic amino acids found. PIII from two strains had identical N-terminal sequence. In contrast to PIII in vivo, purified PIII was highly susceptible to proteolysis. Rabbit antibodies raised with purified antigen reacted with PIII of all strains tested as well as meningococcal protein 4. Furthermore, intact gonococci or meningococci could absorb 80% of antibodies raised by immunization with the purified PIII. The structural gene of PIII was cloned and the DNA sequenced. The predicted primary structure is strongly homologous to the OmpA proteins of Enterobacteria.
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