Purification and characterization of the major iron-regulated protein expressed by pathogenic Neisseriae.
This report describes a method to purify the major iron-regulated protein (MIRP) expressed by N. gonorrhoeae and N. meningitidis. This purification procedure involves maximal expression of the MIRP by growing the organisms on iron-limited media; cellular disruption by sonication followed by centrifugal fractionation; selective solubilization of the MIRP with the cationic detergent hexadecyltrimethylammonium bromide; cation-exchange chromatography in the presence of this detergent; and gel filtration chromatography. The MIRP purified by this technique migrates as a single band when analyzed by SDS-PAGE. The purified MIRP displayed an unusually basic isoelectric point, this value being greater than 9.35. Further biochemical analysis revealed the highly conserved nature of this protein isolated from the two pathogenic species of the genus Neisseria. For example, the amino acid composition of the meningococcal and gonococcal MIRPs were nearly identical and amino terminal sequence analysis showed that both shared the identical primary sequence through residue 48. Surprisingly, the first five NH2-terminal residues of the MIRPs exhibited homology with the first five residues of the gonococcal porin, protein I. Purified preparations of the MIRP exhibited a characteristic pink color reminiscent of the basic iron-binding protein lactoferrin. This observation coupled with the property of iron-regulation prompted us to analyze purified MIRP for iron-content. Approximately 0.5 mol iron per 1 mol of MIRP was detected. This study is the first to show that iron is associated with the MIRP, a property that may implicate this protein as playing a direct role in neisserial iron assimilation. While the precise function of the MIRP is not known, the availability of this protein in pure and biologically relevant quantities will allow further studies to elucidate its pathobiologic function.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002458:Cell Fractionation; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents; D007501:Iron / Q000378:metabolism*; D033862:Iron-Binding Proteins; D007525:Isoelectric Focusing; D009343:Neisseria / Q000032:analysis* / Q000378:metabolism; D033902:Periplasmic Binding Proteins
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002593:Cetrimonium Compounds; D003902:Detergents; D033862:Iron-Binding Proteins; D033902:Periplasmic Binding Proteins; D007501:Iron; D000077286:Cetrimonium