title: Evaluation of cation exchange chromatography for the isolation of M glycoprotein from histoplasmin.
abstract: Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.
authors.affiliation: Laboratório de Micologia, Hospital Evandro Chagas, Fundaçao Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: R M
authors.initials: RM
authors.lastname: Zancopé-Oliveira
authors.forename: S L
authors.initials: SL
authors.lastname: Bragg
authors.forename: S F
authors.initials: SF
authors.lastname: Hurst
authors.forename: J M
authors.initials: JM
authors.lastname: Peralta
authors.forename: E
authors.initials: E
authors.lastname: Reiss
mesh_terms: D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification*; D006658:Histoplasma / Q000276:immunology; D006659:Histoplasmin / Q000737:chemistry* / Q000276:immunology
chemical_list: D000939:Epitopes; D006023:Glycoproteins; D006659:Histoplasmin