title: Recombinant proteinase 3 (Wegener's antigen) expressed in Pichia pastoris is functionally active and is recognized by patient sera.
abstract: The open reading frame of human proteinase 3 (PR3) without the prepro-peptide was cloned and expressed in Escherichia coli (rcPR3) and in Pichia pastoris (rpPR3). The 6-histidine tagged rpPR3 was efficiently secreted into culture supernatant from which it could be purified by immobilized metal chelate chromatography. Purified rpPR3 migrated as a single 32-kD band on SDS-PAGE and harboured protease activity that could be inhibited with inhibitors specific for serine-proteases. By indirect antigen-capture ELISA using rpPR3, 60% of sera from patients with Wegener's granulomatosis bound to the recombinant product, although it was not recognized in ELISA with directly coated rpPR3.
authors.affiliation: Department of Clinical Immunology, University of Groningen, The Netherlands.
authors.forename: M C
authors.initials: MC
authors.lastname: Harmsen
authors.forename: P
authors.initials: P
authors.lastname: Heeringa
authors.forename: Y M
authors.initials: YM
authors.lastname: van der Geld
authors.forename: M G
authors.initials: MG
authors.lastname: Huitema
authors.forename: A
authors.initials: A
authors.lastname: Klimp
authors.forename: A
authors.initials: A
authors.lastname: Tiran
authors.forename: C G
authors.initials: CG
authors.lastname: Kallenberg
mesh_terms: D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D014890:Granulomatosis with Polyangiitis / Q000276:immunology*; D006801:Humans; D053146:Myeloblastin; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053146:Myeloblastin