Open AccessRecombinant proteinase 3 (Wegener's antigen) expressed in Pichia pastoris is functionally active and is recognized by patient sera
Author(s) -
HARMSEN M. C.,
HEERINGA P.,
GELD Y. M.,
HUITEMA M. G.,
KLIMP A.,
TIRAN A.,
KALLENBERG C. G. M.
Publication year - 1997
Publication title -
clinical & experimental immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.329
H-Index - 135
eISSN - 1365-2249
pISSN - 0009-9104
DOI - 10.1111/j.1365-2249.1997.tb08325.x
Subject(s) - pichia pastoris , recombinant dna , proteinase 3 , immunology , antigen , pichia , biology , virology , autoantibody , antibody , biochemistry , gene
Summary 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.