title: Secretion of active human mucus proteinase inhibitor by Aspergillus niger after KEX2-like processing of a glucoamylase-inhibitor fusion protein.
abstract: We report the production of human mucus proteinase inhibitor (MPI) by the filamentous fungus Aspergillus niger to test the ability of this host organism to secrete low molecular weight, highly disulfide-bonded proteins in biologically active conformation. Fungal transformants have been obtained with an expression cassette consisting of a chimeric gene founded on a mpi cDNA, encoding mature MPI, fused in frame to sequences encoding A. niger glucoamylase (glaA), separated by a KEX2-like processing sequence. Expression of the glucoamylase fusion gene in these transformants resulted in secretion of MPI into the growth medium with yields up to 3 mg 1-1. N-terminal sequence analysis of the purified inhibitor confirmed that the glucoamylase-MPI fusion protein was correctly processed to mature MPI by a KEX2-type endopeptidase present in A. niger. Furthermore, recombinant MPI retains full inhibitory activity against chymotrypsin and leukocyte elastase indicating that the protein was folded properly.
authors.affiliation: Institut für Biochemie, Technische Hochschule Darmstadt, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Mikosch
authors.forename: P
authors.initials: P
authors.lastname: Klemm
authors.forename: H G
authors.initials: HG
authors.lastname: Gassen
authors.forename: C A
authors.initials: CA
authors.lastname: van den Hondel
authors.forename: M
authors.initials: M
authors.lastname: Kemme
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003470:Culture Media; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D043484:Proprotein Convertases; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D003470:Culture Media; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005087:Glucan 1,4-alpha-Glucosidase; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae