Secretion of active human mucus proteinase inhibitor by Aspergillus niger after KEX2-like processing of a glucoamylase-inhibitor fusion protein.
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.
Mikosch
T
T
Institut für Biochemie, Technische Hochschule Darmstadt, Germany.
Klemm
P
P
Gassen
H G
HG
van den Hondel
C A
CA
Kemme
M
M
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
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