title: Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor.
abstract: Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.
authors.affiliation: Department of Immunopathology, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. e.debruin@sanquin.nl
authors.forename: Eric C
authors.initials: EC
authors.lastname: de Bruin
authors.forename: Dorina
authors.initials: D
authors.lastname: Roem
authors.forename: Ingrid
authors.initials: I
authors.lastname: Bulder
authors.forename: Miranda
authors.initials: M
authors.lastname: Dieker
authors.forename: Gerard
authors.initials: G
authors.lastname: Voerman
authors.forename: C Erik
authors.initials: CE
authors.lastname: Hack
mesh_terms: D000818:Animals; D007447:Invertebrate Hormones / Q000096:biosynthesis* / Q000235:genetics; D007865:Leeches / Q000737:chemistry*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D007447:Invertebrate Hormones; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C053155:antistasin