title: Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.
abstract: The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity.
authors.affiliation: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Cuba. Electronic address: rossana.gf@gmail.com.
authors.forename: Rossana
authors.initials: R
authors.lastname: García-Fernández
authors.affiliation: Institute of Biochemistry and Molecular Biology, Department of Chemistry, Faculty for Mathematics Informatics and Natural Sciences, University of Hamburg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Ziegelmüller
authors.affiliation: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Cuba.
authors.forename: Lidice
authors.initials: L
authors.lastname: González
authors.affiliation: Elanco Animal Health, Greenfield, IN, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Mansur
authors.affiliation: Centro de Inmunología Molecular, La Habana, Cuba.
authors.forename: Yoan
authors.initials: Y
authors.lastname: Machado
authors.affiliation: Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Redecke
authors.affiliation: Institute of Biochemistry and Molecular Biology, Department of Chemistry, Faculty for Mathematics Informatics and Natural Sciences, University of Hamburg, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hahn
authors.affiliation: Institute of Biochemistry and Molecular Biology, Department of Chemistry, Faculty for Mathematics Informatics and Natural Sciences, University of Hamburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
authors.affiliation: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Cuba. Electronic address: mchavez@fbio.uh.cu.
authors.forename: María de Los Ángeles
authors.initials: Mde L
authors.lastname: Chávez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012615:Sea Anemones / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C080476:ShPI proteinase inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin
keywords: BPTI-Kunitz; Enzyme kinetics; Heterologous expression; Pichia pastoris; Sea anemone; Serine protease; Site-directed mutagenesis; Tight-binding inhibition