title: Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis.
abstract: Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein.
authors.affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic; Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Alan
authors.initials: A
authors.lastname: Kadek
authors.affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic; Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Vyacheslav
authors.initials: V
authors.lastname: Tretyachenko
authors.affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Hynek
authors.initials: H
authors.lastname: Mrazek
authors.affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic; Faculty of Science, Charles University in Prague, Prague, Czech Republic.
authors.forename: Ljubina
authors.initials: L
authors.lastname: Ivanova
authors.affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Halada
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Martial
authors.initials: M
authors.lastname: Rey
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
authors.affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic; Faculty of Science, Charles University in Prague, Prague, Czech Republic. Electronic address: pman@biomed.cas.cz.
authors.forename: Petr
authors.initials: P
authors.lastname: Man
mesh_terms: D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D060435:Carnivory; D004220:Disulfides; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D019684:Magnoliopsida / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019163:Reducing Agents; D013696:Temperature
chemical_list: D004220:Disulfides; D010940:Plant Proteins; D011994:Recombinant Proteins; D019163:Reducing Agents; D057055:Aspartic Acid Proteases
keywords: Carnivorous plant; Nepenthesin; Plant aspartic protease; Protease characterization; Protein stability; Recombinant protein