Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis

Autor: Petr Halada, Vyacheslav Tretyachenko, Martial Rey, Ljubina Ivanova, Alan Kadek, Petr Man, Hynek Mrázek, David C. Schriemer
Přispěvatelé: Institute of Microbiology of the ASCR, v. v. i. [Prague, Czech Republic], Department of Biochemistry and Molecular Biology (BMB)
Jazyk: angličtina
Rok vydání: 2014
Předmět:
MESH: Enzyme Stability
MESH: Hydrogen-Ion Concentration
medicine.medical_treatment
Carnivorous plant
medicine.disease_cause
01 natural sciences
law.invention
MESH: Recombinant Proteins
law
Enzyme Stability
Protein stability
Disulfides
Plant Proteins
chemistry.chemical_classification
0303 health sciences
MESH: Plant Proteins
Temperature
Hydrogen-Ion Concentration
Carnivory
Recombinant Proteins
Nepenthesin
MESH: Temperature
Biochemistry
Reducing Agents
Protease characterization
Recombinant DNA
Biotechnology
Proteases
Aspartic Acid Proteases
Recombinant protein
MESH: Reducing Agents
Biology
MESH: Carnivory
Magnoliopsida
03 medical and health sciences
Plant aspartic protease
medicine
MESH: Disulfides
[SDV.BBM]Life Sciences [q-bio]/Biochemistry
Molecular Biology
MESH: Aspartic Acid Proteases
Escherichia coli
030304 developmental biology
MESH: Magnoliopsida
Protease
010401 analytical chemistry
biology.organism_classification
0104 chemical sciences
Enzyme
Nepenthes gracilis
chemistry
Zdroj: Protein Expression and Purification
Protein Expression and Purification, Elsevier, 2014, 95, pp.121-128. ⟨10.1016/j.pep.2013.12.005⟩
ISSN: 1046-5928
1096-0279
DOI: 10.1016/j.pep.2013.12.005⟩
Popis: International audience; 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.
Databáze: OpenAIRE
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