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

0428463 - MBÚ 2015 RIV US eng J - Journal Article
Kádek, Alan - Tretyachenko, V. - Mrázek, Hynek - Ivanova, Ljubina - Halada, Petr - Rey, M. - Schriemer, D. C. - Man, Petr
Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis.
Protein Expression and Purification. Roč. 95, MAR 2014 (2014), s. 121-128. ISSN 1046-5928. E-ISSN 1096-0279
R&D Projects: GA ČR GAP206/12/0503; GA MŠMT(CZ) EE2.3.30.0003
Institutional support: RVO:61388971
Keywords : Plant aspartic protease * Nepenthesin * Protease characterization
Subject RIV: CE - Biochemistry
Impact factor: 1.695, year: 2014

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 coil. 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
Permanent Link: http://hdl.handle.net/11104/0233839