title: Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry.
abstract: The pitcher secretions of the Nepenthes genus of carnivorous plants contain a proteolytic activity that is very useful for hydrogen/deuterium exchange mass spectrometry (HX-MS). Our efforts to reconstitute pitcher fluid activity using recombinant nepenthesin I (one of two known aspartic proteases in the fluid) revealed a partial cleavage profile and reduced enzymatic stability in certain HX-MS applications. We produced and characterized recombinant nepenthesin II to determine if it complemented nepenthesin I in HX-MS applications. Nepenthesin II shares many properties with nepenthesin I, such as fast digestion at reduced temperature and pH, and broad cleavage specificity, but in addition, it cleaves C-terminal to tryptophan. Neither enzyme reproduces the C-terminal proline cleavage we observed in the natural extract. Nepenthesin II is considerably more resistant to chemical denaturants and reducing agents than nepenthesin I, and it possesses a stability profile that is similar to that of pepsin. Higher stability combined with the slightly broader cleavage specificity makes nepenthesin II a useful alternative to pepsin and a more complete replacement for pitcher fluid in HX-MS applications.
authors.forename: Menglin
authors.initials: M
authors.lastname: Yang
authors.forename: Morgan
authors.initials: M
authors.lastname: Hoeppner
authors.forename: Martial
authors.initials: M
authors.lastname: Rey
authors.affiliation: ‡Institute of Microbiology, Academy of Sciences of the Czech Republic, 117 20 Prague, Czech Republic.|§Department of Biochemistry, Faculty of Science, Charles University in Prague, 116 36 Prague, Czech Republic.
authors.forename: Alan
authors.initials: A
authors.lastname: Kadek
authors.affiliation: ‡Institute of Microbiology, Academy of Sciences of the Czech Republic, 117 20 Prague, Czech Republic.|§Department of Biochemistry, Faculty of Science, Charles University in Prague, 116 36 Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
mesh_terms: D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D003903:Deuterium