Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry

0457205 - MBÚ 2016 RIV US eng J - Článek v odborném periodiku
Yang, M. - Hoeppner, M. - Rey, M. - Kádek, Alan - Man, Petr - Schriemer, D. C.
Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry.
Analytical Chemistry. Roč. 87, č. 13 (2015), s. 6681-6687. ISSN 0003-2700. E-ISSN 1520-6882
Grant ostatní: OPPC(XE) CZ.2.16/3.1.00/24023
Institucionální podpora: RVO:61388971
Klíčová slova: ASPARTIC PROTEASE NEPENTHESIN-1 * UNIQUE MEMBER * STABILITY
Kód oboru RIV: CB - Analytická chemie, separace
Impakt faktor: 5.886, rok: 2015

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 FIX-MS applications. We produced and characterized recombinant nepenthesin II to determine if it complemented nepenthesin I in FIX-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.
Trvalý link: http://hdl.handle.net/11104/0257590