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High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies

Title data

Berkner, Hanna ; Engelhorn, Julia ; Liscombe, David K. ; Schweimer, Kristian ; Wöhrl, Birgitta M. ; Facchini, Peter J. ; Rösch, Paul ; Matečko, Irena:
High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies.
In: Protein Expression and Purification. Vol. 56 (December 2007) Issue 2 . - pp. 197-204.
ISSN 1046-5928
DOI: https://doi.org/10.1016/j.pep.2007.07.010

Abstract in another language

The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Chair Biopolymers - Univ.-Prof. Dr. Paul Rösch
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers > Lehrstuhl Biopolymere - Apl. Prof. Dr. Birgitta Wöhrl
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 29 Jan 2019 10:40
Last Modified: 15 May 2019 08:32
URI: https://eref.uni-bayreuth.de/id/eprint/47103