PUBLICATION
Expression of Zebrafish (Danio rerio) Monoamine Oxidase (MAO) in Pichia pastoris: Purification and Comparison with Human MAO A and MAO B
- Authors
- Arslan, B.K., and Edmondson, D.E.
- ID
- ZDB-PUB-100119-15
- Date
- 2010
- Source
- Protein Expression and Purification 70(2): 290-297 (Journal)
- Registered Authors
- Keywords
- Pichia pastoris, zebrafish, Danio rerio, monoamine oxidase, covalent flavin cofactor
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Benzylamines/metabolism
- Cloning, Molecular
- Electrophoresis, Polyacrylamide Gel
- Enzyme Stability
- Hot Temperature
- Humans
- Kinetics
- Molecular Sequence Data
- Monoamine Oxidase/biosynthesis*
- Monoamine Oxidase/isolation & purification
- Monoamine Oxidase/metabolism
- Monoamine Oxidase Inhibitors/pharmacology
- Phenethylamines/metabolism
- Pichia/metabolism
- Recombinant Proteins/antagonists & inhibitors
- Recombinant Proteins/metabolism
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Zebrafish
- PubMed
- 20079438 Full text @ Protein Expr. Purif.
Citation
Arslan, B.K., and Edmondson, D.E. (2010) Expression of Zebrafish (Danio rerio) Monoamine Oxidase (MAO) in Pichia pastoris: Purification and Comparison with Human MAO A and MAO B. Protein Expression and Purification. 70(2):290-297.
Abstract
The expression, purification and characterization of zebrafish monoamine oxidase (zMAO) using the methylotropic yeast Pichia pastoris expression system is described. A 1 L fermentation culture of Pichia pastoris containing the gene encoding zMAO under control of the methanol oxidase promotor expresses approximately 200 mg of zMAO exhibiting 300 units of total activity. The enzyme is found in the mitochondrial fraction of the expression host and is purified in a 30% yield as a homogenous species with a M(r) of approximately 60,000 on SDS-PAGE and a mass of 58,525+ 40 Da from MALDI-TOF measurements. The zMAO preparation contains one mole of covalent flavin cofactor per mole of enzyme and exhibits >80% functionality. The covalent flavin exhibits fluorescence and EPR spectral properties consistent with known properties of 8alpha-S-cysteinylFAD. Chemical degradation of the flavin peptide results in the liberation of FAD. zMAO exhibits no immuno-chemical cross-reactivity with polyclonal anti-sera raised against human MAO A. The enzyme preparation exhibits reasonable thermostability up to a temperature of 30(o)C. Benzylamine is oxidized with a k(cat) value of 4.7+0.1 min(-1) (K(m) = 82 + 9 muM) and the enzyme oxidizes phenylethylamine with a k(cat) value of 204 min(-1) (K(m) = 86 + 13muM). The K(m) (O(2)) values determined for zMAO using either benzylamine or phenylethylamine as substrates ranges from 108(+5) to 140(+21) muM. The functional behavior of this teleost MAO relative to human MAO A and MAO B is discussed.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping