Expression of zebrafish (Danio rerio) monoamine oxidase (MAO) in Pichia pastoris: Purification and comparison with human MAO A and MAO B

Betül Kacar Arslan, Dale E. Edmondson

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

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 ∼200 mg of zMAO exhibiting 300 U 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 Mr of ∼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 8α-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 °C. Benzylamine is oxidized with a kcat value of 4.7 ± 0.1 min-1 (Km = 82 ± 9 μM) and the enzyme oxidizes phenylethylamine with a kcat value of 204 min-1 (Km = 86 ± 13 μM). The Km (O2) values determined for zMAO using either benzylamine or phenylethylamine as substrates ranges from 108(±5) to 140(±21) μM. The functional behavior of this teleost MAO relative to human MAO A and MAO B is discussed.

Original languageEnglish (US)
Pages (from-to)290-297
Number of pages8
JournalProtein Expression and Purification
Volume70
Issue number2
DOIs
StatePublished - Apr 2010

Keywords

  • Covalent flavin cofactor
  • Danio rerio
  • Monoamine oxidase
  • Pichia pastoris
  • Zebrafish

ASJC Scopus subject areas

  • Biotechnology

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