Expression of Zebrafish (Danio rerio) Monoamine Oxidase (MAO) in Pichia pastoris: Purification and Comparison with Human MAO A and MAO B

摘要

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 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 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-ScysteinylFAD. 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 ± 13μM). 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.