High-level expression of human liver monoamine oxidase A in Pichia pastoris: comparison with the enzyme expressed in Saccharomyces cerevisiae.
The high-level expression, purification, and characterization of recombinant membrane-bound human liver monoamine oxidase A (MAO-A) in Pichia pastoris is described. Two liters of fermentation culture produces 1170 units (660 mg) of MAO-A. The enzyme is purified in a 35% yield, is homogeneous on denaturing gel electrophoresis, and exhibits a single species (60,512 +/- 6 Da) on electrospray mass spectrometry. It contains 1 mol of 8alpha-S-cysteinyl FAD/mole of enzyme and exhibits >95% functionality. In contrast, the Saccharomyces cerevisiae-expressed enzyme is partially processed by C-terminal serine removal as demonstrated by mass spectra. The amino termini of both P. pastoris- and S. cerevisiae-expressed MAO-A are acetylated on the N-terminal methionine. The steady-state kinetic properties of P. pastoris-expressed MAO-A are similar to those of S. cerevisiae-expressed MAO-A using the following substrates: phenethylamine, p-CF(3)-benzylamine, dopamine, serotonin, and kynuramine. Reductive titrations demonstrate that the recombinant enzyme is reduced by 1 mol of substrate or dithionite as expected for the two electron equivalents required for flavin reduction. Absorption and EPR spectra show no radical species in the resting enzyme while the anionic flavin radical is formed in 50% yield during the reductive titration with dithionite. These data demonstrate significant advantages in the heterologous expression of human MAO-A in P. pastoris compared with the published S. cerevisiae system in higher expression level (329 mg/L) and in a higher level of homogeneity of the isolated enzyme.
Li
Min
M
Department of Biochemistry, Emory University, Atlanta, Georgia 30322-3050, USA.
Hubálek
Frantisĕk
F
Newton-Vinson
Paige
P
Edmondson
Dale E
DE
D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D018740:Genetic Heterogeneity; D006801:Humans; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D008995:Monoamine Oxidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature
D008995:Monoamine Oxidase