TY - JOUR
T1 - High-level expression of human liver monoamine oxidase A in Pichia pastoris
T2 - Comparison with the enzyme expressed in Saccharomyces cerevisiae
AU - Li, Min
AU - Hubálek, František
AU - Newton-Vinson, Paige
AU - Edmondson, Dale E.
N1 - Funding Information:
1This work was supported by Grant GM-29433 from the National Institutes of Health (to D.E.E).
PY - 2002
Y1 - 2002
N2 - 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 8α-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-CF3-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.
AB - 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 8α-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-CF3-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.
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U2 - 10.1006/prep.2001.1546
DO - 10.1006/prep.2001.1546
M3 - Article
C2 - 11812236
AN - SCOPUS:0035996741
SN - 1046-5928
VL - 24
SP - 152
EP - 162
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -