High-level expression of human liver monoamine oxidase a in Pichia pastoris:: Comparison with the enzyme expressed in Saccharomyces cerevisiae
High-level expression of human liver monoamine oxidase a in Pichia pastoris:: Comparison with the enzyme expressed in Saccharomyces cerevisiae
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DOI：
10.1006/prep.2001.1546
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发表时间：
2002-02-01
影响因子：
1.6
通讯作者：
Edmondson, DE
中科院分区：
文献类型：
--
作者：
Li, M;Hubálek, F;Edmondson, DE
描述了重组膜结合人肝单胺氧化酶A（MAO-A）在巴斯德毕赤酵母中的高水平表达、纯化和表征。两升发酵培养物产生1170单位（660 mg）的MAO-A。该酶以35%的产率纯化，在变性凝胶电泳上是均一的，并且在电喷雾质谱上显示出单一种类（60，512 +/-6 Da）。它含有1摩尔8 α-S-半胱氨酰FAD/摩尔酶，并表现出>95%的官能度。相比之下，如质谱所示，酵母菌表达的酶通过C-末端丝氨酸去除而部分加工。巴斯德毕赤酵母和S.在N-末端甲硫氨酸上乙酰化表达的MAO-A。毕赤酵母表达的MAO-A的稳态动力学性质与S.使用以下底物表达MAO-A：苯乙胺、p-CF 3-苄胺、多巴胺、血清素和犬尿胺。还原滴定表明，重组酶被1摩尔底物或连二亚硫酸盐还原，如预期的黄素还原所需的两个电子当量。吸收光谱和EPR光谱表明，没有自由基物种的休息酶，而形成的阴离子黄素自由基的产率为50%，在还原滴定与连二亚硫酸盐。这些数据表明，与已发表的S.在酿酒酵母系统中以更高的表达水平（329 mg/L）和更高水平的分离酶的均一性表达。(C)2002 Elsevier Science（美国）。
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-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. (C) 2002 Elsevier Science (USA).