Over-expression in Escherichia coli, functional characterization and refolding of rat dimethylglycine dehydrogenase
Over-expression in Escherichia coli, functional characterization and refolding of rat dimethylglycine dehydrogenase
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DOI：
10.1016/j.pep.2004.06.011
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发表时间：
2004-10-01
影响因子：
1.6
通讯作者：
Barile, M
中科院分区：
文献类型：
--
作者：
Brizio, C;Brandsch, R;Barile, M
二甲基甘氨酸脱氢酶 (Me(2)GlyDH) 是一种线粒体酶，可催化二甲基甘氨酸氧化去甲基化为肌氨酸。该酶需要黄素腺嘌呤二核苷酸 (FAD)，它通过组氨酰 (N3)-(8α)FAD 连接与脱辅基蛋白共价结合。在本研究中，大鼠 Me(2)GlyDH 的成熟形式已在大肠杆菌中作为 N 末端 6-His 标记的融合蛋白过表达。过表达的蛋白质在可溶性和不溶性（包涵体）细胞部分之间几乎均匀分布。通过将可溶性细胞裂解物应用到镍螯合柱上，洗脱了两个级分，在 SDS-PAGE 上，两者均含有分子量为 93 kDa 的几乎均质的蛋白质。第一个蛋白质级分通过蛋白质印迹分析鉴定为共价黄素化的 Me(2)GlyDH。它显示出与天然全酶相似的光学特性和比活性（240 nmol/min/mg 蛋白质）。第二部分被鉴定为 Me2GlyDH 的低黄素化 (apo-) 形式，比活性低 70%。重组全酶在 pH 8.5 时表现出最佳活性，活化能约为 80 kJ/mol，N,N-二甲基甘氨酸有两个 K-M 值（K-M1 = 0.05 mM 和 K-M2 = 9.4 mM），如天然全酶所述。从包涵体开始，通过 SDS 处理溶解未折叠的黄素化酶，并通过 80 倍稀释步骤重新折叠，重新激活率为 50-60%。 (C) 2004 Elsevier Inc. 保留所有权利。
Dimethylglycine dehydrogenase (Me(2)GlyDH) is a mitochondrial enzyme that catalyzes the oxidative demethylation of dimethylglycine to sarcosine. The enzyme requires flavin adenine dinucleotide (FAD), which is covalently bound to the apoprotein via a histidyl(N3)-(8alpha)FAD linkage. In the present study, the mature form of rat Me(2)GlyDH has been over-expressed in Escherichia coli as an N-terminally 6-His-tagged fusion protein. The over-expressed protein distributed almost equally between the soluble and insoluble (inclusion bodies) cell fraction. By applying the soluble cell lysate to a nickel-chelating column, two fractions were eluted, both containing a nearly homogeneous protein with a molecular mass of 93 kDa, on SDS-PAGE. The first protein fraction was identified by Western blotting analysis as the covalently flavinylated Me(2)GlyDH. It showed optical properties and specific activity (240 nmol/min/mg protein) similar to those of the native holoenzyme. The second fraction was identified as an underflavinylated (apo-) form of Me2GIyDH, with a 70% lower specific activity. The recombinant holoenzyme exhibited optimal activity at pH 8.5, an activation energy of about 80 kJ/mol, and two K-M values for N,N-dimethylglycine (K-M1 = 0.05 mM and K-M2 = 9.4 mM), as described for the native holoenzyme. Starting from the inclusion bodies, the unfolded flavinylated enzyme was solubilized by SDS treatment and refolded by an 80-fold dilution step, with a reactivation yield of 50-60%. (C) 2004 Elsevier Inc. All rights reserved.