Departamento de Microbiología y Parasitología y, Universidad Nacional Autónoma de Mexico, Facultad de Medicina, D.F., Mexico.
Exp Parasitol. 2011 Jul;128(3):217-24. doi: 10.1016/j.exppara.2011.03.008. Epub 2011 Mar 31.
We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K(cat) values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56Kb genomic contig assembly is also reported.
我们在此报告猪带绦虫细胞质苹果酸脱氢酶（TscMDH）的完整编码序列。该 cDNA 片段是从猪带绦虫基因组计划数据库中鉴定出来的，编码一个由 332 个氨基酸残基组成的蛋白质，估计分子量为 36517Da。为了进行重组表达，全长编码序列被克隆到 pET23a 中。成功表达和酶纯化后，等电聚焦凝胶电泳证实了从氨基酸序列推断出的计算等电点为 8.1。重组蛋白（r-TscMDH）在还原草酰乙酸时表现出 409U/mg 的 MDH 活性，既没有乳酸脱氢酶活性，也没有 NADPH 选择性。酶活性的最佳 pH 值为 oxaloacetate 还原的 7.6 和 malate 氧化的 9.6。oxaloacetate、malate、NAD 和 NADH 的 k(cat) 值分别为 665、47、385 和 962s(-1)。此外，还报告了分析 1.56Kb 基因组连续序列组装后 TsMDH 基因结构的部分特征。
