Wang Xinying, Inaoka Daniel Ken, Shiba Tomoo, Balogun Emmanuel Oluwadare, Allmann Stefan, Watanabe Yoh-Ichi, Boshart Michael, Kita Kiyoshi, Harada Shigeharu
Department of Biomedical Chemistry, School of International Health, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8523, Japan.
Department of Biomedical Chemistry, School of International Health, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8523, Japan.
Protein Expr Purif. 2017 Oct;138:56-62. doi: 10.1016/j.pep.2017.06.011. Epub 2017 Jun 20.
Isocitrate dehydrogenases (IDHs) are metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate. Depending on the electron acceptor and subcellular localization, these enzymes are classified as NADP-dependent IDH1 in the cytosol or peroxisomes, NADP-dependent IDH2 and NAD-dependent IDH3 in mitochondria. Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and Nagana disease in animals. Here, for the first time, a putative glycosomal T. brucei type 1 IDH (TbIDH1) was expressed in Escherichia coli and purified for crystallographic study. Surprisingly, the putative NADP-dependent TbIDH1 has higher activity with NAD compared with NADP as electron acceptor, a unique characteristic among known eukaryotic IDHs which encouraged us to crystallize TbIDH1 for future biochemical and structural studies. Methods of expression and purification of large amounts of recombinant TbIDH1 with improved solubility to facilitate protein crystallization are presented.
异柠檬酸脱氢酶（IDHs）是一类代谢酶，催化异柠檬酸氧化脱羧生成α-酮戊二酸。根据电子受体和亚细胞定位，这些酶可分为胞质溶胶或过氧化物酶体中的NADP依赖性IDH1、线粒体中的NADP依赖性IDH2和NAD依赖性IDH3。布氏锥虫是一种原生动物寄生虫，可导致人类患非洲昏睡病和动物患那加那病。在此，首次在大肠杆菌中表达了一种推测的布氏锥虫1型糖体IDH（TbIDH1）并进行了纯化，用于晶体学研究。令人惊讶的是，推测的NADP依赖性TbIDH1以NAD作为电子受体时比以NADP时具有更高的活性，这是已知真核IDHs中的一个独特特征，促使我们对TbIDH1进行结晶，以便未来进行生化和结构研究。本文介绍了大量具有改善溶解性以促进蛋白质结晶的重组TbIDH 的表达和纯化方法。
