Department of Clinical Biochemistry, Isfahan Pharmaceutical Sciences Research Center, and Bioinformatics Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran.
Protein J. 2020 Aug;39(4):377-382. doi: 10.1007/s10930-020-09898-w.
Lipoxygenases are non-heme iron-containing lipid dioxygenases enzymes that catalyze the hydroperoxidation of lipids. The Mexican axolotl (Ambystoma mexicanum) is a prominent source of the enzyme with a regeneration capacity in limbs. It has been shown that transfected human osteosarcoma and keratinocyte cells with epidermal lipoxygenase (LOXe) have an increased rate of cell migration. In the present study, LOXe, a peripheral membrane protein, was produced in Escherichia coli. The enzyme was purified using different detergents, anionic solutions, and gel filtration chromatography. Kinetic assay of the enzyme activity was carried out by the spectroscopy method using arachidonic acid as a substrate. Finally, the enzyme was characterized and its growth effect on human fibroblast cells was examined by MTT viability assay. Enzyme kinetic parameters including K of 90.4 µM and V of 2.63 IU were determined for LOXe. The enzyme with 0.1 nM end concentration promoted the growth of 5000 cells/well human fibroblast cells up to 11% (P < 0.01). In the present study, we introduce an E. coli expression system to produce an excessive amount of soluble LOXe and the efficient purification method to provide a soluble and active form of LOXe that is effective in stimulating human fibroblast cell proliferation.
脂氧合酶是一种非血红素铁含脂质双加氧酶，能够催化脂质的加氧水解。墨西哥钝口螈（Ambystoma mexicanum）是该酶的重要来源，具有肢体再生能力。已经表明，转染表皮脂氧合酶（LOXe）的人骨肉瘤和角质形成细胞的细胞迁移率增加。在本研究中，LOXe 作为一种外周膜蛋白，在大肠杆菌中表达。使用不同的去污剂、阴离子溶液和凝胶过滤层析对酶进行纯化。通过使用花生四烯酸作为底物的光谱法进行酶活性的动力学测定。最后，对酶进行了表征，并通过 MTT 细胞活力测定法检查了其对人成纤维细胞的生长作用。LOXe 的酶动力学参数包括 K 值为 90.4 μM 和 V 值为 2.63 IU。终浓度为 0.1 nM 的酶可促进每孔 5000 个人成纤维细胞生长 11%（P<0.01）。在本研究中，我们介绍了一种大肠杆菌表达系统来产生大量可溶性 LOXe，以及有效的纯化方法，以提供一种可溶性和活性形式的 LOXe，可有效刺激人成纤维细胞增殖。
