Department of Biochemistry, Virginia Tech, 111 Engel Hall, West Campus Drive, Blacksburg, VA 24061, USA.
Protein J. 2010 Apr;29(3):204-12. doi: 10.1007/s10930-010-9241-9.
Dopachrome tautomerase (DCT) plays a critical role in lowering the oxidative stress resulting from melanogenesis. Levels of DCT are elevated in melanoma cell lines that are especially resistant to chemotherapy and radiation. DCT is processed as a melanoma antigen and is a potential target for immunotherapy. In order to establish a more complete understanding of the role that DCT may play in the etiology and treatment of melanoma skin cancer, isolation of highly pure and properly processed protein is necessary. Purification of native DCT has been problematic due to a hydrophobic transmembrane anchor and interactions with melanin. In this study, DCT was expressed, without its carboxy-terminal transmembrane region using an Sf9 insect cell protein expression system and its recombinant protein was purified by various chromatographic techniques. Analysis of DCT tryptic peptides by MALDI-TOF/TOF determined N-glycosylation as a primary post-translational modification. Our success in the expression of soluble mammalian DCT and the characterization of N-glycosylation sites is a useful reference toward the comprehensive understanding of the structure/function relationship of mammalian DCT.
多巴色素互变异构酶（DCT）在降低黑色素生成导致的氧化应激方面起着关键作用。在对化疗和放疗特别耐药的黑素瘤细胞系中，DCT 水平升高。DCT 作为一种黑色素瘤抗原被加工，并可能成为免疫治疗的一个潜在靶点。为了更全面地了解 DCT 在黑色素瘤皮肤癌的病因和治疗中的作用，有必要分离出高度纯净和正确处理的蛋白质。由于存在疏水性跨膜锚和与黑色素的相互作用，天然 DCT 的纯化一直存在问题。本研究利用 Sf9 昆虫细胞蛋白表达系统表达了没有羧基末端跨膜区的 DCT，并通过各种色谱技术对其重组蛋白进行了纯化。通过 MALDI-TOF/TOF 对 DCT 胰蛋白酶肽的分析确定了 N-糖基化是主要的翻译后修饰。我们成功地表达了可溶性哺乳动物 DCT，并对 N-糖基化位点进行了表征，这为全面了解哺乳动物 DCT 的结构/功能关系提供了有用的参考。
