Purification and N-glycosylation analysis of melanoma antigen dopachrome tautomerase.
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.
Vavricka
Christopher J
CJ
Department of Biochemistry, Virginia Tech, 111 Engel Hall, West Campus Drive, Blacksburg, VA 24061, USA. cjv@vt.edu
Ray
Keith W
KW
Christensen
Bruce M
BM
Li
Jianyong
J
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000951:Antigens, Neoplasm; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase
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