Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells.
Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells.
复制标题
巴斯德毕赤酵母细胞中表达的 N-糖基化突变小鼠和人 P-糖蛋白的纯化和表征。
DOI：
10.1006/abbi.2001.2299
复制
发表时间：
2001
影响因子：
3.9
通讯作者：
Senior,AE
中科院分区：
文献类型：
--
作者：
Urbatsch,IL;Wilke-Mounts,S;Gimi,K;Senior,AE
P-糖蛋白在哺乳动物细胞中赋予多药耐药性，其基本结构-功能研究与抗肿瘤和抗艾滋病治疗密切相关。最近已经获得了足够数量的纯的、去污剂可溶的小鼠MDR 3和人MDR 1 P-糖蛋白，用于在巴斯德毕赤酵母（Pichia pastoris）中表达后的高分辨率结构分析（N. Lerner-Marmarosh等人（1999）J. Biol. Chem. 274，34711-34718）。这些制剂的糖基化程度未知，与结晶研究相关。因此，在巴斯德毕赤酵母中表达其中N-糖基化位点被消除的突变蛋白（小鼠MDR 3 Pgp中的Asn → Gln，人MDR 1 Pgp中的Asn → Gln或Ala），并纯化至均一。突变体Pgp的产量与亲本野生型蛋白相同。核苷酸结合和催化（ATP酶）的特点是完全正常的突变蛋白质。质谱分析表明，突变体和野生型蛋白质在质量上没有显著差异，表明野生型蛋白质不含N-糖基化。
P-glycoprotein confers multidrug resistance in mammalian cells and basic structure–function studies of it are germane to anti-cancer and anti-AIDS therapy. Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N. Lerner-Marmarosh et al. (1999) J. Biol. Chem. 274, 34711–34718). The degree of glycosylation of these preparations was unknown, and was of relevance for crystallization studies. Therefore mutant proteins in which the N-glycosylation sites were eliminated (Asn → Gln in mouse MDR3 Pgp, Asn → Gln or Ala in human MDR1 Pgp) were expressed in P. pastoris and purified to homogeneity. Yields of mutant Pgp were the same as for parent wild-type proteins. Nucleotide-binding and catalytic (ATPase) characteristics were completely normal in the mutant proteins. Mass spectrometry indicated that mutant and wild-type proteins did not differ significantly in mass, demonstrating that the wild-type proteins contain no N-glycosylation.