X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32
X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32
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DOI：
10.1021/bi9824543
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发表时间：
1999-02-23
期刊：
影响因子：
2.9
通讯作者：
MacGillivray, RTA
中科院分区：
文献类型：
--
作者：
Bewley, MC;Tam, BM;MacGillivray, RTA
人血清转铁蛋白 N 叶的三价铁 (Fe(III)-hTF/2N) 在毕赤酵母中高水平表达。 Fe(III)-hTF/2N在空间群P4(1)2(1)2中结晶，并使用X射线晶体学以2.5埃分辨率解析重组蛋白的结构。这代表了迄今为止确定的第二个源自毕赤酵母的蛋白质结构，并且允许将在毕赤酵母和哺乳动物细胞中表达的重组 Fe(III)-hTF/2N 的结构与血清来源的转铁蛋白进行比较。所有三种蛋白质的多肽折叠模式基本相同。对巴斯德毕赤酵母-hTF/2N 和蛋白水解衍生片段的质谱分析揭示了 Ser-32 与单个己糖的糖基化。这代表了 O-连接聚糖在巴斯德毕赤酵母衍生蛋白中的首次定位。由于其与铁结合位点的距离，Ser-32 的糖基化不应影响巴斯德毕赤酵母中表达的 hTF/2N 的铁结合特性，使其成为生产 hTF/2N 的优异表达系统。
The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P4(1)2(1)2, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 Angstrom resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.