Zahn R, von Schroetter C, Wüthrich K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland.
FEBS Lett. 1997 Nov 17;417(3):400-4. doi: 10.1016/s0014-5793(97)01330-6.
An efficient method is presented for the production of intact mammalian prion proteins and partial sequences thereof. As an illustration we describe the production of polypeptides comprising residues 23-231, 81-231, 90-231 and 121-231 of the human prion protein (hPrP). Polypeptides were expressed as histidine tail fusion proteins into inclusion bodies in the cytoplasm of Escherichia coli and refolded and oxidized while N-terminally immobilized on a nickel-NTA agarose resin. This 'high-affinity column refolding' facilitates the preparation of prion proteins by preventing protein aggregation and intermolecular disulfide formation. After elution from the resin the histidine tail can be removed using thrombin without cleaving the prion protein polypeptide chain. The same protocol as used here for hPrP has been successfully applied with bovine and murine prion proteins. The protein preparations are stable for weeks at room temperature in concentrated solution and are thus suitable for detailed structural studies. Preliminary biophysical characterization of hPrP(23-231) suggests that the C-terminal half of the polypeptide chain forms a well-structured globular domain, and that the N-terminal half does not form extensive regular secondary structures.
本文介绍了一种生产完整哺乳动物朊病毒蛋白及其部分序列的有效方法。作为示例，我们描述了包含人朊病毒蛋白（hPrP）第23 - 231、81 - 231、90 - 231和121 - 231位残基的多肽的生产。多肽以组氨酸尾融合蛋白的形式表达于大肠杆菌细胞质中的包涵体中，在N端固定于镍 - NTA琼脂糖树脂上的同时进行重折叠和氧化。这种“高亲和力柱重折叠”通过防止蛋白质聚集和分子间二硫键形成，促进了朊病毒蛋白的制备。从树脂上洗脱后，可使用凝血酶去除组氨酸尾，而不切割朊病毒蛋白多肽链。此处用于hPrP的相同方案已成功应用于牛和鼠的朊病毒蛋白。蛋白质制剂在室温下于浓缩溶液中可稳定保存数周，因此适用于详细的结构研究。hPrP(23 - 231)的初步生物物理表征表明，多肽链的C端一半形成了结构良好的球状结构域，而N端一半未形成广泛的规则二级结构。
