Departments of Biochemistry, Hanover, New Hampshire 03755, USA.
Prep Biochem Biotechnol. 2012;42(1):77-86. doi: 10.1080/10826068.2011.564256.
Mature prion protein (PrP) is a 208-residue polypeptide that contains a single disulfide bond. We report an alternative method to purify recombinant mouse PrP produced in Escherichia coli. Bacterial inclusion bodies were solubilized in a buffer containing 2 M urea at pH 12.5. The solubilized protein was rapidly purified on a nickel affinity column without a chaotrope gradient, followed by ion-exchange chromatography. The yield and purity of PrP produced by this alternative approach was similar to that obtained using a conventional solubilization and on-column refolding protocol. Recombinant PrP produced using the non-reducing purification protocol is properly folded, as determined by circular dichroism, and a competent substrate for amyloid fibril formation, as determined by Thoflavin-T dye binding assays. In summary, this report describes a rapid method for producing properly folded recombinant PrP without reducing agents or a chaotrope gradient.
成熟的朊病毒蛋白（PrP）是一种含有一个二硫键的 208 个残基的多肽。我们报告了一种从大肠杆菌中生产的重组小鼠 PrP 的纯化替代方法。细菌包涵体在 pH 值为 12.5 的含 2 M 尿素的缓冲液中溶解。溶解的蛋白质在镍亲和柱上快速纯化，而无需使用变性剂梯度，然后进行离子交换层析。这种替代方法生产的 PrP 的产量和纯度与使用传统的溶解和柱上复性方案获得的产量和纯度相似。通过圆二色性测定，使用非还原纯化方案生产的重组 PrP 是正确折叠的，并且通过 Thoflavin-T 染料结合测定，它是一种有能力形成淀粉样纤维的底物。总之，本报告描述了一种快速生产正确折叠的重组 PrP 的方法，无需还原剂或变性剂梯度。
