Yin Shao-Man, Zheng Yi, Tien Po
Department of Molecular Virology, Institute of Microbiology, Chinese Academy of Science, P.O. Box 2714, Beijing 100080, PR China.
Protein Expr Purif. 2003 Nov;32(1):104-9. doi: 10.1016/S1046-5928(03)00195-5.
Prion protein has a key role in the occurrence of transmissible spongiform encephalopathy (TSE) and development of these diseases. Here, we provide a convenient procedure for on-column purification and refolding of the full-length mature bovine prion protein (bPrP) from Escherichia coli using immobilized metal (Ni) affinity chromatography, based on the metal-binding property of its unusual octarepeat sequences containing six tandem copies. Following extensive washing, the bPrP pellet was solubilized by guanidine hydrochloride and subjected to Ni-NTA agarose column. Purification and refolding were achieved by stepwise gradient washing with reduced guanidine hydrochloride concentrations. Triton X-100 and beta-mercaptoethanol were required in this rapid refolding process. The isolated prion protein was identified by monoclonal antibodies and its integrity was monitored by mass spectroscopy. Its correct folding was confirmed from circular dichroism (CD) experiments. Moreover, thioflavin T-binding assay showed that the recombinant bPrP could be transformed into amyloid fiber structures like that of the infectious prion isoform PrP(sc).
朊病毒蛋白在传染性海绵状脑病（TSE）的发生及这些疾病的发展过程中起关键作用。在此，我们基于全长成熟牛朊病毒蛋白（bPrP）中含有六个串联拷贝的异常八肽重复序列的金属结合特性，提供了一种利用固定化金属（镍）亲和色谱从大肠杆菌中对其进行柱上纯化和重折叠的简便方法。经过大量洗涤后，将bPrP沉淀用盐酸胍溶解并上样到镍-亚氨基二乙酸琼脂糖柱上。通过用浓度递减的盐酸胍进行逐步梯度洗脱实现纯化和重折叠。在这个快速重折叠过程中需要Triton X-100和β-巯基乙醇。用单克隆抗体鉴定分离出的朊病毒蛋白，并用质谱监测其完整性。通过圆二色性（CD）实验证实其正确折叠。此外，硫黄素T结合试验表明，重组bPrP可转化为类似传染性朊病毒异构体PrP(sc)的淀粉样纤维结构。
