Mehlhorn I, Groth D, Stöckel J, Moffat B, Reilly D, Yansura D, Willett W S, Baldwin M, Fletterick R, Cohen F E, Vandlen R, Henner D, Prusiner S B
Department of Neurology, University of California, San Francisco 94143, USA.
Biochemistry. 1996 Apr 30;35(17):5528-37. doi: 10.1021/bi952965e.
The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.
传染性朊病毒的主要成分，也可能是唯一成分，是瘙痒病朊病毒蛋白（PrPSc）；PrPSc的蛋白酶抗性核心是PrP 27-30，一种约142个氨基酸的蛋白质。PrPSc是通过翻译后过程从细胞PrP异构体（PrPC）衍生而来，在此过程中发生了深刻的构象变化。将不同长度的叙利亚仓鼠（SHa）PrP基因（从N端和C端截短的90-228到全长成熟蛋白23-231）插入各种分泌和细胞内表达载体中，这些载体被转化到缺乏蛋白酶的大肠杆菌中。对于包含90-231位残基的截短SHaPrP获得了最大表达，该残基对应于PrP 27-30的序列；使用微流化器破坏细菌产生了该蛋白（称为rPrP）的最高产量。rPrP在8 M盐酸胍中溶解后，通过尺寸排阻色谱和反相色谱进行纯化。纯化过程中的回收率约为50%，从每升大肠杆菌培养物中可获得约50 mg纯化的rPrP。包含碱性N端区域的较长物种的表达不太成功，未进一步研究。rPrP的一级结构通过埃德曼测序和质谱验证，二级结构通过圆二色性和傅里叶变换红外光谱确定。当rPrP在还原条件下纯化时，它具有高β-折叠含量和相对低的溶解度，类似于PrPSc，特别是在pH值>7时。通过氧化使rPrP重折叠以在该多肽的两个半胱氨酸残基之间形成二硫键，产生了具有高α-螺旋含量的可溶性蛋白，类似于PrPC。rPrP的这些多种构象让人想起天然存在的PrP异构体所具有的结构多样性。现在能够获得的高纯度rPrP应该有助于确定Prp可以采用的多种三级结构。
