High-level expression and characterization of a purified 142-residue polypeptide of the prion protein
High-level expression and characterization of a purified 142-residue polypeptide of the prion protein
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DOI：
10.1021/bi952965e
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发表时间：
1996-04-30
期刊：
影响因子：
2.9
通讯作者：
Prusiner, SB
中科院分区：
文献类型：
--
作者：
Mehlhorn, I;Groth, D;Prusiner, SB
感染性朊病毒的主要成分，也可能是唯一的成分是羊瘙痒症朊病毒蛋白（PrPSc）; PrPSc的蛋白酶抗性核心是PrP 27-30，一种类似于142个氨基酸的蛋白质。PrPSc是由细胞PrP同种型（PrPC）通过翻译后过程衍生而来的，在翻译后过程中发生深刻的构象变化。将从N-和C-末端截短的90-228到全长成熟蛋白23-231的不同长度的叙利亚仓鼠（SHa）PrP基因插入到各种分泌和细胞内表达载体中，所述载体转化到蛋白酶缺陷的大肠杆菌中。最大的表达获得了截短SHaPrP含有残基90-231，这对应于PrP 27-30的序列;使用微流化器的细菌的破坏产生了最高产量的这种蛋白质命名为rPrP。rPrP在8 M GdnHCl中溶解后，通过尺寸排阻色谱和反相色谱纯化。在纯化过程中，回收率接近50%，并且从每升E.大肠杆菌培养物中，获得类似于50 mg纯化的rPrP。含有碱性N末端区域的较长物种的表达不太成功，因此没有进一步研究。通过Edman测序和质谱法验证了rPrP的一级结构，并通过圆二色谱和傅里叶变换红外光谱法确定了二级结构。当rPrP在还原条件下纯化时，它具有类似于PrPSc的高β-折叠含量和相对低的溶解度，特别是在pH值>7时。通过氧化再折叠rPrP以在该多肽的两个Cys残基之间形成二硫键，产生具有类似于PrPC的高Ct-螺旋含量的可溶性蛋白。rPrP的这些多种构象使人想起表征天然存在的PrP同种型的结构多样性。现在可以获得的高水平的纯化rPrP应该有助于确定PrP可以采用的多种三级结构。
The major, and possibly 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 similar to 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 GdnHCl, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was similar to 50%, and from each liter of E. coli culture, similar to 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 ct-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occuring 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.