SYNTHETIC AND RECOMBINANT PRP PEPTIDES AND PROTEINS

PrP的性质及其在大脑中的低丰度使其非常 难以分离足够量的细胞或瘙痒病 进行高分辨率的构造研究。PRP 27-30， 最容易提纯的形式，是多相和不溶的，因此不是 适用于结晶、X-射线衍射或核磁共振分析。 重组形式的蛋白质可能很快就会出现，但这些将会 需要稳定的同位素掺入以进行核磁共振分析。现有方法 允许表达掺入重同位素的重组蛋白 在所有一种氨基酸中。然而，固相样品的核磁共振 要求在选定的位置特定地结合同位素标记， 可以使用以下技术参与磁化交换 旋转共振。化学合成提供了另一种选择 重组过表达的策略，但即使是构建 PrP 27-30对应的142个氨基酸片段超出范围 当前的方法论。多肽的一种化学合成组合 然而，酶连接确实提供了一个现实的前景 获得足够数量的纯的、均一的蛋白质。 已经为一个为期一年的项目提供了资金，以开发合成 在对应于残基90-231的142个氨基酸的PrP片段中。 初步结果令人鼓舞，这很可能是 将在来年内完成。在下一年，我们将 提纯并提高此合成的产率。我们还将设计和 合成仅含两个标记的同位素标记类似物 固体核磁共振的位置。作为完全合成的附属品 多肽，我们将制定连接人工合成多肽的方案 多肽转化为更大的重组PrP片段。最新的研究结果 重组PrP大片段的表达和纯化 非常鼓舞人心。多肽的连接及重组表达 将是加州大学旧金山分校和科学家之间持续合作的一部分 在基因泰克。因此，使用多种方法的组合，我们将获得 适用于液体和固体核磁共振研究的样品。 随后，这些研究将扩展到含有已知的 致病突变。我们还将合成包含其他 用于其他核磁共振测量的基团，如氟原子和自旋标记。 我们将开发含有以下区域的PrP类似物的合成 一种受约束的结构，迫使分子采用一定的 构象可能特别有助于确定 有助于将PrPC转换为PrPSc的PrP的特征。 所有人工合成的物种都将受到各种各样的 为鼓励PrPSc的发展而选择的复性条件。 他们将在叙利亚仓鼠身上或在 合适的转基因宿主。将选择后者以最小化 由于初级序列的差异造成的物种障碍。在 如果可能产生传染性，我们将确定最低限度 致病所需的β-折叠区域。最重要的 这些物种将被同位素标记为结构 核磁共振研究。所有这些物种的折叠也将被研究。 与结构分析技术相结合，这些技术将包括 CD、FTIR和荧光光谱，作为核磁共振研究的辅助手段。

The properties of PrP and its low abundance in brain have made it very difficult to isolate sufficient amounts of the cellular or scrapie isoforms to undertake high resolution structural studies. PrP 27-30, the most easily purified form, is heterogeneous and insoluble, thus is not suited to crystallization and X-ray diffraction or analysis by NMR. Recombinant forms of the protein may soon be available but these will require stable isotope incorporation for NMR analysis. Existing methods allow expression of recombinant proteins with heavy isotopes incorporated in all amino acids of one type. NMR on solid phase samples however requires specific incorporation of isotope labels at selected sites that can participate in magnetization exchange using techniques such as rotational resonance. Chemical synthesis provides an alternative strategy to recombinant over-expression but the construction of even the 142 amino acid fragment corresponding to PrP 27-30 is beyond the scope of current methodology. A combination of chemical synthesis of peptides and enzymatic ligation does however offer a realistic prospect of obtaining sufficient quantities of pure, homogeneous, protein. Funding has been provided for a one-year project to develop the synthesis of the 142 amino acid PrP fragment corresponding to residues 90-231. Preliminary results are encouraging and it is very probable that this will be completed within the coming year. In the following year we shall refine and improve the yield of this synthesis. We shall also design and synthesize isotopically labelled analogs containing only two labelled sites for solid state NMR. As an adjunct to making entirely synthetic peptides, we shall develop protocols for the ligation of synthetic peptides to larger recombinant PrP fragments. Recent results on the expression and purification of a large recombinant PrP fragment have been very encouraging. The peptide ligations and the recombinant expression will be part of a continuing collaboration between UCSF and scientists at Genentech. Thus, using a combination of approaches we shall obtain samples suitable for NMR studies in both the liquid and the solid states. Subsequently these studies will be extended to molecules containing known pathogenic mutations. We shall also synthesize analogs containing other groups such as fluorine atoms and spin labels for other NMR measurements. We shall develop the synthesis of PrP analogs containing regions of constrained structure which force the molecules to adopt certain conformations which may be particularly helpful in determining the features of PrP that contribute to the conversion of PrPC to PrPSc. All the synthetic species will be subjected to a wide variety of refolding conditions selected to encourage the development of PrPSc. They will be assayed for prion infectivity in Syrian hamsters or in appropriate transgenic hosts. The latter will be selected to minimize species barriers due to differences in the primary sequences. In the event that infectivity can be generated, we shall determine the minimum regions of beta-sheet required for pathogenicity. The most significant of these species will then be isotopically labelled for structural studies by NMR. The refolding of all these species will also be studied in conjunction with structural analyses by techniques that will include CD, FTIR and fluorescence spectroscopy, as adjuncts to the NMR studies.