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 很难分离出足够数量的细胞或羊瘙痒症 异构体进行高分辨率的结构研究。 PrP 27-30， 最容易纯化的形式，是异质的和不溶的，因此不是 适用于结晶和X射线衍射或NMR分析。 重组形式的蛋白质可能很快就会出现，但这些将 需要稳定的同位素掺入用于NMR分析。 现有方法 允许重组蛋白的表达， 一种类型的所有氨基酸。 然而，固相样品的NMR 需要在选定的位点特异性结合同位素标记， 可以使用诸如以下技术参与磁化交换 旋转共振 化学合成提供了一种替代方法 策略重组过表达，但即使是构建 与PrP 27-30对应的142个氨基酸片段超出范围 目前的方法论。 化学合成多肽的组合 而酶促连接确实提供了一个现实的前景， 获得足够量的纯的、均质的蛋白质。 已为一个为期一年的项目提供了资金， 对应于残基90-231的142个氨基酸的PrP片段。 初步结果是令人鼓舞的，这很可能是 将于明年内完成。 明年，我们将 改进和提高该合成的产率。 我们还将设计和 合成仅含有两个标记的同位素标记的类似物 固态NMR的位置。 作为一种辅助手段， 肽，我们将开发用于连接合成的 重组PrP片段。 最近的结果 大的重组PrP片段的表达和纯化已经被 非常鼓舞人心。 肽连接和重组表达 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，使用方法的组合，我们将获得 样品适合在液体和固体状态下进行NMR研究。 随后，这些研究将扩展到含有已知的 致病突变 我们还将合成含有其他 基团，如氟原子和自旋标记，用于其他NMR测量。 我们将开发含有PrP类似物的区域的合成， 约束结构，迫使分子采取某些 构象，这可能是特别有助于确定 有助于PrPC转化为PrPSc的PrP特征。 所有的合成物种都将受到各种各样的 选择重折叠条件以鼓励PrPSc的发展。 他们将在叙利亚仓鼠或 合适的转基因宿主。 将选择后者，以尽量减少 由于一级序列的差异而形成的物种屏障。 在 如果可以产生传染性，我们将确定最小值 致病性所需的β折叠区域。 最重要的 这些物种的结构将被同位素标记， NMR研究。 所有这些物种的重折叠也将进行研究 结合结构分析技术，包括 CD，FTIR和荧光光谱，作为NMR研究的补充。

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.