The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

1.代质粒和Shadoo表达注:基因编码鼠Shadoo蛋白（Shadoo 25-122），亚克隆到表达载体pET-28 11。该克隆转化 E. 大肠杆菌 SoluBL21菌株，表达Shadoo蛋白质与N-末端六组氨酸标签，（HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG）如先前11说明。质粒可以从作者索取。 Shadoo的​​分子量从它的氨基酸序列计算出是12,243.2沓。 注:执行下一个分层的流罩或接近本生灯火焰与细菌的所有操作。 <ol><li>转化感受SoluBL21细菌与PET-28-他6 -Shadoo质粒，使用标准的热休克协议。对于这种混合料1〜5微升质粒（通常为10pg至100纳克）到50微升的细菌在管的。进行热休克在42℃为45秒。 </li><li>培养转化的细菌，在37℃，直至在的Luria-Bertani培养基A 600 0.5-0.7补充有40毫克/毫升卡那霉素（ 图1B）。 </li><li>诱导Shadoo表达O / N通过加入240微克/毫升异丙基β-D-硫代半乳糖苷，ITPG，向培养。下以150rpm摇动，在37℃培养细菌。注:通常情况下，细菌培养达到600 3 O / N中。 注意:转化的细菌可以被储存在-80℃下以供将来的蛋白质生产。对于这一点，添加10％-50％（体积/体积）的无菌甘油进细菌培养等分试样并冷冻在-80℃。 </li></ol><img alt="图1" src="/files/ftp_upload/53432/53432fig1.jpg" /> 图1.方案Shadoo表达Shadoo表达构建，细菌转化和诱导的（如步骤1所述）。 </stro纳克&gt;（A），参考 Shadoo的表达构建体编码融合到蛋白质，Nt个的N-末端六组氨酸标签。该方案表明Shadoo蛋白含有碱性精氨酸/甘氨酸重复（绿色气缸），疏水性结构域（HD）和一个单一的N-糖基化位点（CHO），接着是C末端数（Ct）。 （B）的PET-28-他6 -Shadoo质粒在42℃下转化到SoluBL21细菌通过热休克45秒。转化的细菌铺板于含有选择性琼脂40微克/毫升卡那霉素。从板的单菌落被用于接种培养在1,000-3,000毫升摇瓶中。重组蛋白的表达诱发用1mM IPTG。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.净化Shadoo蛋白的低压液相色谱（LPLC） <ol><li>细胞裂解和在包涵体机构编制<ol><li>收集细菌悬浮液，并将其转移到离心管中（图2）。 </li><li>自旋在2500 XG，20分钟，在4℃。通过倾析和在15ml缓冲液（50mM的Tris，10mM EDTA的，pH值8与抗蛋白酶混合物）重悬粒料为从500 ml的细胞培养物衍生的每个颗粒除去上清液。 注意:重​​悬粒料可以冷冻并储存在-20℃下数天。冷冻利于细菌细胞裂解。 </li><li>检查重组蛋白的使用粗细菌粒料通过SDS-PAGE分析和考马斯染色的过表达。 <ol><li>对于这一点，离心机1 ml的细菌培养物以沉淀​​细胞。加入100微升的Laemmli变性溶液（277.8毫摩尔Tris盐酸，pH值6.8，4.4％SDS中，44.4％w / v的甘油，0.02％溴酚蓝）以沉淀和热在100℃下5分钟。 </li><li>负载10微升样品至12％的丙烯酰胺凝胶，运行SDS-PAGE和VISU艾莉婕考马斯亮蓝染色分离的蛋白质。 </li></ol></li><li>加入的Triton X-100至0.5％的终浓度为完全再悬浮颗粒从2.1.2）。 </li><li>孵育悬浮液在水浴中于37℃进行30分钟。 </li><li>超声处理该悬浮液5分钟，在6-12微米的冰冷水峰 - 峰振幅以裂解细菌细胞。 </li><li>转移超声悬浮到50 mL的干净离心管。 </li><li>离心管以15,000rpm×g离心15分钟，在4℃。 </li><li>通过倾析除去上清液，加入5 ml的每管的结合缓冲液（20mM的Tris-HCl缓冲液，pH 7.4，0.5M氯化钠，8M尿素，5mM咪唑）的。 </li><li>在旋转轮O / N将管完全悬浮的包涵体蛋白。 </li></ol><img alt="图2" src="/files/ftp_upload/53432/53432fig2.jpg" /> 夹杂物的纯化图2.方案体（如在步骤2.1中所述）。转化的细菌表达Shadoo通过离心收集，并再悬浮于裂解缓冲液。将悬浮液在37℃下进行30分钟，然后超声处理以机械方式破坏细菌细胞。超声处理完成在冰上，以防止样品加热。破碎的细胞通过离心收集并重新悬浮于含8M尿素溶解的包涵体蛋白的缓冲。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </li><li> 镍离子-affinity色谱 注意:色谱是使用Ni 2+ -charged，加入5ml琼脂糖螯合柱（图3A）上的FPLC系统进行。所有解决方案，是与1毫升/分钟的流速的柱上。 <ol><li>注入溶于10ml 0.2M的制备NiSO 4水溶液放入琼脂糖柱，以充电与镍离子-离子。 </li><li>通过注入30〜50毫升含有咪唑的低浓度（20毫摩尔Tris-HCl缓冲液，pH值7.4，0.5M氯化钠，8M尿素，5mM咪唑）的结合缓冲液平衡装入镍2+ -离子的柱。 </li><li>加载脲溶解出的蛋白用50毫升的注射环。 </li><li>通过彻底清洗，用10ml低咪唑结合缓冲液（20mM的Tris-HCl缓冲液，pH 7.4，0.5M氯化钠，8M尿素，5mM咪唑）柱以除去最大未结合的蛋白质的回收流出级分从柱。 </li><li>用50-100毫升的洗涤缓冲液（20mM的Tris-HCl，pH值7.4，0.5M氯化钠，8M尿素，80 mM咪唑）洗脱蛋白质非特异性结合到柱上的洗柱。 </li><li>在含有20mM的Tris-HCl，pH值7.4，0.5M氯化钠，8M尿素的缓冲液适用的80-800 mM咪唑一个15分钟线性梯度。梯度应用过程中收取1毫升级分。注意:His-标记的蛋白被洗脱机智的含咪唑的缓冲公顷梯度（用作竞争者）。 </li><li>取每个级分的8微升等分试样，并添加4μl的4倍的Laemmli变性溶液（见2.1.3.1）。热溶液在100℃下5分钟。 </li><li>负载10微升蛋白大小标记物和10μl各样品的12％丙烯酰胺凝胶上，运行SDS-PAGE和可视化分离的蛋白质通过考马斯染色。注意:Shadoo的​​分子量计算出形成其氨基酸序列是12,243.2沓。 </li><li>池包含Shadoo的​​所有分数。 </li></ol><img alt="图3" src="/files/ftp_upload/53432/53432fig3.jpg" /> 图3. 纯化步骤（如步骤2.2-2.4中描述）。 （一） 镍离子-离子可以通过组氨酸残基，这使得多组氨酸标记蛋白的选择性纯化进行协调。咪唑来洗脱蛋白质，通过其协调与镍离子-离子和取代结合蛋白质的能力。（二）不同流体力学半径被保留对Ni 2+ -column分离到一个尺寸排阻柱的蛋白质。蛋白从最大到最小的那些洗脱。（C）的使用前，含有Shadoo级分汇集和脱盐以除去尿素，咪唑和盐。质量控制实验包括SDS-PAGE /考马斯染色，免疫印迹。纯化的Shadoo可以冷冻干燥。冻干Shadoo稳定了几个月储存在-20°C下。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </li><li>尺寸排阻色谱法 注意:第二个纯化步骤包括在尺寸排阻色谱法从该其它蛋白质在第一个纯化步骤中共同洗脱分离Shadoo蛋白（图3B）。 <ol><li>平衡120ml的总床体积的Superdex柱通过注入250个毫升的20mM的Tris-HCl缓冲液，pH 7.4，0.5M氯化钠，8M的尿素以1.5毫升/分钟的流速。 </li><li>负载合并的级分含Shadoo从2.2.9上的平衡列。为40ml时上述缓冲液的流量恒定的柱空隙容积后收集1毫升级分。 </li><li>每次取分数的10微升等份与上述找出哪些部分包含Shadoo进行SDS-PAGE分析和考马斯亮蓝染色。 </li><li>池部分含Shadoo。 </li></ol></li><li>脱盐 注意:脱盐过程删除的盐，咪唑和尿素以获得重组Shadoo蛋白在适合于进一步的生物物理和生物化学研究（图3C）的缓冲器。相同的结果可通过透析样品对缓冲器来获得。 <ol><li>平衡53 ml的用150ml脱盐柱的10mM乙酸铵缓冲液，pH 5中，在5毫升/分钟的流速。 </li><li>负荷集中在柱上Shadoo馏分。在注射的乙酸铵缓冲的手动收集，显示增加了信号的280nm处的UV检测器的输出的级分。 </li><li>冷冻干燥该脱盐Shadoo并储存在-20℃。 </li><li>溶解冻干Shadoo在适当的缓冲液，如10mM醋酸钠缓冲液，pH 5，在使用之前。 注意:聚组氨酸标签可以用肠激酶除去。 </li></ol></li></ol> 3.在体外组装Shadoo进入淀粉样蛋白原纤维在生理pH值注:Shadoo蛋白聚集体和自发形成纤维在pH≥7月11日。 <img alt="图4" src="/files/ftp_upload/53432/53432fig4.jpg" /> 图4. 计划Shadoo复性淀粉样纤维（如descri床在步骤3）。将纯化的Shadoo自发转换成淀粉样蛋白纤维溶解在pH值≥7的缓冲器。在酸性溶液时，Shadoo温育后用1M GDN盐酸转换为淀粉样蛋白原纤维，同时摇动。质量控制检测包括电子显微镜和ThT的染色。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>稀Shadoo至2微米的20mM的Tris-HCl缓冲液，pH 7.4的终浓度。通过测量溶液在280nm处的光密度，并使用从20970 Shadoo氨基酸组成推断的消光系数测量的浓度。 </li><li>孵育500微升在锥形塑料管将溶液在4℃下为几个星期，以允许自发蛋白质转化为淀粉样蛋白的结构（图4）。 </li><li>加入新鲜制备的硫磺素T（ThT的）到最终conce为10μMntration到Shadoo溶液的100μl等分的。孵育5分钟，在室温的溶液中。使用的435纳米的激发波长，以验证淀粉样结构 17的存在下测量荧光发射从460到520纳米。 </li></ol> 4.在Shadoo 体外组装到淀粉样纤维酸性pH <ol><li>在1M盐酸胍，20mM的醋酸钠缓冲液，pH 5稀释Shadoo到的2μM的终浓度。 </li><li>孵育将500μl此溶液在锥形管连续振荡的，在37℃，3-7天。 </li><li>通过加入的ThT到该溶液的等分试样在3.3检查淀粉样蛋白原纤维的存在。 </li><li>透析得到的悬浮液对10毫乙酸钠缓冲液，pH 5.0，以纯化Shadoo淀粉样蛋白原纤维。 </li><li>存储纯化纤维在+ 4℃。 </li></ol><img alt="图5" src="/files/ftp_upload/53432/53432fig5.jpg"/&gt; 选自Ni 2+洗脱图5 代表性的结果。蛋白馏分-charged螯合柱（A）和尺寸排阻柱（ 乙 ），使用SDS-PAGE和考马斯染色进行分析。使用SPRN抗Shadoo抗体（C）纯化Shadoo的鉴定评估。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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<li>Sorensen, H. P., Mortensen, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+genetic+strategies+for+recombinant+protein+expression+in+Escherichia+coli%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">Advanced genetic strategies for recombinant protein expression in Escherichia coli.</a> Journal of Biotechnology. 115, 113-128 (2005).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+glycosylation+enhances+ubiquitin-mediated+proteasomal+degradation+of+Shadoo+in+Scrapie-infected+rodents+and+cultured+cells%5BTitle%5D)+AND+%22Molecular+Neurobiology%22%5BJournal%5D)">Disruption of glycosylation enhances ubiquitin-mediated proteasomal degradation of Shadoo in Scrapie-infected rodents and cultured cells.</a> Molecular Neurobiology. 49, 1373-1384 (2014).</li>
<li>Englund, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+and+biosynthesis+of+glycosyl+phosphatidylinositol+protein+anchors%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors.</a> Annual Review of Biochemistry. 62, 121-138 (1993).</li>
<li>Puig, B., Altmeppen, H., Glatzel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+GPI-anchoring+of+PrP%3A+implications+in+sorting+and+pathogenesis%5BTitle%5D)+AND+%22Prion%22%5BJournal%5D)">The GPI-anchoring of PrP: implications in sorting and pathogenesis.</a> Prion. 8, 11-18 (2014).</li>
</ol>

The authors have nothing to disclose.

一个简单的，高效的协议为表达和大量重组小鼠Shadoo蛋白的纯化给出。所描述的方法可以成功溶解和（他的6）-tagged重组Shadoo蛋白的纯化。重要的是，如在标题所指出的，当在原核细菌大肠杆菌表达大肠杆菌 Shadoo积聚在包涵体。之前Shadoo纯化，细菌必须通过超声处理在含有的Triton X-100的缓冲液裂解。接着包涵体可溶于8M尿素变性溶液。此外，整个纯化过程必须在8M尿素缓冲液?...

朊病毒神经变性疾病，其中包括牛海绵状脑病在牛，羊瘙痒在绵羊和Creutzfeldt-Jakob病在人类，是致命的，无法治愈的。朊病毒病的特征在于细胞朊病毒蛋白的构象变化主要由α螺旋的，进入交叉β-片富含淀粉样蛋白构象1,2-。交叉β-结构包含密集和高度有序的结晶状β片具有稳定的氢键3,4。朊病毒淀粉样蛋白可以自我复制由于β链的生长边缘提供招募的模板和转换单体蛋白质单元。 据"唯一蛋白质"假说，朊病毒蛋白的淀粉样蛋白构象是唯一的感染因子。然而，一些其它生物分子已经被提出是必不可少的朊病毒病。例如，细胞膜被认为是一个地方，conversi上发生与一些负电荷的脂质已经显示出增强了转换过程5,6。此外，某些蛋白质也可以是参与朊病毒病症。具体来说，有朊蛋白家族的另外两名成员:Doppel和Shadoo 7,8。 Doppel具有相对刚性的结构稳定化与二硫醚桥，未能自聚合和聚合到淀粉样蛋白原纤维9。与此相反，类似于朊病毒蛋白，Shadoo可以采用一个β片层富集结构和自缔成淀粉样蛋白纤维的结构。它表明，Shadoo可在天然条件下或在结合带负电荷的膜10,11原纤化。转换Shadoo成淀粉样纤维可以与病理相关联。事实上，通过淀粉样蛋白样结构引起疾病的机理知之甚少。 Shadoo熊疏水结构域（HD）等一系列串联精氨酸/甘氨酸的重复类似的牛逼Ø朊蛋白（ 图1A）的N末端 ​​部分。如朊病毒蛋白的N-端部分，Shadoo高度正电，似乎是一个本地非结构化蛋白11,12。 Shadoo似乎是功能上相关的朊病毒疾病，因为它可以直接绑定朊病毒蛋白。另外，它的表达被向下期间朊病毒病理13,14调节。然而，Shadoo的​​朊病毒疾病中的作用尚未建立。 我们开发了一种质粒携带鼠标Shadoo基因的编码序列。将质粒用于转化大肠杆菌大肠杆菌生产N端他的6融合Shadoo蛋白质。该表达系统是公认的在我们的实验室和我们正在进行的项目6,15,16常用。为Shadoo的表达的一个重要问题是大肠杆菌的选择大肠杆菌菌株 。而感受态BL21细菌菌株通常用于重组亲表达teins Shadoo已成功表达并只从转化SoluBL21菌株纯化。 SoluBL21主管E.大肠杆菌是BL21宿主菌株的改进的突变体，用于生产蛋白质的表达在父BL21给没有可检测的可溶性产物开发的。 Shadoo的SoluBL21 大肠杆菌高效表达大肠杆菌导致的蛋白质的积累中包涵体。作为一般特征，当非天然蛋白质高表达在大肠杆菌大肠杆菌 ，这种蛋白质往往积聚在不溶性包涵体。 Shadoo大概粒料通过非共价的疏水性或离子相互作用（或两者的组合），以通过折叠到不同程度的蛋白质形成的高度富集的动态结构。由此，纯化包括至少两个步骤:（i）从在变性介质其它生物分子的蛋白的选择性分离，以及（ii） 使用体外折叠纯化蛋白的复性技术。 Shadoo的​​选择性分离物在含有8M尿素（或者6M胍-HCl）的缓冲溶液来实现。 （ⅰ）复性在酸性pH溶液中，得到的非结构化单体Shadoo蛋白质，或（ii）复性pH值≥的溶液7，得到Shadoo聚合:尿素去除和蛋白质的复性可以通过应用不同的协议来实现成稳定的淀粉样蛋白纤维特性交叉β折叠基序。

<table><tbody><tr><td>AKTA FPLC&amp;nbsp;</td><td>GE, Amersham</td><td># 1363</td><td></td></tr><tr><td>HiLoad 16/60 Superdex</td><td>GE Healthcare</td><td>17-1069-01</td><td></td></tr><tr><td>HiTrap IMAC</td><td>GE Healthcare</td><td>GE17-0920-05</td><td></td></tr><tr><td>HiPrep26/10 Desalting column</td><td>GE Healthcare</td><td>GE17-5087-01</td><td></td></tr><tr><td>SoluBL21 Competent E. coli</td><td>Genlantis</td><td>C700200</td><td></td></tr><tr><td>pET-28b+</td><td>Novogen</td><td>69865-3</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I6758</td><td></td></tr><tr><td>LB-Broth medium</td><td>Sigma-Aldrich</td><td>L3022</td><td></td></tr><tr><td>Eppendorf Biophotometar</td><td>Eppendorf</td><td>550507804</td><td></td></tr><tr><td>Trito X-100</td><td>Life Technologies</td><td>85111</td><td></td></tr><tr><td>NiSO4</td><td>Sigma-Aldrich</td><td>656895</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>RES3098T</td><td></td></tr><tr><td>Protease Inhibitor Cocktail Tablets</td><td>Roche</td><td>4693116001</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>746398</td><td></td></tr><tr><td>Imidazol</td><td>Sigma-Aldrich</td><td>I5513</td><td></td></tr><tr><td>Gdn-HCl</td><td>Sigma-Aldrich</td><td>G4505</td><td></td></tr><tr><td>protein size marker&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>26620</td><td></td></tr></tbody></table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

大肠杆菌表达系统是一种强大的工具，用于生产重组真核蛋白。我们用它来生产Shadoo，属于家庭朊病毒蛋白。作为包涵体被描述为（他）6 -tagged重组Shadoo纯化的色谱法表示。包涵体溶解于8M尿素和结合到镍离子-charged柱进行离子亲和层析。结合的蛋白质通过咪唑的梯度洗脱。含有Shadoo蛋白质级分进行尺寸排阻色谱法，以获得高度纯化的蛋白质。在最后的步骤纯化Shadoo脱盐以除去盐，脲和咪唑。重组Shadoo蛋白是用于在朊病毒病发生的蛋白质构象疾病的生物物理和生物化学研究的重要试剂。许多报告表明，朊病毒神经退行性疾病，从刺的沉积起源乐，有序的淀粉样纤维。描述如何纤化Shadoo成淀粉样纤维在酸性和中性样品协议/碱性pH值呈现。关于如何产生和原纤化Shadoo可以促进研究在实验室中工作的朊病毒疾病，因为它允许生产的快速和低成本的方式大量蛋白质的方法。

每升细菌培养物中获得约5-10毫克纯化Shadoo蛋白质。两步净化需要获得高纯度的重组Shadoo。第一步骤是通过亲和层析利用Ni 2+ -charged柱，其保存His-标记的蛋白进行的。洗脱级分进行SDS-PAGE和用考马斯亮蓝（图5A）。含有Shadoo蛋白的级分汇集并通过大小排阻层析其中根据其大小分离蛋白质（Shadoo分子量为约12 kDa的）进一步纯化。在洗脱收集的级分通过SDS-PAGE /考马斯分析（</st...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

纯化及复性的淀粉样纤维（他）<sub&gt; 6</sub&gt; -tagged重组Shadoo蛋白表达的包涵体<em&gt;大肠杆菌</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

purification-refolding-to-amyloid-fibrils-his-6-tagged-recombinant

Research

JoVE Journal

Biology

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

14.1K 次观看.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 

视频: Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

淀粉样β-蛋白，阿尔茨海默氏病的病原体适配的选择

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

科研

神经科学

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

生物化学

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

生物学

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

淀粉样前体蛋白胞内结构域的分离纯化和聚集

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

医学

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

从脑组织提取物中分离淀粉样蛋白原纤维

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

纯化及复性的淀粉样纤维（他）

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来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

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