这项工作是由加拿大健康研究学会的支持。 CSC是由一个从自然科学和工程研究理事会，加拿大博士后奖学金。 FD是Tier II加拿大研究主席。

<html>整体重建过程重整过程开始用纯化的MalFGK 2复合物中的洗涤剂溶解的磷脂存在下通过混合膜支架蛋白（MSP）。该步骤之后是由吸附剂的聚苯乙烯材料称为生物珠或Amberlite（ 图1），由缓慢除去洗涤剂。的自动装配过程的发生是最有可能的，因为非极性疏水磷脂，的MalFGK 2配合物和表面的两亲性的MSP蛋白之间的相互作用。最终产物是一种盘状的颗粒制成的两个分子的MSP包装周围MalFGK 2配合物 。颗粒的加合物和聚集体，通过超离心分离和分析的体积排阻色谱法分离。该颗粒的特征在于由母语的凝胶电泳和动态光散射光谱。 标题“&gt; 1。制备的膜支架蛋白，MSP <ol><li>的his标记MSP（版本MSP1D1 1）产生从质粒pMSP1D1在大肠杆菌大肠杆菌 BL21（DE3），在OD 600〜0.5诱导与0.5mM的异丙基-β-D-1-硫代半乳糖苷3小时，在37℃下</li><li>以5,000 xg离心10分钟，在4℃通过离心收获细胞，并再悬浮在TSG10缓冲器含有100μM苯甲基磺酰氟。 </li><li>细胞裂解与法国媒体（3次），在8,000 psi的，并通过离心除去不溶性物质在4℃以5,000 xg离心10分钟含可溶组分的MSP通过超离心分离，以125,000 xg离心45分钟。 </li><li> MSP是纯化通过使用镍螯合层析〜1.5毫升Ni Sepharose 4B柱HP TSG10缓冲。污染物被冲走TSG10缓冲液含有50mM咪唑。 MSP洗脱TSG10缓冲区，其中包含600毫米咪唑。纯化的蛋白质TSG10缓冲区中透析，并储存在-70℃下在〜10-15毫克/毫升的蛋白质浓度。 </li></ol> 2。制备的MalFGK 2复杂<ol><li>该MalFGK 2复合物，他在C-末端的MalK标签的，表示从质粒在大肠杆菌 pBAD22-FGK 大肠杆菌 BL21（DE3）中，并在OD 600诱导〜0.5，在37℃下3小时用0.2％的L-阿拉伯糖</li><li>之后的细胞裂解和离心步骤1.3中，该膜的颗粒再悬浮TSG20缓冲器中的最终浓度为5毫克/毫升。该材料溶解用1％w / v的正十二烷基-β-吡喃麦芽糖苷（DDM）的3小时，在4℃下轻轻摇动。 </li><li>步骤1.3中，通过超离心除去不溶性物质和上清液含有溶解 MalFGK 2复合物的收集和纯化的步骤1.4中，但没有透析。 </li><li> Furtheŕ纯化通过凝胶过滤色谱法在TSGD缓冲器实现的Superdex 200 HR 10/300柱的流速为0.5毫升/分钟。 </li></ol> 3。磷脂制备<ol><li>一个E.大肠杆菌总脂质提取物溶解在氯仿中1000纳摩尔的等分试样，在螺旋盖的微量离心管中被分离成。蒸去溶剂，在温和的氮气流中，并在真空干燥器中进一步干燥过夜。 </li><li>脂质膜溶解在TS缓冲液5 nM的最终浓度和涡旋大力和超声处理。溶解脂肪，由于其不溶于水TS缓冲会出现轻微的不透明，但他们应该保持悬浮状态。 DDM被添加至终浓度为0.5％（〜10毫摩尔），在该溶液变得清晰。 </li><li>的脂类混合物被放置在水浴中和脉冲超声处理5倍〜5秒。脂质混合物在4℃下的最大的1凌晨存储K。 </li></ol> 4。下游的生物珠<ol><li>大约为10-15毫升（干体积）生物珠放入50毫升的试管。 </li><li>的珠子依次用50ml 100％的甲醇，95％乙醇中的MilliQ H 2 O，和最后TS缓冲器（两次，每次）。 </li><li>洗过的小珠被存储在4℃下在〜10毫升TS缓冲器。 </li></ol> 5。 Nanodisc重建<ol><li>在TSGD缓冲液稀释的MSP〜7毫克/毫升（〜0.3毫摩尔）在终浓度为。 </li><li> 〜2 nmol的纯化MalFGK 2配合物进行混合的蛋白质：MSP：类脂比1:3:60或1:3:400 TSGD缓冲区。最终浓度为6的μMMalFGK 2，18μM的MSP和360μM的脂质（1:3:60）或2.4mM脂质（1:3:400）。的最终体积为300微升。 DDM的最终浓度为0.08％（〜1.6毫摩尔）。甘油的最终浓度取决于脂质的量添加，但仍然大约5-10％V / V。 </lI&gt; <li> 〜50微升生物珠子悬架被添加到所述管，将混合物摇椅表上在4℃下孵育过夜</li><li>珠粒沉淀通过重力和该溶液用移液管通过一个狭窄的尖端尽可能多的生物珠尽可能避免。 </li><li>大的析出物以100,000 xg离心20分钟超离心除去。光盘TSG10缓冲区在步骤2.4中，通过凝胶过滤层析纯化。包含光盘的馏分被汇集，并使用其等分应重新注入相同的凝胶过滤柱，来测试该制剂的稳定性。 </li><li>将纯化的光盘可以存储在-70℃下在较长的一段时间。在冰上解冻，光盘应进行超离心在步骤5.5去除潜在的沉淀物。的等分试样应通过凝胶过滤色谱法进行分析，以确保，解冻期间未发生显着的聚集或沉淀的过程。 </li></ol> 6。变性凝胶电泳<ol><li>奈米圆盘（1μM〜0.2毫克/毫升），以英语为母语的第6，第7页的Tris-HCl pH值为8.8，（4-12％）的重组（ 图3A）的质量进行评估分析。 </li><li>奈米圆盘的结合男性（1μM）的MalFGK 2复杂的重组，也进行了评估以英语为母语-PAGE（ 图3A）。 </li><li>电泳后，将凝胶染色10分钟，用考马斯亮蓝，和脱色〜1小时。 </li></ol> 7。动态光散射（DLS） <ol><li>奈米圆盘TSG10缓冲区使用的Superdex 200 HR 10/300柱的流速为0.1毫升/分钟，在步骤2.4中，通过凝胶过滤色谱法进行纯化。含有奈米圆盘的馏分被汇集，并浓缩至约10毫克/毫升的Amicon离心过滤。 </li><li>将样品过滤两次（0.22μm过滤器）进行分析之前通过DLS使用一个DynaPro NANOSTAR仪器（悦科技）在1微升内卷石英比色皿。数据使用动力学软件（Wyatt技术）来估计重量的颗粒的直径和分子嵌合。 </li></ol> 8。 ATP酶活性测量<ol><li> ATPase活性的重组MalFGK 2奈米圆盘确定用比色法检测8。 </li><li> 1μM的纯化的光盘和1mM ATP的混合在一起，与递增量的MalE和孵育在37℃下20分钟。无机磷酸盐的释放在660nm处测量。 </li><li>释放的磷酸盐的量进行比较磷标准溶液与产生的标准曲线。 </li></ol> 9。代表性的成果奈米圆盘被纯化，通过凝胶过滤色谱法（ 图2A，左 ）。的色谱图显示，大多数的重组ðISCS（黑色线）洗脱作为一个单一的峰，而用过量的脂质（红色迹线）的空隙体积中洗脱，并作为一个系列的宽峰的光盘。进一步分析非变性凝胶电泳和动态光散射光谱（DLS）的奈米圆盘的质量。正确重组的光盘作为一个尖锐的能带在凝胶上迁移而那些重组脂质过量的存在下在作为涂抹（ 图2A，右 ）迁移。利用动态光散射（DLS）分析显示该光盘的人口是均匀的，其平均粒径为11.4纳米（ 图2B）。重组后的光盘具有的表观分子量215 kDa的基于上的DLS近似。过剩的脂质的存在下，在重组的样片显示器广泛分布半径约100 nm，这是典型的非均质样本。 母语的凝胶电泳和其活性由ATP酶的MalFGK 2配合物的质量的评估是通过测量（ 图3）。麦芽糖结合蛋白的男性结合的高亲和性的MalFGK 2转运9，10。使用非变性凝胶电泳，它能够检测2 MALE和MalFGK的（ 图3A）之间的复合物。刺激的MalK ATP酶活性的男性在图3（b）所示。 <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="图1"/> 图1。典型的流程重组协议。 <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="图2"/> 图2。质量控制的nanodisc准备。凝胶过滤分析（Superdex 200 HR 10/300柱）的奈米圆盘重组，低脂肪比率（60年1月3日，黑色迹线）或高血脂比率（1/3/400的红色曲线）。非变性凝胶电泳同一张光盘的准备。摩尔以kDa表示ecular重量标记。 B.动态光散射分析同一张光盘的准备。 <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="图3"/> 图3。的MalFGK的2 - nanodisc颗粒分析A.。培养的男性越来越多的MalFGK 2-nanodisc的颗粒凝胶移位分析。 B. ATPase活性的MalFGK 2 nanodisc颗粒作为功能的MalE浓度。

<ol>
	<li>Denisov, I. G., Ginkova, Y. V., Lazarides, A. A., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+self-assembly+of+monodisperse+phospholipid+bilayer+Nanodiscs+with+controlled+size.">Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.</a> J. Am. Chem. Soc. 126, 3477-3487 (2004).</li><li>Boldog, T., Grimme, S., Li, M., Sligar, S. G., Hazelbauer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+separate+chemoreceptor+oligomeric+states+and+reveal+their+signaling+properties.">Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.</a> Proc. Natl. Acad. Sci. USA. 103, 11509-11514 (2006).</li><li>Bass, B. J., Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homotropic+cooperativity+of+monomeric+cytochrome+P450+3A4+in+a+nanoscale+native+bilayer+environment.">Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment.</a> J. Biol. Chem. 282, 7066-7076 (2007).</li><li>Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S. G., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+unravel+the+interaction+between+the+SecYEG+channel+and+its+cytosolic+partner+SecA.">Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.</a> EMBO J. 26, 1995-2004 (2007).</li><li>Mi, L. -. Z., Grey, M. J., Nishida, N., Walz, T., Lu, C., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+stability+of+the+epidermal+growth+factor+receptor+in+detergent+micelles+and+phospholipid+nanodiscs.">Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs.</a> Biochemistry. 47, 10314-10323 (2008).</li><li>Sch&#228;gger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217, 220-230 (1994).</li><li>Dalal, K., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+SecY+translocon+in+Nanodiscs.">Reconstitution of the SecY translocon in Nanodiscs.</a> Methods Mol. Biol. 619, 145-156 (2010).</li><li>Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., Candia, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+assay+for+nanomole+amounts+of+inorganic+phosphate.">An improved assay for nanomole amounts of inorganic phosphate.</a> Anal. Biochem. 100, 95-97 (1979).</li><li>Davidson, A. L., Dassa, E., Orelle, C., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+evolution+of+bacterial+ATP-binding+cassette+systems.">Structure, function and evolution of bacterial ATP-binding cassette systems.</a> Microbiol. Mol. Biol. Rev. 72, 317-364 (2008).</li><li>Bordignon, E., Grote, M., Schneider, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maltose+ATP-binding+cassette+transporter+in+the+21st+century-towards+a+structural+dynamic+perspective+on+its+mode+of+action.">The maltose ATP-binding cassette transporter in the 21st century-towards a structural dynamic perspective on its mode of action.</a> Mol. Microbiol. 77, 1354-1366 (2010).</li><li>Alvarez, F. J., Orelle, C., Davidson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+an+ABC+transporter+for+use+in+electron+paramagnetic+resonance+spectroscopy.">Functional reconstitution of an ABC transporter for use in electron paramagnetic resonance spectroscopy.</a> J. Am. Chem. Soc. 132, 9513-9515 (2010).</li><li>Ritchie, T. K., Grinkova, Y. V., Bayburt, T. H., Denisov, I. G., Zolnerciks, J. K., Atkins, W. M., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+in+phospholipid+bilayer+Nanodiscs.">Reconstitution of membrane proteins in phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 464, 211-231 (2009).</li><li>Gl&#252;ck, J. M., Koenig, B. W., Willbold, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+allow+the+use+of+integral+membrane+proteins+as+analytes+in+surface+plasmon+resonance+studies.">Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.</a> Anal. Biochem. 408, 46-52 (2011).</li><li>Wan, C. -. P. L., Chiu, M. H., Wu, X., Lee, S. K., Prenner, E. J., Weers, P. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein-induced+conversion+of+phosphatidylcholine+bilayer+vesicles+into+nanodisks.">Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks.</a> Biochim. Biophys. Acta (BBA). 1808, 606-613 (2011).</li><li>Nath, A., Trexler, A. J., Koo, P. K., Miranker, A. D., Atkins, W. M., Rhoades, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+spectroscopy+using+phospholipid+bilayer+Nanodiscs.">Single-molecule fluorescence spectroscopy using phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 472, 89-117 (2010).</li><li>Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochromes+P450+in+Nanodiscs.">Cytochromes P450 in Nanodiscs.</a> Biochim. Biophys. Acta. 1814, 223-229 (2011).</li><li>Zhang, X. X., Chan, C. S., Bao, H., Fang, Y., Foster, L. J., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+and+SILAC-based+mass+spectrometry+to+identify+a+membrane+protein+interactome.">Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome.</a> J. Proteome Res. , (2011).</li></ol>

没有利益冲突的声明。

我们描述了一个简单的程序，重组的麦芽糖转运到奈米圆盘。转运体ATPase活性和与可溶性的结合配偶体的男性的相互作用（ 图3）可以重新创建。成功重组的转运到奈米圆盘开启方式为更多的生物物理和生物化学分析。特别令人感兴趣的是系统的分析MalK ATP酶和麦芽糖运输活动的清洁剂，脂质体和奈米圆盘。 ABC转运过程中改变构象的运输周期，但脂质，这些构象变化的贡献仍有待探讨...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的Amicon Ultra-50K离心式过滤器</td><td>密理博</td><td> UFC805008 </td><td>按照制造商的协议，为正确使用</td></tr><tr><td>生物珠SM-2吸附剂</td><td> Bio-Rad公司</td><td> 152-3920 </td><td></td></tr><tr><td> 大肠杆菌总脂质</td><td>阿凡提极性脂质</td><td> 100500C </td><td>溶解在氯仿中，处理以适合的有机溶剂的</td></tr><tr><td> Ni Sepharose 4B柱HP树脂</td><td> GE医疗集团</td><td> 17-5268-01 </td><td></td></tr><tr><td>磷标准溶液</td><td>Sigma-Aldrich公司</td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE医疗集团</td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表一特定的试剂。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 名称 </td><td> 组成 </td><td> 评论 </td></tr><tr><td> DDM股票</td><td> 10％w / v的DDM </td><td>重悬在MilliQ水和存储在-20°C </td></tr><tr><td> MalFGK 2股票</td><td> 1-2毫克/毫升 的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油 0.03％w / v的DDM </td><td>商店纯化后，在-70℃下</td></tr><tr><td> MSP股票</td><td> 10-15毫克/毫升 的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油</td><td>储存于-70℃的纯化后，在&lt;1毫升的等分试样，并避免过度的冷冻/解冻循环</td></tr><tr><td>磷脂股票</td><td> 5纳米E.大肠杆菌总脂质 0.5％w / v的（10毫摩尔）DDM 的50mM Tris-HCl，pH值7.9; 50mM NaCl的</td><td>在4℃下储存1周</td></tr><tr><td> TS缓冲</td><td>的50mM Tris-HCl，pH值7.9; 50mM NaCl的</td><td>储存于4°C </td></tr><tr><td> TSG10缓冲</td><td>的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油</td><td>储存于4°C </td></tr><tr><td> TSG20缓冲</td><td>的50mM的Tris-HCl，PH8 100mM NaCl的 20％v / v甘油</td><td>储存于4°C </td></tr><tr><td> TSGD缓冲</td><td>的50mM Tris-盐酸，pH7.9 100mM NaCl的 10％v / v甘油 0.03％w / v的DDM </td><td>商店在4°C，并添加之前使用的DDM </td></tr><tr><td colspan="3"> 表二。解决方案的食谱。 </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

nanodisc是一个圆盘状的颗粒（〜10-12纳米大），成一小片的磷脂双分子层的陷阱膜蛋白。该nanodisc的研究，尤其是膜蛋白的配体 - 受体相互作用的上下文中的一个特别有吸引力的选择。该方法率先由Sligar和同事的两亲性质的基础上设计高度α-螺旋支架蛋白载脂蛋白A1来自。支架蛋白的疏水性表面的相互作用与脂肪酰基侧链的脂双层，而极性的区域面对的含水环境。奈米圆盘膜蛋白的分析有显着的优势脂质体，因为颗粒是小的，均匀的和水溶性的。此外，通常保留以可溶性蛋白的生物化学和生物物理的方法可以被应用，并从膜的任一侧。在这种可视化的协议，我们提出了一个一步一步的重组的良好特征在于细菌ABC转运，男性，MalFGK的的2复杂。在盘的形成是一个自组装过程中，依赖于疏水相互作用发生期间逐步除去洗涤剂。我们描述的基本步骤，我们强调的重要性选择正确的蛋白质 - 类脂比，以限制形成的聚集体和较大的多分散性的脂质体样颗粒。简单的质量控制，例如凝胶过滤色谱法，天然凝胶电泳和动态光散射光谱，确保光盘已正确重组。

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

奈米圆盘是小盘状颗粒结合成一小片的磷脂双分子层膜蛋白。我们提供了一个可视化的协议，一步一步注册成立的MalFGK2转运到光盘。

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

a-step-step-method-for-reconstitution-an-abc-transporter-into

Research

JoVE Journal

Biology

25.3K 次观看.  University of British Columbia. 奈米圆盘是小盘状颗粒结合成一小片的磷脂双分子层膜蛋白。我们提供了一个可视化的协议，一步一步注册成立的MalFGK2转运到光盘。

视频: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

科研

生物学

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

过程评价絮凝剂的效率的分散粒子的从植物提取物的去除

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...