支持这项工作是由威康信托基金资助[077108]荣获GJW。

1。编译诱饵和猎物质粒表达载体的图书馆<ol><li>编译的细胞表面受体的列表和筛选相互作用的蛋白质分泌的兴趣，并从一个合适的蛋白质序列数据库检索它们的DNA序列。适合大多数蛋白质含有N-末端分泌信号肽（其中包括I型，磷脂挂钩和分泌的蛋白质）。通常情况下，蛋白质家族（如免疫球蛋白超家族或限制到特定的细胞类型，如红细胞的所有受体）会被选中。 </li><li>通过识别信号肽和跨膜区的蛋白质功能预测软件的位置确定外地区的程度。我们通常使用SignalP V3.0 5和TMHMM V2.0 6。 </li><li>设计的引物，扩增整个每个受体胞外片段。我们有一个套件，是为这个目的，这是一个合适的诱饵和猎物载体从Addgene（vailable <a href="http://www.addgene.org/">http://www.addgene.org/~~V</a> ）。设计感底漆周围蛋氨酸开始，包括NotI双酶切位点和最佳Kozak序列，这是由25个碱基对精确匹配特定的基因序列（图2A）。 </li><li>设计反义引物，只是之前预测的跨膜区，以作为一种可溶性的重组蛋白表达的整个胞外截断I型受体蛋白。在此底漆包括的ASCI限制的网站应该翻译框架的C-末端大鼠Cd4d3 +4蛋白标记7（图2A）。 </li><li>放大胞外所有使用这些引物的基因片段，并克隆到诱饵或猎物载体。我们有时会发现它有助于他们首先克隆到穿梭载体亚克隆到一个适当的哺乳动物表达载体之前，我们使用衍生pTT3载体3,4。诱饵和猎物载体contai为n C-端蛋白标签（如下图2B）。 <ol><li>诱饵：一个标签包含域的第3和第4的大鼠CD4（Cd4d3 +4），其次是大肠杆菌的BirA酶的底物的一种肽序列，因此可以在一个特定的赖氨酸残基monobiotinylated的蛋白（图2B） 。 OX68单克隆抗体的Cd4d3 +4标签被识别和量化表达诱饵蛋白的酶联免疫吸附试验。诱饵蛋白因此，单体和monobiotinylated的。附加诱饵蛋白更换为净化6-His标签的载体，其中biotinylatable肽。 </li><li>猎物：猎物还包含1大鼠Cd4d3的+4从大鼠软骨寡聚基质蛋白（COMP）和C-末端β-内酰胺酶的肽序列标签。 COMP肽确保猎物的五聚体形式的一次表达（图2B）。 </li></ol></li></ol> 2。猎物和诱饵蛋白表达我们使用方便的高层次表达系统使用悬浮培养4号线的HEK293E细胞产生的蛋白质库，但任何哺乳动物表达系统应该是合适的。市售的替代方案是自由式系统。细胞常规培养用颤抖的平台（125转）在37°C，5％CO 2，相对湿度70％。正反两方面的控制蛋白质也必须表达。大鼠Cd4d3 +4标签片段作为诱饵和猎物是合适的阴性对照。同样，诱饵和猎物载体编码阳性对照（Addgene）;我们通常使用的大鼠CD200-Cd200R互动的8。 <ol><li>种子的HEK293E细胞转染前一天在一个2.5×10 5细胞密度毫升-1。我们日常文化细胞在50毫升Freestyle293媒体制造商的建议量。为了确保有效的生物素，补充ţ他用来制造诱饵蛋白与D-生物素细胞培养液中终浓度为100μm。用来表达猎物蛋白中等不需要额外的D-生物素补充。 </li><li>翌日，使用合适的转染试剂转染细胞（如293fectin）。使用25微克的诱饵或猎物质粒结构，转染50毫升的文化。 为了生产生物素饵，共同转染质粒构建诱饵质粒编码的大肠杆菌生物素蛋白连接酶的分泌形式（ BIRA）是从Addgene提供。在10:1的比例（2.5微克）的分泌BirA蛋白的质粒共同转染的诱饵质粒。 </li><li>收获文化在3000×Ğ离心20分钟，上清液过滤通过0.22微米过滤器由第一制粒细胞转染后5天。 </li></ol> [提示：诱饵和猎物的蛋白质S应该在4°C储存稀释，作为一般指引，应在一年内使用。加入bacteriostatics如50微克/毫升多粘菌素B硫酸或10毫米南三上清，以防止细菌污染。 3。正常化的诱饵和猎物样本的的AVEXIS法的主要特点之一是，重组蛋白的分泌，因为他们可以稀释或集中（归）内检测中被使用之前设立的门槛活动。我们已经发现，在重组蛋白的表达水平跨越了广泛 - 高达4个数量级 - 等正常化的步骤，在屏幕上显着降低误报数量。 3.1诱饵正常化注：D-生物素未结合，必须从媒体（通常是通过透析），因为它会与生物素白ţ蛋白结合链亲和素结合位点。 <ol><li>诱饵上清转移到透析管，标签，安全夹和反对，如PBS合适的缓冲，以去除游离生物素广泛透析。 </li></ol> [提示：我们发现，通常为6×50毫升对体积的PBS，4.5大号诱饵，有4个在24小时内的变化上清达到充分透析。不足透析可以观察到在诱饵正常化的步骤（图3A）。抑制蛋白结合低稀释<ol start="2"><li>执行酶联免疫吸附试验，以确定其中诱饵蛋白表达的相对水平。准备系列稀释的生物素诱饵蛋白和捕捉微孔板上的链亲和素涂层。使用大鼠Cd4d3 +4标签的检测和量化的捕获诱饵蛋白的单克隆抗体（OX68）。诱饵蛋白浓缩或稀释到所需的最低金额，所有的Biot饱和在一个链霉亲和涂层板以及结合位点。 </li><li>与磷酸盐的链霉亲和涂层板座，水井缓冲saline/0.1％吐温20（PBST）/ 2％的牛血清白蛋白（BSA）为15分钟。 </li><li>在一个单独的板块，使PBST / 2％BSA和一个链霉亲和涂层板转移到水井中的诱饵蛋白系列稀释（通常就足够了四个1时03稀释）。在室温下孵育1小时。 </li><li>在PBST洗3倍。加入100μL2μg/ mL的鼠标灭鼠Cd4d3 +4抗体（OX68）1小时室温孵育。 </li><li>在PBST洗3倍。加入PBST / 2％牛血清白蛋白在1:5,000稀释100μL抗鼠碱性磷酸酶孵育1小时室温。 </li><li>在PBST洗3倍和执行最终在PBS洗，以去除任何残留的洗涤剂。加入100μL1毫克/毫升基质在乙醇胺缓冲区（二乙醇胺10％（V / V），MgCl 2的 0.5毫米，10毫米为NaN 3，pH9.8，存放避光4 104℃）。 </li><li>一个小时后，测量在405 nm处的吸光度。代表的诱饵正常化结果显示在图3A。 </li><li>诱饵蛋白质表达水平不足以饱和的链霉亲和涂层板的所有生物素结合位点，用稀释时，应集中使用Vivaspin集中（Vivascience，截留分子量10kDa）。为指导，我们估计，一个典型的诱饵蛋白（〜80 kDa的）〜5微克是足够饱和的96孔板。 <ol><li>在高水平表达诱饵蛋白可稀释PBST / 2％BSA的浓度足以饱和的链霉亲和涂层板。诱饵的活动，应稀释或浓缩后复查，以确保他们所有的生物素结合位点的链霉亲和涂层板饱和。 </li></ol></li></ol> 3.2捕食正常化<ol><li>量化相对猎物蛋白的表达水平，使用β-内酰胺酶的活性。 </li><li>首先，一个包含猎物蛋白在PBST / 2％BSA的缓冲上清稀释系列。然后加入20μL的稀释至60μL242微米（0.125毫克/毫升）硝噻吩溶液（Calbiochem公司）。 </li><li> 立即转移到微孔板阅读器，需要20分钟，在室温下在每分钟485 nm处的吸光度测量板。代表的猎物正常化结果如图3B所示。 </li><li>集中使用离心浓缩的样品或稀释PBST / 2％BSA〜2 nmol分钟-1硝噻吩的营业额达到一个阈值活动。实际上，这是实现如果所有的硝噻吩〜7分钟内水解。 </li></ol> 4。 AVEXIS筛选程序<ol><li>在PBST洗链霉亲和涂层板。 </li><li>每孔加入100μLPBST / 2％BSA孵育30分钟，以阻止任何虚假的蛋白质结合位点，在每口井。 </li&gt; </ol> [提示：去除任何残留的洗涤缓冲液洗涤步骤后，挖掘板块 - 大力 - 纸巾] <ol start="3"><li>取出阻塞的解决方案与智能多片板轻弹，添加100μL，规范化的生物素化的单体诱饵，以适当的井和1小时室温孵育。 </li><li>从板的诱饵样品上翻板和巧妙超过水槽弹，并在PBST洗3次。 </li><li>加入100μL的归pentamerized猎物每口井的建设，并在室温孵育1小时，洗净，在步骤4.4，只执行最后用PBS洗。 </li><li>加入60μL242微米硝噻吩溶液（硝噻吩5毫克溶于500μL二甲基亚砜，调匀，然后进一步稀释在40毫升PBS硝噻吩并通过0.22μm过滤器前通过增加板），并在室温下孵育。可以观察到良性互动通过眼睛，因为黄色的硝噻吩水解红色。量化阅读使用酶标仪在485 nm处的吸光度。一个典型的板块和定量图所示。 4。 </li></ol> [提示：良性互动通常在室温下观察，在2小时内，虽然板应放置16小时，在4°C，以确保所有潜在的相互作用检测。我们还发现偶尔会导致沉淀nitrocefin和塑料如划痕/指纹微孔板上的缺陷可以在485 nm处的不真实误报，​​尽管没有明显的硝噻吩水解。因此，建议采取板块的照片直观地记录在井硝噻吩的营业额。] 5。代表结果一个有代表性的AVEXIS筛选板的一个例子如图4所示。每个人都应该遵守水解硝噻吩在基板（黄色到红色）阳性对照井（抗体介导的捕获猎物的控制和阳性对照互动）大约在十几分钟内。在屏幕的某些相互作用还观察到在这段时间内的规模，但其他人可能需要更长的时间（几个小时）出现。通常情况下，命中率约0.4-0.6％（为被甄别每150-250相互作用的良性互动）是典型的，取决于正在筛选蛋白质库。 <img alt="图1" src="/files/ftp_upload/3881/3881fig1.jpg" /> 图1。受体-配体相互作用介导细胞识别过程AVEXIS鉴定 。示意图显示了两个相互作用的细胞（A和B）交换信息通过嵌入膜受体蛋白之间的相互作用。 AVEXIS是专门设计，以确定新的受体 - 配体的特点是非常弱相互作用优势对一个可伸缩的蛋白质相互作用检测。重组，以便luble蛋白库，占整个胞外两种细胞的细胞表面受体的剧目被编译成五聚体β-内酰胺酶标记的猎物（细胞A型）或单体生物素标记的诱饵（细胞B型）。猎物的pentamerisation增加当地的胞外区的浓度，对整体的亲和力增益，所以可以发现，即使是很短暂的相互作用。链霉亲和涂层钢板上摆着诱饵库和系统探讨与猎物蛋白相互作用。经过短暂的洗涤，任何捕获猎物被发现使用β-内酰胺酶的活性与猎物的关联。 <img alt="图2" src="/files/ftp_upload/3881/3881fig2.jpg" /> 图2。诱饵和猎物的结构设计。 （一）提供大鼠CD200受体蛋白作为整个细胞表面的受体蛋白的胞外区是如何确定的，并引物的设计，使两个诱饵的例子和猎物的表达结构。从N-端信号肽跨膜大鼠CD200跨膜区的cDNA，并翻译成蛋白质序列。感引物的设计，包括5&#39;NotI双酶切网站和CD200 cDNA序列的25个基地的精确匹配序列的最佳Kozak序列。反义引物包含一个的ASCI网站和25基地位于立即上游选定的胞外截断残留精确匹配序列。为了让与大鼠Cd4d3 +4标签帧的胞外，ASCI网站应转化为甘氨酸，丙氨酸-PRO。 （二）诱饵和猎物的表达结构的示意图表示。每一个都包含NotI双和ASCI地点和Cd4d3的+4标记两侧的受体胞外区。诱饵包含一个C-末端肽序列表达质粒的分泌的BirA酶，可以专门染monobiotinylated。其次由pentamerisation序列的猎物软骨寡聚基质蛋白（COMP）和β-内酰胺酶。 <img alt="图3" src="/files/ftp_upload/3881/3881fig3.jpg" /> 图3。诱饵和猎物的蛋白质正常化。诱饵正常化（ 一 ）代表性的例子。稀释系列的四种不同透析诱饵上清用ELISA法检测，使用抗CD4标记单克隆抗体。四诱饵是1&gt; 2&gt; 3&gt; 4不同程度的表达。毒饵应正常化饱和阈值水平，在每口井的生物素结合位点。高表达的诱饵可稀释节约蛋白质（如诱饵1可以稀释〜1:100），不善表达的诱饵集中。有时也观察到一些诱饵（如诱饵2），这是由于游离D-生物素的存在，由于不完整的透析减少在低稀释的读数。 （二）在哪个猎物蛋白的水平是前按下的量化使用β-内酰胺酶的底物，硝噻吩水解率。在所示的例子中，在不同层面表达三个猎物：猎物1&gt; 2&gt; 3。应正常化〜2 nmol -1分钟，相当于在7分钟内完成14.5 nmol硝噻吩水解（如猎物1）活动的猎物。在这个例子中，其他的猎物，应集中使用前筛查。 <img alt="图4" src="/files/ftp_upload/3881/3881fig4.jpg" /> 图4。代表AVEXIS筛选结果。 （A）一个有代表性的AVEXIS筛选板的照片。猎物蛋白探讨对41个不同的饵料和检测以及E2的良性互动。在筛查板使用的控制包括：生物素化的抗CD4抗体捕获猎物蛋白添加到井（G6），控制互动：大鼠CD200（诱饵）Cd200R（猎物）与Cd200R的的猎物在门槛稀释使用（高3），1:10（H4）和1:100型（H5）。阴性对照：1 Cd4d3 +4诱饵探测与猎物蛋白（H1）的筛选和Cd200R猎物（H2）。 （二）一式三份执行相同的屏幕吸光度值量化。数据点的平均值±标准差。

<ol>
	<li>Fagerberg, L., Jonasson, K., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+the+human+membrane+proteome.">Prediction of the human membrane proteome.</a> Proteomics. 10, 1141-1141 (2010).</li><li>Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+initiation+in+biological+systems:+the+properties+and+detection+of+transient+extracellular+protein+interactions.">Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions.</a> Molecular bioSystems. 5, 1405-1405 (2009).</li><li>Bushell, K. M., Sollner, C., Schuster-Boeckler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+screening+for+novel+low-affinity+extracellular+protein+interactions.">Large-scale screening for novel low-affinity extracellular protein interactions.</a> Genome research. 18, 622-622 (2008).</li><li>Durocher, Y., Perret, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+and+high-throughput+recombinant+protein+production+by+transient+transfection+of+suspension-growing+human+293-EBNA1+cells.">High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.</a> Nucleic acids research. 30, e9-e9 (2002).</li><li>Bendtsen, J. D., Nielsen, H., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Prediction+of+Signal+Peptides:+SignalP+3.0.">Improved Prediction of Signal Peptides: SignalP 3.0.</a> Journal of molecular biology. 340, 783-783 (2004).</li><li>Krogh, A., Larsson, B., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.</a> Journal of molecular biology. 305, 567-567 (2001).</li><li>Brown, M. H., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+immunoglobulin+and+scavenger+receptor+superfamily+domains+as+chimeric+proteins+with+domains+3+and+4+of+CD4+for+ligand+analysis.">Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis.</a> Protein engineering. 7, 515-515 (1994).</li><li>Wright, G. J., Puklavec, M. J., Willis, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid/neuronal+cell+surface+OX2+glycoprotein+recognizes+a+novel+receptor+on+macrophages+implicated+in+the+control+of+their+function.">Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.</a> Immunity. 13, 233-23 (2000).</li><li>Dustin, M. L., Golan, D. E., Zhu, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+affinity+interaction+of+human+or+rat+T+cell+adhesion+molecule+CD2+with+its+ligand+aligns+adhering+membranes+to+achieve+high+physiological+affinity.">Low affinity interaction of human or rat T cell adhesion molecule CD2 with its ligand aligns adhering membranes to achieve high physiological affinity.</a> The Journal of biological chemistry. 272, (1997).</li><li>Wright, G. J., Martin, S., Bushell, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+identification+of+transient+extracellular+protein+interactions.">High-throughput identification of transient extracellular protein interactions.</a> Biochemical Society transactions. 38, (2010).</li><li>de Wildt, R. M., Tomlinson, I. M., Ong, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+receptor-ligand+pairs+by+capture+of+long-lived+multivalent+interaction+complexes.">Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, (2002).</li><li>Jiang, L., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+leucocyte+surface+protein+interactions+by+high-throughput+screening+with+multivalent+reagents.">Identification of leucocyte surface protein interactions by high-throughput screening with multivalent reagents.</a> Immunology. 129, 55-55 (2010).</li><li>Urech, D. M., Lichtlen, P., Barberis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+growth+selection+system+to+detect+extracellular+and+transmembrane+protein+interactions.">Cell growth selection system to detect extracellular and transmembrane protein interactions.</a> Biochimica et biophysica acta. 1622, 117-117 (2003).</li><li>Voulgaraki, D., Mitnacht-Kraus, R., Letarte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivalent+recombinant+proteins+for+probing+functions+of+leucocyte+surface+proteins+such+as+the+CD200+receptor.">Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor.</a> Immunology. 115, 337-337 (2005).</li><li>Wojtowicz, W. M., Wu, W., Andre, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vast+repertoire+of+Dscam+binding+specificities+arises+from+modular+interactions+of+variable+Ig+domains.">A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains.</a> Cell. 130, 1134-1134 (2007).</li><li>Martin, S., Sollner, C., Charoensawan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+Large+Extracellular+Protein+Interaction+Network+and+Its+Resolution+by+Spatiotemporal+Expression+Profiling.">Construction of a Large Extracellular Protein Interaction Network and Its Resolution by Spatiotemporal Expression Profiling.</a> Mol. Cell. Proteomics. 9, 2654-2654 (2010).</li><li>Crosnier, C., Bustamante, L. Y., Bartholdson, S. J., Bei, A. K., Theron, M., Uchikawa, M., Mboup, S., Ndir, O., Kwiatkowski, D. P., Duraisingh, M. T., Rayner, J. C., Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basigin+is+a+receptor+essential+for+erythrocyte+invasion+by+Plasmodium+falciparum.">Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.</a> Nature. 480, 534-537 (2011).</li><li>Sollner, C., Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+surface+interaction+network+of+neural+leucine-rich+repeat+receptors.">A cell surface interaction network of neural leucine-rich repeat receptors.</a> Genome biology. 10, R99-R99 (2009).</li></ol>

作者没有透露。

当体内观察到活细胞的细胞膜表现出高度的动态行为：扩展和回缩膜过程中，为他们联系和沟通，与他们的邻居2。膜嵌入的受体蛋白之间的物理相互作用，调解这些接触很多已经发展到极度虚弱，以便允许这个高度能动的行为。据估计，单体50μM弱相互作用的优势可能足以推动在生理上的受体密度9，这使得检测新颖的互动，极具挑战性的2,10自发的相互作用。 AVEXIS这里描述的方法提供了一个敏感的方法检测瞬态胞外蛋白：蛋白具有较低的假阳性率，可以在一个可扩展的方式实现的交互。而其他可伸缩的方法已发展到检测外的相互作用11-15一AVEXIS优势是诱饵和猎物的活动，如实验参数已被量化，甚至检测最弱的相互作用（T 1/2≤0.1秒），同时保持较低的假阳性率3。此外，精简和健壮的样品制备程序已经启用此法可实现更大的规模上比其他实验，我们最近描述了系统的互动屏幕，共计超过16500 16〜潜在的相互作用。 该法可以对任何蛋白质，它是可能的表达可溶性重组蛋白片段。因此，这包括蛋白质，含有如类型我的GPI挂钩和分泌的蛋白质N-末端的信号肽。最近，我们设计了一个载体，现在让我们来表达II型蛋白作为诱饵17套房。该法一般不适合多通道的膜蛋白，如离子转运或泵，因为它们是不可能被正确折叠外质膜中。我们已申请到各种不同的系统，并已成功地表达胞外蛋白3,16,18斑马鱼，人类，小鼠和P.恶性的互动屏幕。 设立AVEXIS屏幕所需的资源方面，我们已经找到了一个重要的瓶颈是选择，建设，充分表达质粒测序。这方面的方法，可以采取长达一年〜100诱饵和猎物的结构，然而，我们现在用的基因合成降低成本，有利于商业这方面的项目外包。大摇组织培养孵化器（我们使用一个INFORS的高温Multitron细胞）的哺乳动物表达系统，使一个单一的研究员，表达和正常化〜100诱饵和猎物的蛋白质一个月。一旦生产和规范化的蛋白质的互动creening是快速板被方便地每天处理高达12 96。只有定制的设备，我们已经制定促进生产如此大量的蛋白质，是压缩空气驱动活塞，这是用来传递五上清，0.22μm的过滤器通过并行。 上课的时候相比，预计从文学（假阴性）的相互作用可能错过的相互作用主要是同种的相互作用，尽管这些相互作用可以检测3。在没有检测到预期的相互作用的情况下，我们相信，这是由于猎物蛋白（猎物“掩蔽效应”）的同种相互作用的形成，从而防止进一步固定化诱饵蛋白相互作用。相互作用检测的频率取决于被筛选蛋白质库的内容。作为一个读者的指南，屏幕&gt; 30，000个斑马鱼的免疫球蛋白超家族内的相互作用，导致188正片- 3,16,18频率为0.6％。可以进行验证任何积极的命中是非常迅速的检查，以确定是否相互作用可以检测两个诱饵猎物方向;即可以相同的相互作用被发现不论是否结合蛋白作为诱饵或猎物。每当我们观察到了这种回报行为，随后被绑定已证明是由证明直接利用表面等离子体共振的纯化蛋白饱和约束力的具体。应当强调的，因为我们增加人工pentamerising猎物蛋白结合的亲和力，我们不考虑适合的互动优势定量比较分析。为了获得结合生物物理参数，我们使用单体蛋白质和表面等离子体共振的相互作用。最后，一​​旦互动已经确定，您好检测GH吞吐量性质使得它比较容易适应的检测试剂，如抗体或小分子阻止互动屏幕。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 猫。数 </td><td> 评论 </td></tr><tr><td>自由式媒体</td><td> Invitrogen公司</td><td> 12338018 </td><td></td></tr><tr><td>硝基噻吩</td><td> Calbiochem公司</td><td> 484400 </td><td></td></tr><tr><td> SA涂层微量板</td><td> NUNC </td><td> 734-1284 </td><td></td></tr><tr><td> OX68抗体</td><td> AbDSerotec </td><td> MCA1022R </td><td></td></tr><tr><td>透析管</td><td>刺穿</td><td> PN68100 </td><td></td></tr><tr><td>多粘菌素B </td><td>西格玛</td><td> P4932 </td><td></td></tr><tr><td> D-生物素</td><td>西格玛</td><td> B4639-5G </td><td></td></tr><tr><td>基板-104 </td><td>西格玛</td><td> 1040-506 </td><td></td></tr><tr><td> vivaspin-20离心浓缩</td><td>赛多利斯</td><td> VS2002 </td><td></td></tr><tr><td> 0.22微米的过滤器</td><td> NALGENE </td><td> 190-2520 </td><td></td></tr></tbody></table>

avidity-based extracellular interaction screening (avexis) for the scalable detection of low-affinity extracellular receptor-ligand interactions

胞外蛋白：蛋白质分泌或栓膜蛋白之间的相互作用是同时启动间的沟通，并确保在多细胞有机体的凝聚力的关键。蛋白质预计将形成约四分之一的人类基因1编码外的相互作用，但尽管其重要性和丰富，这些蛋白质大多数没有记载约束力的合作伙伴。这主要是由于其生化棘手：膜嵌入蛋白是难以solubilise在其天然构象，并包含结构最重要的翻译后修饰。此外，受体蛋白之间的相互作用的亲和力的特点往往是极低的互动优势，解除他们的检测与许多常用的高通量方法2（半衰期&lt;1秒）。 在这里，我们描述一个试验，AVEXIS（基于亲和力的细胞外LAR互动屏幕），克服这些技术挑战，使检测非常微弱的蛋白质相互作用，具有较低的假阳性率3（T 1/2≤0.1秒）。通常实施该法在高通量格式，使成千上万在一个方便的微孔板格式的相互作用（图1）系统的筛选。它依赖于可溶性细胞表面的受体或分泌的蛋白质内筛选相互作用的胞外片段重组蛋白库包含的生产;因此，这种做法是合适的类型，II型，GPI相连的细胞表面受体和分泌蛋白质，但不为多通道的膜蛋白，如离子通道或转运。 重组蛋白库是使用一个方便的和高层次的哺乳动物表达系统 ，以确保重要的翻译后修饰，如glycosyla添加TION和二硫键。表达的重组蛋白分泌到中期和生产有两种形式：可上的链亲和素涂层的固相筛选适合捕获生物素诱饵，和pentamerised酶标记（β-内酰胺）的猎物。诱饵和猎物的蛋白质是彼此在二进制的方式来检测它们之间的直接相互作用，类似于传统的酶联免疫吸附试验（图1）。在猎物的蛋白质pentamerisation是通过从肽序列的软骨寡聚基质蛋白（COMP）和局部浓度增加，从而提供了大量的亲和力收益，使被检测到即使非常短暂的相互作用的胞外区。诱饵和猎物筛选之前预定水平的活动正常化，我们已经表明，单体的半衰期为0.1秒的相互作用可以用较低的假阳性率检测。

Watch this Scientific Journal Video about Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions at JoVE.com

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS小说外，受体 - 配体对细胞识别过程中所涉及的系统屏幕开发的一种高通量蛋白质相互作用检测。它是专门用于检测短暂的蛋白质相互作用，难以确定使用其他高通量方法。

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

avidity-based-extracellular-interaction-screening-avexis-for-scalable

Research

JoVE Journal

Biology

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

21.4K 次观看.  Wellcome Trust Sanger Institute. AVEXIS小说外，受体 - 配体对细胞识别过程中所涉及的系统屏幕开发的一种高通量蛋白质相互作用检测。它是专门用于检测短暂的蛋白质相互作用，难以确定使用其他高通量方法。

视频: Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

科研

生物学

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

细胞外离子流的使用离子选择性自引用的微电极技术测量

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

一种研究受体配体相互作用的 bw 报告系统

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

细胞外囊泡的量化和尺寸测定纳米粒子跟踪分析

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

通过共聚焦显微镜成像分析进行细胞外囊泡摄取测定

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...