这项研究是由国立新电德拉RECHERCHE（项目ANR-2010-BLAN-1535），并从一个地区法兰西岛大区法国（DIM-Malinf 110058）的资助。

1。蛋白生产与纯化<ol><li> MEXA，MexB 铜绿假单胞菌 <ol><li>变换窝藏MEXA和MexB基因在大肠杆菌的质粒大肠杆菌生产菌株（ 如 C43 DE3）。板的LB琼脂培养基中补充有合适的抗生素。挑取单个菌落接种的O / N预培养。第二天早上，接种预培养1升的LB，并在37℃下搅拌（​​240转）增长。 </li><li>一旦细胞达到对数生长期（OD 600 = 0.6-0.8）诱导用1mM IPTG诱导。在搅拌下进行诱导2-3小时，在30℃。 </li><li>离心细胞（9,000×g离心20分钟），重悬在30毫升的Tris-HCl的20mM，pH为8，氯化钠150mM的。 </li><li>使用法国媒体（10,000 psi的，4通）破坏细胞。通过在9,000 XG离心20分钟不间断的倒掉细胞和包涵体。 </li><li>颗粒膜1小时，在100,000×g离心和resuspend每个粒料在30毫升的Bis-Tris 10mM的pH为7.4，甘油20％（重量/体积），咪唑的10mM，NaCl的500mM的。 </li><li>确定使用BCA比色测定膜的总蛋白浓度和溶解的膜O / N，用2％十二烷基麦芽糖苷（DDM）中所测定的终体积，使得洗涤剂：蛋白质为1:1.5（W / W）。 </li><li>超速离心机在100,000×g离心1小时，以丢弃不溶解的和聚集的材料。 </li><li>培养上清液中2小时，在4℃下与镍树脂（MEXA纯化）或钴类树脂（MexB纯化）平衡过的Bis-Tris 10mM的pH为7.4，甘油5％（重量/体积），咪唑的10mM，NaCl的500毫米，DDM 0.2％。 </li><li>倒入树脂在一个空列。通过收集流量。 </li><li>用50ml的Bis-Tris 10mM的pH为7.4，甘油20％（重量/体积），咪唑的10mM，NaCl的500mM的，DDM 0.2％，然后用25毫升的Bis-Tris 10mM的pH值为6，甘油洗涤树脂5％（重量/体积），咪唑的10mM，NaCl的500mM的，DDM 0.2％。 </li><li>洗脱的Protein用20毫升的Bis-Tris 10mM的pH为7.4，甘油5％（重量/体积），咪唑300mM的，氯化钠的500mM，DDM 0.2％。 </li><li>浓缩的蛋白质与超滤装置向下至3ml和负载平衡的咪唑的缓冲液脱盐柱。只是重建前（见下文），超速离心机的溶解蛋白（100,000×g离心20分钟）摆脱脂类和未溶解的蛋白质。然后再次浓缩蛋白至0.3毫升</li></ol></li><li>从Halobacter salinarium细菌视紫红质<ol><li>在含NaCl的4M，用MgSO 4 150mM的盐，柠檬酸盐的10mM，氯化钾的30mM，酵母提取物5g / L，蛋白胨和为5g / L的液体生长培养基中生长Halobacter salinarium细胞在光照下在37℃下10天</li><li>通过对透析用去离子水破坏的细胞。 </li><li>纯化的30-40％的紫膜（重量/体积）蔗糖梯度（100,000 xg离心17小时）。 </li><li>溶解的膜悬浮液，用2％octylglucoside放置24小时，在37℃（体积为以重悬膜以2:1（W / W）的洗涤剂的溶解：蛋白质比率）。用ε（570纳米）= 54,000 M -1厘米-1估算溶解BR的浓度。只是重建前（见下文），超速离心机溶解的BR（100,000×g离心20分钟）摆脱脂类和未溶解的蛋白质。 注：本机膜是天然富含BR所以没有进一步纯化是必要的。 </li></ol></li></ol> 2。脂蛋白体的制备<ol><li>脂质体的形成：将400微升DOPC的溶解在氯仿（25毫克/毫升）在玻璃烧杯中，并加入1.5毫克胆固醇作为粉末贮存。干他们只是在氮气氛下或真空的蒸汽处理前至少1小时，在玻璃烧杯中。的DOPC：胆固醇的摩尔比为3.3:1。 <ol><li>他们已经干燥后，水合脂质用1ml HEPES 25mM的pH为7，K 2 SO 4 100毫米，氯化镁2 2毫米，羟基芘磺酸2毫米。暂停应该在这个阶段出现浑浊。 </li><li>加热10分钟，将溶液在37℃下超声处理在40瓦与30秒脉冲，30秒的暂停周期在室温10分钟。暂停应该在这个阶段出现明显的。 </li><li>为了获得脂质体的单分散的人口，进行挤压的两个周期，并为每个周期，通过过滤器至少11倍通过脂质体混悬液。对于第一次循环中，使用200nm的膜孔径和第二周期中，使用100nm的膜孔径。动态光散射测量可以在这一步骤被执行以检查该悬浮液的均匀性。 </li></ol></li><li>蛋白质结合 原理：膜蛋白可以重新溶解在脂质体由于清洁剂的增溶作用。洗涤剂溶解的脂质体孵育与p的洗涤剂溶解的膜蛋白和形成roteoliposomes被迅速消除与聚苯乙烯珠9洗涤剂的触发。 <ol><li>加入28毫克的Triton X-100（0.56％最终）和孵化O / N在4℃（溶解温度可适应被研究的蛋白质）。 </li><li>添加洗涤剂溶解的蛋白质（BR，MexB和MEXA）以溶解的脂质体，并培育15分钟，在4℃下蛋白质添加到溶解的脂质体悬浮液在以下比例（w / w）的：脂质/ MexB = 20，MexB / MEXA = 2.5和脂质/ BR = 30。 </li><li>加聚苯乙烯珠以30:1珠：洗涤剂比（w / w的，考虑到洗涤剂的总重量计，包括洗涤剂用纯化的蛋白质中加入），孵育5小时（因为BR的），在暗处在室温下温和搅拌。在此之前的过程中，激活用甲醇和乙醇孵育biobeads并用水充分洗涤。 </li><li>使用PD-10纯化的蛋白脂质蛋白质和游离底物平衡与HEPES 25 mM的pH值为7，K 2脱盐柱SO 4 100毫米和MgCl 2 2毫米。 </li><li>存储脂蛋白在18℃下在黑暗中长达2-3周。 </li></ol></li><li>在蔗糖梯度重建的疗效评估。 要检查，无论是MexB和BR已重组于脂质体，净化上不连续蔗糖梯度的脂蛋白体悬浮液。 <ol><li>轻轻地定居于蔗糖的五层梯度的蛋白脂质体（60％，20％，10％，5％，2.5％，重量/体积）。 </li><li>超速17小时为100,000 XG（以最小的加速度和减速度）。 </li><li>小心地收集各梯度组分（尤其是蔗糖的各层之间的界面），用考马斯亮蓝染色或Western印迹分析它们在SDS-PAGE（10％）。聚集蛋白被发现在管的底部，而nonincorporated，洗涤剂溶解的蛋白质被发现在顶部管。蛋白脂质体和脂质体被发现在对应于它们的固有密度蔗糖接口。空脂质体更远回收在比脂蛋白体的梯度。 </li></ol></li></ol> 3。荧光测量<ol><li>使用荧光分光光度计允许双波长测量（氙灯，150瓦）进行荧光测量在25.0°C。备用照明周期（激活BR为550nm / 550nm的激发/发射波长）荧光测定用455毫微米/ 509毫微米为2秒来衡量羟基芘磺酸荧光激发/发射波长。设置的激发和发射带宽至5nm。执行中的50nM的缬氨霉素存在下的测量结果，以防止反向膜电位ΔΨ的形成。 注：事实上，由于BR的质子泵，还有脂质体内部更积极的收费比外面。这种电荷梯度可以用缬氨霉素被丢弃，这是一种钾离子选择性离子载体。一旦加入到脂质体悬浮液缬氨霉素，作为疏水性的分子，将插入到脂质体膜上，被动运输K +，因此消散膜电位ΔΨ。 </li></ol>

<ol>
	<li>Granseth, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+structural+biology+-+How+far+can+the+bugs+take+us?+(Review).">Membrane protein structural biology - How far can the bugs take us? (Review).</a> Molecular Membrane Biology. 24 (5-6), 329 (2007).</li><li>Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+resistance+in+bacteria.">Multidrug resistance in bacteria.</a> Annu Rev Biochem. 78, 119 (2009).</li><li>Pos, 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=Drug+transport+mechanism+of+the+AcrB+efflux+pump.">Drug transport mechanism of the AcrB efflux pump.</a> Biochim Biophys Acta. 1794 (5), 782 (2009).</li><li>Murakami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+a+multidrug+transporter+reveal+a+functionally+rotating+mechanism.">Crystal structures of a multidrug transporter reveal a functionally rotating mechanism.</a> Nature. 443 (7108), 173 (2006).</li><li>Seeger, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Asymmetry+of+AcrB+Trimer+Suggests+a+Peristaltic+Pump+Mechanism.">Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism.</a> Science. 313 (5791), 1295 (2006).</li><li>Sennhauser, G., Bukowska, M. A., Briand, C., Grutter, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+multidrug+exporter+MexB+from+Pseudomonas+aeruginosa.">Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa.</a> J Mol Biol. 389 (1), 134 (2009).</li><li>Su, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+CusBA+heavy-metal+efflux+complex+of+Escherichia+coli.">Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli.</a> Nature. 470 (7335), (2011).</li><li>Nikaido, H., Takatsuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+RND+multidrug+efflux+pumps.">Mechanisms of RND multidrug efflux pumps.</a> Biochim Biophys Acta. 1794 (5), 769 (2009).</li><li>Rigaud, J. L., Levy, D. <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+into+liposomes.">Reconstitution of membrane proteins into liposomes.</a> Methods in enzymology. 372, 65 (2003).</li><li>Aires, J. R., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycosides+are+captured+from+both+periplasm+and+cytoplasm+by+the+AcrD+multidrug+efflux+transporter+of+Escherichia+coli.">Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli.</a> J Bacteriol. 187 (6), 1923 (2005).</li><li>Picard, M., Verchere, A., Broutin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+active+transport+of+efflux+pumps+after+their+reconstitution+into+proteoliposomes:+caveats+and+keys.">Monitoring the active transport of efflux pumps after their reconstitution into proteoliposomes: caveats and keys.</a> Anal Biochem. 420 (2), 194 (2012).</li><li>Verchere, A., Dezi, M., Broutin, I., Picard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Protein+Purification+and+Analysis,+edited+by+iConcept+Press.">Basic Methods in Protein Purification and Analysis, edited by iConcept Press.</a> , (2012).</li><li>Verchere, A., Broutin, I., Picard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+proton+gradients+for+the+in+vitro+investigation+of+bacterial+efflux+pumps.">Photo-induced proton gradients for the in vitro investigation of bacterial efflux pumps.</a> Sci Rep. 2, 306 (2012).</li></ol>

没有什么可以透露。

我们描述了一个重建的过程设计为测定膜蛋白转运。一旦建立，蛋白质重组过程导致的测量是非常可重复的。然而，对于重构的蛋白质的精确条件将随蛋白质的其他。多，必须小心在优化参数如下： 纯化 （纯度和不存在聚集的材料） 的 ⅰ） 质量 ; 洗涤剂增溶步骤 （如果可能的ⅱ）效率，低临界胶束浓度的洗涤剂，应优选的，因为它们是容易得到除掉）;ⅲ） 解吸洗涤剂 （它是例如可以使用聚苯乙烯珠与透析步骤结合起来，但这样做可能影响解吸的速率，用于所述蛋白质的后续活动的一个关键参数，参见文献[ 9]）;ⅳ） 脂质重构 （脂质的化学性质，所述脂质与蛋白质的比率，额外amphiph存在尔斯是必须改变和优化）的关键参数。除了温度和保温时间影响所有这些问题。 质量控制是这样始基在重建过程中的每个步骤。从这个角度来看，光散射是一个非常方便的方法，因为它是快速的，非破坏性的，并且只需要20微升样品中的浓度在微摩尔范围。一旦脂蛋白形成，蔗糖梯度也有很大帮助，因为它打开对重组进行系统的核查方式：定性（是蛋白质确实嵌入脂质体膜）和定量（多少蛋白质在一个脂质体）。后者将通过精确地测量所述蛋白质和脂质的浓度加以解决。 我们的分析并不依赖于基板本身的滴定和这避免了关于可能的伪迹被动扩散不安因素<html>的分子由于在膜的疏水性划分。该实验利用羟基芘磺酸作为一种可靠的和定量的探针，用于监测pH值的变化特性。我们的结果与使用霉素10在其中执行质子梯度的形成另一个程序得到的结果一致，但它允许一个更强大的和可重复的梯度11。此外，该质子梯度可以精确调整，简单地通过改变缓冲液的浓度（对于进一步的细节见Verchere 等人 12）。 在首先在不存在衬底的执行的控制测量过程（这可以访问该蛋白质的被动活动）。后的基板已在同一比色杯中添加实际的膜蛋白转运蛋白活性，然后测量。这是真正的资产，因为该实验可以被认为是一个真正的，非模糊的，底物诱导的结果。 ENT“&gt;的协议可以适用于高介质的吞吐量（例如96孔测量）自动化，因为照射样本触发反应。自动化和并行化将包括在制备大批量的脂蛋白体，并在一个96等份分装井板。衬底（以及还可能的抑制剂）将被添加并在黑暗中孵育45分钟后，该板会受到光照/羟基芘磺酸荧光测量周期上酶标仪。 有关协议的更多信息，欢迎读者阅读Verchere 等 。12,13

整合膜蛋白表示编码在真核基因组基因的高达30％。他们分享举足轻重的角色，如守门（受体），运送营养物质，离子或有毒化合物（运输）或维护跨双层膜（通道）的渗透率在所有活细胞1。外排泵具有抵抗抗生素治疗2核心作用。在革兰氏阴性菌绿脓杆菌 ，它是由一外保护膜，外排转运体被组织为三方系统中MexB，外排泵位于内膜，工程与MEXA，周质蛋白，和OprM的同时，一个外膜通道。胞质内的膜蛋白充当能量依赖性泵具有广泛的底物特异性。外膜蛋白充当孔蛋白，而第三个是位于周质空间和被认为是稳定的整体复杂<s了&gt; 3。在下文中，我们集中在功能测定法对MEXA MexB组件的设计。 结构信息也累积了过去5年由于从大肠杆菌 4-7的同源蛋白ACRB的X射线测定。虽然这显然是重要的夫妇这样的信息与动态和动力学数据，设计一个功能测定该转运是一个真正的挑战8。事实上，膜蛋白，必须保持在一个膜质环境和封闭的隔室必须保持基底的矢量传输来实现。 膜蛋白为蛋白脂质体的重组已被广泛介绍和审查（见里戈和Levy 9）。这些协议允许通过在脂质体膜后的基板的监测膜蛋白活性的，例如。在这种情况下的限制产生的一个滴定基板vailability（荧光，放射性等 ），并从该很多时候，这些底物是疏水性的，有一种倾向，越过膜而与转运蛋白的存在的事实。作为一种替代方法，可以跟随一个报告染料对所发生的一种运输后果化学变化敏感的光谱变化。如上所述，质子通过MexB通电运输。因此，一个相关的方法是监控一个pH敏感的报告染料跟随由于质子驱动的基板传输pH值的变化的光谱变化。荧光染料的选择范围可避免由于衬底的疏水性质的任何伪迹影响。 一个额外的困难是质子梯度的产生。多种协议可在文献中找到。其中，利用霉素技术是普遍，但我们已经表明，它是乏味的执行10-11。此外，它是不能再现，这是不可逆的。我们已经使出了利用细菌视紫红质（BR），从Halobacter salinarium光激活的质子泵，一种嗜盐的海洋革兰氏阴性需氧预留古菌的。这种蛋白质是coreconstituted到脂质体与MexB，一经光照，BR泵的脂质体的内部的质子，从而创建ΔPH（酸性内）运送基板12-13所需要的蛋白质。

<table border="0" cellpadding="0" cellspacing="0" width="1051">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine)</td>
			<td style="width:344px;">Avanti Polar Lipids</td>
			<td style="width:344px;">850375C</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Cholesterol</td>
			<td style="width:344px;">Sigma Aldrich</td>
			<td style="width:344px;">C8667</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Hoechst 33342</td>
			<td style="width:344px;">Sigma Aldrich</td>
			<td style="width:344px;">B2261</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Triton X-100</td>
			<td style="width:344px;">Sigma Aldrich</td>
			<td style="width:344px;">93427</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Valinomycin</td>
			<td style="width:344px;">Invitrogen</td>
			<td style="width:344px;">V-1644</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid)</td>
			<td style="width:344px;">Invitrogen</td>
			<td style="width:344px;">H-348</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">DDM (n-Dodecyl-&#946;-D-maltopyranoside)</td>
			<td style="width:344px;">Affymetrix</td>
			<td style="width:344px;">D310LA</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Extruder kit (extruder, Hamilton gas-tight syringes)</td>
			<td style="width:344px;">Avanti Polar Lipids</td>
			<td style="width:344px;">610000</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Biobeads SM-2 Adsorbents</td>
			<td style="width:344px;">Bio-Rad</td>
			<td style="width:344px;">152-3920</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Econo-Pac Chromatography empty column</td>
			<td style="width:344px;">Biorad</td>
			<td style="width:344px;">732-1010</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Desalting columns</td>
			<td style="width:344px;">Bio-Rad</td>
			<td style="width:344px;">54805</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:363px;">Dynamic light scattering instrument</td>
			<td style="width:344px;">Wyatt Technology</td>
			<td style="width:344px;">DynaPro NanoStar</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:363px;">Fluorometer JASCO FP-6200</td>
			<td style="width:344px;">JASCO</td>
			<td style="width:344px;">6816-J002A</td>
		</tr>
	</tbody>
</table>

in vitro investigation of the mexab efflux pump from pseudomonas aeruginosa

有一个新兴的科学需要可靠的工具，用于监测膜蛋白转运。我们提出了一种方法，从而导致外排泵的重构来自于一个仿生环境中的革兰氏阴性细菌铜绿假单胞菌 ，允许其传输的活动的准确调查。三个先决条件得到满足：隔在脂质环境中，用于交通运输相关的索引，并产生质子梯度。膜蛋白转运重组到脂质体与细菌视紫红质，光活化的质子泵，其产生的质子梯度是健壮以及可逆的和可调谐在一起。该蛋白质的活性，从脂质体内部发生的pH值的变化推导出，用pyranin，pH依赖的荧光探针。我们描述一个一步一步的过程，其中的膜蛋白的纯化，脂质体形成，重组蛋白，和运输nalysis得到解决。尽管它们对于RND转运是专门设计的，所描述的方法可能适于与任何其他膜蛋白转运蛋白由一个质子梯度通电使用。

该测定由羟基芘磺酸监测荧光作为时间的函数而产生质子梯度。到该目的，样品交替进行照明（因此质子通过BR泵送被触发），然后用激发波长和发射羟基芘磺酸的波长进行荧光测量（λEX = 455 nm和λ 发射 = 509纳米）。 图1显示了该测定得到的，在没有MEXA / MexB的代表控制，验证通过BR产生的质子梯度是可逆的。酸化的一个周期之后，脂蛋白体被保持在黑暗中45分钟，以使BR停止泵送质子（质子扩散缓慢和被动跨脂质双层以下的浓度梯度）。在这之后的恢复时间，活化的BR是可能的，仅仅通过再次照射相同的悬浮液。 图2对应于实际传输的测量。的阴性对照无蛋白脂质体表明，正如所料，pH值是在光照不变（ 图2A和2B，橙色圆圈）。然而，含有BR在它们的膜蛋白脂质体做质子泵，从而内部的脂质体减少，pH值和稳定的梯度是建立（ 图2A和2B，紫色正方形）。灰色三角形和红色菱形（ 图2A）表示的pH含有BR和MexB蛋白脂质体的内部，在存在或不存在的Hoechst 33342的分别。我们观察到一个基板无关的活动，其中的质子梯度仅部分由MexB逆运输消散。我们认为这种观察到MexB的基础活性。 为了测试MEXA对MexB的活性的影响，MEXA加入到重建，首先在没有任何基材（ <st荣&gt;图2B，蓝钻石）。再次，由BR中产生的质子梯度仅部分消退。实际的传输测量是实现对同样的样品。事先，质子梯度必须被重新初始化，并在基片必须在悬浮液中添加以达到在其蛋白结合位点。该目的将悬浮液温育在黑暗中在衬底45分钟的存在。在随后的照度，人们可以看到，通过对BR产生的质子梯度，现在完全由MexAB（ 图2B，绿色三角形）消散，作为底物的运输由泵造成的。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50894/50894fig1highres.jpg" src="/files/ftp_upload/50894/50894fig1.jpg" /> 图1：通过可逆性BR照明建立了质子梯度 ：羟基芘磺酸FLuorescence测量作为时间的函数的BR蛋白脂质体的。从0-15分钟，BR质子泵作为光活化的结果。从15-60分钟，蛋白脂质体孵育在黑暗中，在这段时间，质子被动地移出脂质体，直至囊泡内的pH值和extravesicular pH值是相等的。从60-75分钟，BR仍然是功能和质子泵时的照明。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50894/50894fig2highres.jpg" src="/files/ftp_upload/50894/50894fig2.jpg" /> 图2：运输试验：羟基芘磺酸荧光，转化成相应的pH值的变化，作为时间的 ）pH为对照脂质体（橙色圆圈）内的函数 ; pH值包含BR的膜（紫色正方形）脂蛋白体的内部; pH值内。含有Br和MexB在膜无赫斯特33342（红D脂酶的iamonds）; pH值包含BR和MexB在膜用Hoechst 33342（灰色三角形）B）pH值的控制脂质体（橙色圆圈）内蛋白脂质体的内部; pH值的BR包含在膜（紫色方块）蛋白脂质体内部;内pH值含BR，MexB和MEXA在膜无赫斯特33342（蓝钻石）脂酶的;含pH值的BR，MexB和MEXA在用Hoechst 33342（绿色三角形）的膜蛋白脂质体的内部。

Watch this Scientific Journal Video about In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa at JoVE.com

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

我们提出了一个协议，用于在体外研究铜绿假单胞菌外排泵。该协议允许对脂质体膜中的健壮的，可逆的，可调谐的质子梯度的产生，因此，应能适应由protomotive力激励任何膜蛋白。

在 MexAB外排泵从铜绿假单胞菌的体外研究

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

in-vitro-investigation-mexab-efflux-pump-from-pseudomonas

Research

JoVE Journal

Biology

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

11.8K 次观看.  CNRS & Université Paris Descartes. 我们提出了一个协议，用于在体外研究铜绿假单胞菌外排泵。该协议允许对脂质体膜中的健壮的，可逆的，可调谐的质子梯度的产生，因此，应能适应由protomotive力激励任何膜蛋白。 

视频: In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

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 ...

科研

生物学

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 ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

从成年小鼠肾脏中分离细胞核以进行单核RNA测序

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

从成年、幼年、幼虫和胚胎斑马鱼中分离心脏和血管平滑肌细胞以进行电生理研究

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

丁香假单胞菌基因在植物组织中表达的单细胞分析

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

使用 FluoZin-3 测定研究哺乳动物细胞中的锌转运机制

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

作者聚焦：通过 ZnT1 功能探索细胞锌调节 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...