这项工作是由NSERC经费资助 386634.我们想感谢Screaton实验室成员，有益的讨论。

1.准备试剂，板，和细胞<ol><li>制成500 ml的裂解缓冲液：25毫摩尔Tris pH值7.5，150mM的氯化钠，50mM的氟化钠，0.5mM EDTA的pH为8.0，0.5％的TritonX-100，5mM的β-内甘油，5％甘油。保存在4℃。使用，添加1mM的二硫苏糖醇（DTT），1mM的苯甲基磺酰氟（PMSF），和1mM钒酸钠之前立即。在该步骤之后，PMSF中不需要任何漂洗缓冲液中。 </li><li>使将20ml 10×激酶缓冲液：200毫摩尔Tris pH为7.5，50mM的β-内甘油（FW 216），2mM的钒酸钠。分装在1ml试管并储存在-20℃。使用，加上5 mm DTT前夕。 </li><li>使20ml中10倍的M-ATP的缓冲液：300μM三磷酸腺苷（ATP）的66mM的MgCl 2，33毫摩尔的MnCl 2，分装在1ml试管并储存在-20℃。 </li><li>使100个毫升2×十二烷基硫酸钠（SDS）裂解缓冲液：1.5克的TRIS碱，将20ml甘油，加入30ml 的 H 2 O.溶解并调整pH至6.8，用HCl。添加40毫升10％SDS中，音量调节至100毫升。加入25毫克的溴酚蓝。 </li><li>现货4微升25毫微克/微升哺乳动物表达质粒套96孔板和标签板相应的编码每一个GST激酶14家 。密封和冷冻板在-20℃下直到使用。 </li><li> 1-2天前，转染，培养HEK293T细胞中完整的Dulbecco改良的Eagle培养基（DMEM）+ 10％胎牛血清（FBS）和抗生素在37℃培养箱中补充有二氧化碳 （最终5％）。通道与胰蛋白酶和不允许的股票成为&gt; 80％汇合膨胀的过程中。至少6×10 7个细胞的所需屏幕（约3×15厘米，在80％汇合HEK293T细胞的碗碟）。 </li></ol> 2.转染注： 图 1所示为整个协议的流程图。 <ol><li>如果包含激酶质粒板已被冻结，在室温下解冻一次离心在1900×g离心3分钟以收集任何湿气在孔的底部。 </li><li>拌8.6 ml的低血清培养基（例如，OPTI-MEM）与312.7微升基于脂质的转染试剂的。让我们坐5分钟。 </li><li>加10微升减少血清的培养基，每孔使用自动化液体分配器装配有小体积盒。 </li><li>添加每降低血清培养基/转染试剂混合物从步骤2.2的井10微升用自动液体分配器装配有小体积盒。让我们坐20〜45分钟。 </li><li>重悬293T细胞在在80ml完全DMEM的7.5×10 5个细胞/ ml。加入100微升细胞悬浮液（即7.5×10 4个细胞）的每使用自动化液体分配器装有标准量盒式良好。 </li><li>检查井显微镜均匀细胞分布下并返回到培养箱24小时。 </li></ol> 3. GST激酶下拉<ol><li>让˚FRESH：4mM的通过混合60微升的0.2M钒酸钠与540微升H 2 O过钒酸盐溶液在第二管混合2.7微升过氧化氢的30％和1.4毫升的PBS。添加两种方法结合，让坐在使用前15分钟。 </li><li>使用多道移液器（因为它在井产生太多的湍流不使用自动化液体分配器）分配2微升0.25M的氯化钙 2在各孔中，接着2.5微升在步骤3.1中制备的过钒酸盐的。孵育各板在37℃下进行10分钟，然后放置在冰上。 </li><li>通过添加DTT，PMSF，和钒酸钠如第1（试剂的配制）所示制备的35ml裂解缓冲液。 </li><li>保持板置于冰上，利用真空抽吸每孔删除网上平台。立即用自动液体分配器装有标准盒式添加50μl/孔的冰冷的裂解缓冲液。让我们坐30分钟，在冰上裂解（可选：可以在这里如果有必要通过密封高原停止e和储存在-80℃。要继续，解冻板冰）。 </li><li>旋板在1900×g离心3分钟，在4℃。 </li><li>使用多道移液器向各孔刮去细胞和转移所有内容以适当标记的V型底96孔板中。旋板在1900 xg离心在4℃下10分钟。 </li><li>期间的旋转，填充谷胱甘肽包被的板（每个96孔板）用100μl/孔的冰冷裂解缓冲液（无PMSF），为漂洗。置于冰上板。 </li><li>从离心机中取出V形底平板。一次一个，反转谷胱甘肽板在水槽上方，以摇出的纸巾裂解液和印迹。通过倾斜板，并使用多道移液器，小心不要在底部打扰颗粒移植自V-底板的谷胱甘肽板裂解液。盖板和置于冰上至少2小时进行绑定。 </li><li>接近2小时的结合步骤的结束时，准备一个放射性工作站，确保吨他必要的安全防范措施到位放射性工作。设置杂交炉至30℃。 </li><li>如在第1节PMSF表明不需要在该阶段制得200毫升裂解缓冲液加入DTT和钒酸钠。 </li><li>反转的谷胱甘肽板在水槽上方，以摇出裂解液涂抹在纸巾上。冲洗孔3倍用100μl裂解缓冲液（无PMSF）。不要让井坐干 - 让他们在漂洗，直到准备进行。 </li><li>通过稀释10倍的库存和添加DTT作为用自动液体分配器装有标准体积盒在第1指示的冲洗板一次用50μl的1×KB的制备55毫升1×激酶缓冲液（KB）。留1个KB的井，直到溶液中的准备： <ol><li>通过添加500至530微克的兴趣将基片，500微克的髓鞘碱性蛋白（MBP），2.65毫升10×KB，13.25微升的1M DTT和H 2 O的高达15.9 ml的制备溶液A。 </li></ol></LI&gt; <li>一次一个，翻转板在水槽上方，除去1个KB冲洗，涂抹在纸巾上，并立即使用自动液体分配器配有体积小纸盒加30微升溶液A。置于冰上板。 </li><li>制备溶液B在放射性工作区域通过加入2.5 ml的10倍的M-ATP，500微居里的γ-32 p ATP和H 2 O的至10ml。 <ol><li>加入20微升溶液B每使用中继器吸管，它有助于混合，由于喷射力很好。盖上盖子进行培养在30℃杂交炉30分钟。 </li></ol></li><li> 30分钟后，回转板，以冰。加入50微升2×SDS裂解缓冲液，使用多道移液器每个孔中。可以进行在这个点到下一个步骤，或密封用铝箔和存储所述板在-20℃直至方便。 </li></ol> 4.运行，染色，干燥凝胶注：所有工作都应该在一个地区DESIGNA进行泰德放射性。 <ol><li>打开杂交炉和设定为85℃。解冻板在室温下。一旦烘箱达到温度​​，传热板到烤箱并孵育10分钟以变性样品。 </li><li>负载26孔预制胶与15微升使用多道移液器每一个反应，以填补多口井一次。必须注意，所有的技巧配合以及相对应先加样品。运行凝胶在150五，不要让示踪染料（蓝线）跑出凝胶的底部，因为这包含了未结合的ATP。 </li><li>拆除凝胶和切断使用手术刀或直边，因为它会曝光过度，影片的未结合的ATP（蓝线）。将凝胶在标签的容器和盖用考马斯染色15分钟。 </li><li>取出考马斯染色，简单地用清水冲洗凝胶，并添加脱色的解决方案。脱色的凝胶直至蛋白质都清晰可见。 A波段的MBP和基材带应对于每个样品可见。 </li><li>以干燥的凝胶： <ol><li>切滤波器一张大纸，并把它放在烘干机。 </li><li>湿蒸馏水玻璃纸片，直到它是光滑，无皱，并将其放置在纸张的顶端。 </li><li>躺在玻璃纸片材的顶部的凝胶，使得记凝胶的量级。湿的第二玻璃纸片和放在凝胶的顶部。 </li><li>铺开所有的泡沫（一板的密封辊可以很好地用于本）的一个很好的均匀的表面。关闭挡板，打开真空，并在80℃干燥该凝胶3小时。 </li></ol></li><li>一旦凝胶干燥，用丝网加紧信号，将其暴露在XAR胶片。用保鲜保鲜膜或塑料袋磁带，并用胶带密封，保持了霜冻。存储盒在-80℃下过夜。 </li></ol> 5.制定XAR电影<ol><li>翌日，从冷冻库取出暗盒，并让在室温下解冻。发展中的膜使用薄膜处理器根据制造商的说明暗室。 注意：检查薄膜激酶底物对的证据。阿秒更长的曝光也可以是有用的检测更弱的磷酸化事件。 </li></ol>

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The authors have nothing to disclose.

由于原来的出版物描述的方法14,15，180 GST-激酶原库已扩大到420件，或约80％的人类蛋白质激酶组的。与膨胀的文库，如所描述的协议需要4-5天，然后1-4天来开发薄膜（认为必要），这可以通过使用磷光和数字信号增强的缩短。有些情况必须注意几个关键步骤（ 见图 1协议的概述）。第一，是细胞的股票的健康（无支原体，决不允许在膨胀过程中达到汇合，并用胰蛋白酶在每次...

蛋白质的翻译后修饰（翻译后修饰）是细胞内的通信是必不可少的。也许最好的研究了所有翻译后修饰是磷酸化，由蛋白激酶，从而调节的蛋白质的功能，包括它们的生化活性，亚细胞定位，构象和稳定性无数催化。关于靶蛋白磷酸化位点的鉴定可以通过胰蛋白酶磷酸肽映射或通过使用富含磷酸化肽1,2-样品现在标准蛋白质组技术来完成。而四分之三的表达蛋白质组预计被磷酸和3所识别200000磷酸化位点5，据估计高达1 亿 6，许多这些没有分配的生物学，信号传导途径，或蛋白激酶。 虽然识别磷酸化位点是相对简单的，相对更大的挑战是，鉴定同源激酶（s）表示，针对这些网站，我们称为映射激酶的方法：基片对。几种方法用于鉴定激酶：基板对已经描述，无论是在开始用感兴趣的激酶和寻找其底物或开始与感兴趣的基板，并试图找到改性激酶实验7-11或计算12。为了鉴定激酶为已知的磷酸化底物，生物信息学可以用来识别包含氨基酸侧翼磷酸化残基（共识位点），以及识别形成沉淀络合物与基板激酶短保守序列的蛋白质。然而，这些方法是耗时，经常不与成功。 我们开发了一个系统功能的方法来迅速查明激酶磷酸化可以在给定基片13。屏幕试验产生优良的比性，具有非常明确的选择潜在的同源激酶。鉴于磷酸化生物信号的中心地位，画面中几乎所有的细胞信号通路14-16用于发现有用的。屏幕包括与人蛋白激酶的文库进行大规模激酶测定。所述激酶已被标记为细菌谷胱甘肽-S-转移酶（GST）蛋白和从哺乳动物细胞提取物纯化的，这意味着该重组酶 - 不同于那些来自细菌制备 - 将在上游蛋白激酶经常所需的存在而产生重组酶具有体外活性。实际上，虽然所需的下游激酶活化丝氨酸，苏氨酸，和酪氨酸激酶活性是存在于酵母10中，酵母基因组编码122蛋白激酶，指示所述哺乳动物激酶组，有超过500个基因17，已成为显著更加复杂，以边条忒独特高阶生物体的过程。此外，相关的细胞生物学和人类疾病（如小分子，生长因子，激素， 等等）不同的刺激的效果可以用来14,15调节激酶活性在一个适当的范围内。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lysis buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.1</td>
		</tr>
		<tr>
			<td>10x kinase buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.2</td>
		</tr>
		<tr>
			<td>10x M-ATP</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.3</td>
		</tr>
		<tr>
			<td>Human kinase plasmids</td>
			<td>Orfeome, Invitrogen, Origene</td>
			<td></td>
			<td>GST-tagged in house</td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Fisher Scientific</td>
			<td>CS003595</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Fisher Scientific</td>
			<td>SH3002201</td>
			<td>supplement with 100U/ml penicillin, 100ug/ml streptomycin, 10% fetal calf serum.</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture dishes</td>
			<td>Fisher Scientific</td>
			<td>877224</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Invitrogen</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated liquid dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>14072670</td>
			<td></td>
		</tr>
		<tr>
			<td>4mM pervanadate</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 3.1</td>
		</tr>
		<tr>
			<td>0.25 M CaCl2</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20-200 uL)</td>
			<td>Labnet</td>
			<td>p4812-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (1-10 uL)</td>
			<td>Thermo Scientific</td>
			<td>4661040</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 6-well plates</td>
			<td>Evergreen Scientific</td>
			<td>290-8116-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione coated 96-well plates</td>
			<td>Fisher Scientific</td>
			<td>PI-15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Biostad</td>
			<td>350355</td>
			<td></td>
		</tr>
		<tr>
			<td>GST tagged substrate</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP)</td>
			<td>Sigma</td>
			<td>M1891</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette (1 mL)</td>
			<td>Eppendorf</td>
			<td>22266209</td>
			<td></td>
		</tr>
		<tr>
			<td>32P gamma-ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU502Z500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>2X SDS lysis buffer (100 mL)</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.4</td>
		</tr>
		<tr>
			<td>26-well precast TGX gels</td>
			<td>BioRad</td>
			<td>567-1045</td>
			<td>gel percentage required is dependent on the molecular weight of the substrate of interest</td>
		</tr>
		<tr>
			<td>Coomassie stain</td>
			<td>Made in house</td>
			<td></td>
			<td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td>
		</tr>
		<tr>
			<td>Coomassie destain</td>
			<td>Made in house</td>
			<td></td>
			<td>10% acetic acid, 20% methanol</td>
		</tr>
		<tr>
			<td>Labeled gel containers</td>
			<td>Made in house</td>
			<td></td>
			<td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>Fisher Scientific</td>
			<td>57144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane sheets (2)</td>
			<td>BioRad</td>
			<td>165-0963</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Labconco</td>
			<td>4330150</td>
			<td></td>
		</tr>
		<tr>
			<td>Double emulsion autoradiography film</td>
			<td>VWR</td>
			<td>IB1651454</td>
			<td></td>
		</tr>
		<tr>
			<td>Film cassette</td>
			<td>Fisher Scientific</td>
			<td>FBAC-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Intensifying screen</td>
			<td>Fisher Scientific</td>
			<td>FBIS-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealing rubber roller</td>
			<td>Sigma</td>
			<td>R1275</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of kinase-substrate pairs using high throughput screening

我们已经开发出一种筛选平台，以确定专用人蛋白激酶磷酸化的底物，可用于阐明新的信号转导途径。我们的方法的特征是使用纯化的GST标记的人蛋白激酶的文库和感兴趣的重组蛋白衬底。我们已经用这种技术来识别的MAP /微管亲合性 - 调节激酶2（MARK2）作为激酶的葡萄糖调节站点上CREB稳压转录辅激活2（CRTC2），所需的β细胞增殖的蛋白质，以及AXL家族酪氨酸激酶的作为细胞转移的磷酸化的衔接蛋白ELMO的调节剂。我们描述这种技术，并讨论如何帮助建立细胞对环境刺激如何应对一个全面的地图。

来自屏幕代表性的结果示于图2。180激酶使用来自CRTC2以及经典激酶测定衬底髓鞘碱性蛋白（MBP）相应于氨基酸268-283一个GST-标记的肽底物进行筛选。只有两个激酶MARK2和高度相关激酶磷酸化MARK3的CRTC2肽。 MBP被包括在所有测定中的内部对照，因为它包含了许多可磷酸化的残基，并运行在18 kDa的，朝向所述凝胶的底部。这允许特异性的解释：有些激酶将有力磷酸化底物和MBP。...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

identification-kinase-substrate-pairs-using-high-throughput

Research

JoVE Journal

Biology

8.1K 次观看.  Children's Hospital of Eastern Ontario Research Institute. Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

视频: Identification of Kinase-substrate Pairs Using High Throughput Screening

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

科研

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...