我们要感谢支持这项工作的创新，不列颠哥伦比亚省知识发展基金和卫生研究所（CIHR）加拿大研究所，加拿大基金会。 MT是从微生物的多样性和进化（CMDE）图拉基金会资助中心的奖学金支持。

重组蛋白在甲醇毕赤酵母中的表达在开始此协议，你应该有你感兴趣的基因在体育帧克隆酵母父载体和它测序检查的正确插入的基因载体的利益。 第一步：生成electrocompetent酵母细胞，线性的兴建和改造成体育酵母 对于这一步，你将需要有以下媒体和板： <ul><li> YPDS板</li><li> 600毫升YPD培养基</li><li>冰冷的无菌水</li><li> 1中号山梨醇</li><li> Amicon超4离心过滤装置</li><li>单片机YM - 30离心式过滤器单元</li><li> 0.2厘米电比色皿</li><li> 15毫升无菌玻璃管</li><li>无菌玻璃巴斯德吸管</li><li> YPDS板含有Zeocin </li></ul>第1部分：electrocompetent酵母细胞的制备<ol><li> 4天前拟出体育的转型连胜没有Zeocin YPDS板的酵母细胞，让他们成长为1-2天，或直至形成单菌落30℃ 。 </li><li>拟改造前两天增长5毫升您的体育酵母株50毫升猎鹰管在YPD培养基在30 ° C过夜。猎鹰管放置在一个烧瓶固定在振动筛。 </li><li>改造前新鲜的YPD培养基接种500毫升在2公升烧瓶中，用0.25毫升过夜培养，让成长在摇床一夜之间再次在30 ° C至OD 600 = 1.3-1.5天。 注：稀释你的样品前分光光度计测量，使您得到准确的结果。 </li><li>当天的改造，有冰冷的无菌水和1米在手山梨糖醇。离心5分钟，在1500 XG的细胞在4 ° C。冰冷，无菌水500毫升，重悬沉淀。 </li><li>第4步，再次离心细胞。冰冷，无菌水250毫升，重悬沉淀。 </li><li>离心机的细胞再次在第4步。重悬在冰冷的1米山梨糖醇20毫升沉淀。 </li><li>离心机的细胞再次在第4步。在冰冷的1〜1.5毫升的最终体积山梨糖醇中号的1毫升，重悬沉淀。在冰上或保存在4 ° C，直到进一步利用细胞。 注：我们准备electrocompetent比需要的细胞，并存储在80μL离心管分装于-80 ° C。我们使用不超过一个月的存储单元格。 </li></ol>第2部分：线性和浓度pPICZαA构建改造你感兴趣的基因载体酶切线性化。我们使用的酶PmeI。其他限制的网站是可能的。你必须确保你插入不包含您要使用你的载体线性化的限制，网站。对于改造成体育酵母 ，您将需要5-20微克5-10μL无菌水的线性DNA。 线性，集中和转移到P.纯向量（没有插入） 酵母 。没有插入父向量为背景的细胞内表达的控制，并允许你解释你的表达结果。 <ol><li>在冰上解冻所有试剂，结合下面的试剂，并让所有液体底部短暂离心管，自来水管和旋转一次： <ul><li> xμL无菌水</li><li> 5.0μL10X NEBuffer 4 </li><li> 100X BSA 0.5μL </li><li> 2微克的载体DNA（〜100毫微克/μL） </li><li> 2.0μLPME我酶混合物</li></ul> →50μL总反应体积 注：要获得足够的线性载体DNA准备的上述组合，在不同的管3或4次，并通过Amicon超离心设备集中消化的DNA结合的解决方案时。 </li><li>在37 ° C孵育3小时，热灭活20分钟，在65 ° C的酶混合物。 </li><li>的平均时间，冲洗前用1毫升Milli - Q水Amicon超4离心过滤装置，在4000 XG旋转6-8分钟管和丢弃的流量通过。 注意：不要让膜干燥一次湿。如果你不使用该设备的预冲洗后，留在膜的水，直到设备使用。 </li><li>在4000 XG热灭活的解决方案，从第2步转移到预冲洗Amicon离心过滤单元，旋转管为6-8分钟，并丢弃流经。 注意：如果您已经准备好了一个以上的线性组合，组合成一个Amicon离心过滤单元所有的解决方案。 </li><li>离心机，直到解决方案Amicon过滤量减少到约100〜150＆MU;湖从上面添加1.5毫升无菌水的另一个空管和传输解决方案的Amicon管和重复离心步骤。第二次用1.5毫升无菌水清洗，并通过再次丢弃流量。减少〜150μL的最终体积。洗涤步骤删除的缓冲区，以防止拱脉冲的细胞时，剩下的盐。 请注意：我们使用摆动桶转子离心步骤，只需放置在一个50毫升猎鹰管的上限Amicon离心过滤装置在离心过程中，它举行的地方。 Amicon过滤装置上，保留50μL的解决方案，即使延长离心后的过滤器。 </li><li> DNA制备的进一步集中转移Amicon过滤装置的预冲洗单片机YM - 30离心式过滤器单元，剩余的DNA溶液冲洗额外的50μL无菌水，收回所有的DNA Amicon管。直到过滤器仍然存在轻微的液体覆盖在10000 XG离心离心过滤单元在微量的单片机。只有很短的时间旋转，检查每分钟，如果只有少量的液体留在过滤器上。要恢复您的DNA溶液，放置过滤器倒挂在新的离心管和旋转3分钟，在1000 XG在第二离心步骤。最终体积不得超过总额的10-15μL，否则你的DNA可能会过于稀释的改革前进了一步。 注：不旋转过滤装置过长，请不要完全干燥，以防止潜在的样品损失。如果你的膜干燥，膜添加到10-15μL无菌水，轻轻搅拌为30秒，如上所述，恢复你的DNA。 </li></ol>第3部分：改造成体育酵母由电<ol><li>线性处理和集中pPICZαA的DNA，包含您插入现在准备转变到electrocompetent体育酵母细胞（见我一步，第2部分）。改造前约15分钟准备以下试剂和设备：填写1毫升的1 M山梨醇离心管置于冰上;对冰电0.2厘米比色皿;标签无菌的15 mL玻璃试管中，并有无菌玻璃巴斯德吸管就在眼前。 </li><li>将80μL一步，我的一部分，2.7细胞冰冷的0.2厘米电比色皿。我一步，部分3.6和混合添加，从一侧到另一侧的电比色皿的枪头集中的线性pPICZαADNA溶液。 注：加入的DNA时，只把吸管的第一站。混合后的DNA与细胞推吸管的第二站，慢慢地从试管中删除。 </li><li> 5分钟就冰细胞培养的试管。 </li><li>的比色皿中，用纸巾擦去外面和脉冲的细胞，并根据参数的具体电正在使用的设备制造商的建议酵母（酿酒酵母）。 注：我们用用Bio - Rad公司GenePulser下列条件： <ul><li>充电电压（V）：1500; </li><li>电容（μF）：25; </li><li>电阻（Ω）：200。 </li></ul>使用一个0.2厘米的电比色皿，该协议将产生一个〜7500 V / cm时的磁场强度的〜10毫秒脉冲长度。 </li><li>立即加入1毫升冰冷1米，山梨糖醇试管。试管内容转移至15毫升无菌试管使用无菌玻璃巴斯德吸管。 </li><li>在30 ° C，不晃动为1.5小时，让管孵化。 </li><li>新增四个标记YPDS板含有100微克/毫升Zeocin 5-7灭菌的玻璃珠。传播250μL每盘15毫升的玻璃管电混合。摇板水平均匀分布的细胞。让板干燥15分钟，然后取出琼脂珠反相板。 请注意：我们使用，转化为100微克/毫升Zeocin选择使用GS115毕赤酵母株。如果您使用的是另一种毕赤酵母株，选择条件可能会有所不同。 </li><li>直到菌落形成2至3天在30 ° C孵育板倒置。包裹在一个黑色的塑料板，以防止光敏感Zeocin退化。板块的抗生素量较少，可能会导致假阳性克隆。 </li><li>已形成的殖民地之后，挑选12个殖民地和纯化含100微克/毫升的Zeocin的新鲜YPDS板条纹的克隆。 </li></ol>步骤二： 在毕赤酵母蛋白表达对于这一步，你将需要手边有下列媒体，板和试剂： <ul><li> BMGY培养基</li><li>甘油，消毒</li><li> BMMY培养基</li><li>甲醇，消毒</li><li>震中酵母DNA纯化试剂盒</li></ul>您还需要以下烧瓶： <ul><li> 250 ml容量瓶中，蒸压</li><li> 1升百思不得其解的烧瓶中，蒸压</li><li> 200 mL烧杯中，蒸压</li></ul>第1部分： 在毕赤酵母蛋白表达以下所有步骤，也为转换后的控制向量（没有插入）。 <ol><li>接一步，我的一部分，3.9纯化毕赤酵母殖民地单个菌落，接种于25 mL BMGY，在无菌的250 mL容量瓶中。成长在30 °一个摇床（250-300转），直到文化中的C 到达一个外径600 2-6（约16-18小时）。 注：稀释你的样品前分光光度计测量，使您得到准确的结果。这些细胞会在登录阶段的增长。 </li><li>当细胞达到相应的OD 600准备一份甘油。转移800 25 mL细胞培养液2毫升康宁cyrogenic小瓶，加200μL灭菌甘油。 &#39;冻结在-80甘油和存储° C。 </li><li>酵母DNA纯化，转移25 mL细胞培养的1.5毫升离心管。收获细胞，离心1分钟，在室温下于1300 XG。这些细胞用于分析毕赤酵母 integrants仔细检查，如果感兴趣的基因整合到毕赤酵母的基因组（见步骤二，第2部分）。商店细胞沉淀于4 ° C，直到进一步的分析。 </li><li>其余的25 mL培养转移至50 mL猎鹰管和收获的细胞，离心5分钟，在室温3000 XG。倒出上清液和地方管，倒挂在组织，以消除任何残留的媒体。洗细胞的颗粒悬浮在20毫升BMMY删除任何剩余BMGY培养基，从BMGY介质甘油可以抑制以后的表达。离心机再次在3000 XG在室温下5分钟，倒出上清液。 </li><li>悬浮细胞沉淀到一个外径600 BMMY培养基1.0诱导表达（约100-200毫升）和转让的文化，在1升百思不得其解的烧瓶。封面用200 mL烧杯烧瓶中，并返回到孵化器继续增长30 ° C。 注：重要的是，温度不超过30 ° C。如果您的孵化器波动的温度，设定温度在28 ° C。充足的曝气也是一个重要参数，在甲醇诱导高效表达（诱导表达时，从来没有超过一个文化卷&gt;总烧瓶量的10-30％）。我们强烈建议使用百思不得其解烧瓶的，因为他们在文化传媒介绍更多的氧气。 </li><li>到终浓度为0.5％的甲醇添加灭菌纯甲醇，每24小时保持诱导。 </li><li>在一定的时间点启动后的表达的文化转移1毫升到1.5 mL离心管。在1300 XG为2.5分钟，在室温下离心。将上清转移至一个单独的1.5 ml离心管。商店，直到进一步的分析上清，细胞沉淀在-80 ° C。来自不同时间点的样品进行分析，以建立诱导后蛋白表达的最佳时间期限。 注意：我们选择6H，12H，24小时，36小时和48小时的时间点采取样品。长达4天的进一步增长是可能的。之间不同表达的蛋白质的最佳时间点。 </li><li>分析蛋白表达和细胞颗粒的上清，考马斯染色的SDS - PAGE和免疫印迹（未在本协议中所述）。 </li></ol>第2部分：酵母DNA纯化和PCR分析毕赤酵母 integrant BMGY 毕赤酵母培养酵母的DNA纯化步骤所需的所有试剂，包括从震中在MasterPure酵母DNA纯化试剂盒。 注：此分析是额外的执行，以确定是否感兴趣的基因已整合到毕赤酵母基因组。 BMGY文化（见步骤二，部分1.3）采取从1.5毫升样品细胞沉淀。 按照震中MasterPure酵母DNA纯化手册中的说明和运行特异性引物插入酵母DNA PCR。分析3μLPCR产物琼脂糖凝胶含有1μL溴化乙锭。包括在一个井1 KB +尺寸标记解释插入的大小和可视化的DNA，使用凝胶文档站。 可以分析重组蛋白免疫印迹分析。可以进行蛋白质纯化His - tag的净化上的带电金属树脂（未在本协议中所述）。 附录，配方清单步骤一： <ol><li> YPD培养基 ： 要准备600毫升YPD培养基（酵母提取蛋白胨葡萄糖中等），溶于水540毫升5克酵母提取物和蛋白胨12克。高压灭菌液体周期为20分钟。 与此同时，准备70毫升20％葡萄糖和过滤器在使用前消毒。 让蒸压解决方案，酷〜60 ° C和添加60毫升20％葡萄糖过滤灭菌。 </li><li> YPDS（+ Zeocin）板 ： 要准备500毫升YPDS + Zeocin琼脂（酵母提取蛋白胨葡萄糖中等山梨醇）溶于5克酵母提取物，山梨醇和91.1克，10克，水450毫升蛋白胨。加入琼脂10克和一个磁力搅拌棒和液体循环20分钟高压灭菌。 与此同时，准备60毫升20％葡萄糖和过滤器在使用前消毒。 让蒸压解决方案，酷〜60 ° C，加入50毫升20％葡萄糖过滤灭菌。 倒入前几个板块加入YPDS板没有Zeocin抗生素。然后从100毫克/毫升原液添加500μLZeocin获得琼脂终浓度为100微克/毫升Zeocin。让旺火上一个磁板时加入抗生素，并让约增加2分钟，同样混合搅拌。 倒入培养皿中的介质，并用黑色塑料覆盖。让板在实验台上干通宵。商店YPD板含有Zeocin 4 ° C。保质期为一到两周。 注：与塑料覆盖面是必要的，因为Zeocin对光敏感。 </li></ol>步骤二： <ol><li> BMGY（缓冲甘油复杂介质）和BMMY（缓冲甲醇-复杂介质）： 每个中等8克酵母提取物和蛋白胨16克，560毫升的水溶解。高压灭菌液体周期为20分钟。 在平均时间，准备了以下解决方案： <ul><li> 1 M磷酸钾缓冲液，pH值 6.0 ： </li></ul>结合1M K 2 HPO 4和156毫升的1M K 2 HPO 4 24毫升，并确认在pH = 6.0（如果需要调整pH值，使用磷酸或氢氧化钾）。过滤消毒，并在室温下保存。这种解决方案的保质期大于一年。 <ul><li> 10X鼎贞（13.4％酵母氮基与无氨基酸铵硫酸）： </li></ul>溶解26.8克酵母氮基（YNB），硫酸铵，并没有在200毫升的水和过滤消毒氨基酸。热的解决方案，完全溶于水YNB。保存在4 ° C。这种解决方案的保质期约为一年。注：另外，使用未经硫酸铵和3.4克YNB无氨基酸，并添加10克硫酸铵。 <ul><li> 500X B（0.02％生物素）： </li></ul>溶解在50毫升水和过滤消毒10毫克生物素。保存在4 ° C。这种解决方案的保质期约为一年。 <ul><li> 10X中号（5％甲醇）： </li></ul>混合95毫升水5毫升甲醇。过滤消毒，并储存于4 ° C。这种解决方案的保质期大约两个月。 <ul><li> 10X庚寅（10％甘油）： </li></ul>甘油10毫升90毫升水混合。过滤消毒。在室温下储存。这种解决方案的保质期大于一年。 让灭菌溶液冷却到室温，然后添加以下拌匀： <ul><li> 80毫升1个M钾磷酸盐缓冲液，pH值6.0; </li><li> 80毫升的10X YNB </li><li> 0.16毫升500X乙</li><li> 80毫升的10X的GY </li></ul> BMMY准备，添加80毫升10X米而不是甘油。 店内媒体在4 ° C这种解决方案的保质期大约两个月。 </li></ol>代表性的成果<img src="/files/ftp_upload/1862/1862fig1.jpg" alt="图1" /> 图1免疫印迹图像显示后24小时表达四个表达的蛋白质（标记为1605，0537，1228年，和1682）。为免疫抗体是一种c - myc基因的辣根过氧化物酶抗体。第二线（N）是一种消极的控制和与不表达的重组蛋白的细胞上清加载，因为它们与父（纯）载体转化。

<ol>
	<li>Cereghino, G. P., Cregg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+yeast+in+biotechnology:+protein+production+and+genetic+analysis.">Applications of yeast in biotechnology: protein production and genetic analysis.</a> Curr Opin Biotechnol. 10, 422-427 (1999).</li><li>Cregg, J. M., Barringer, K. J., Hessler, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pichia+pastoris+as+a+Host+System+for+Transformations.">Pichia pastoris as a Host System for Transformations.</a> Mol. Cell. Biol. 5, 3376-3385 (1985).</li><li>Cregg, J. M., Cereghino, J. L., Shi, J., Higgins, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+in+Pichia+pastoris.">Recombinant protein expression in Pichia pastoris.</a> Mol Biotechnol. 16, 23-52 (2000).</li><li>Cereghino, G. P., Cereghino, J. L., Ilgen, C., Cregg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+in+fermenter+cultures+of+the+yeast+Pichia+pastoris.">Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris.</a> Curr Opin Biotechnol. 4, 329-332 (2002).</li></ol>

蛋白的表达，使用甲醇毕赤酵母作为主机系统，是在本议定书。蛋白可分泌到根据使用的克隆载体介质。重组蛋白的分泌，使后续的纯化更容易。然而，所显示的条件可能要为不同的蛋白质和条件和表达时间的变化表达优化，可能导致蛋白质水平增加。在本议定书中所使用的向量有一个c - myc基因的抗原表位，并可以购买此抗原表位的抗体的功能。其中有没有抗体，这使得蛋白质的分...

expression of recombinant proteins in the methylotrophic yeast pichia pastoris

蛋白质在微生物的真核宿主毕赤酵母中的表达提供了可能产生一个快速且易于使用的表达系统的重组蛋白的高含量。 作为一种单细胞微生物体育酵母是容易操纵的，并在高细胞密度的廉价媒体迅速成长。作为真核细胞，P。酵母是能够执行许多更高的真核细胞中获得的重组蛋白进行翻译后修饰进行蛋白质折叠，蛋白加工，形成二硫键和糖基化[1 ]。 由于甲醇酵母P.酵母是甲醇作为唯一的碳源代谢。强启动醇氧化酶AOX1，严格监管和甲醇诱导和它感兴趣的基因的表达。因此，外源蛋白的表达可诱导培养基中[2加入甲醇; 3]。 另一个重要优势是重组蛋白分泌到培养基中，使用一个信号序列为目标的外源蛋白毕赤酵母的分泌途径。媒体酵母本身分泌的内源性蛋白和媒体没有添加蛋白质水平低，外源蛋白建立在介质中的总蛋白的多数，有利于蛋白质纯化步骤[3，4]。 这里所用的载体（pPICZαA）含有AOX1启动子的严格监管，甲醇诱导感兴趣的基因的表达，一个可供选择的Zeocin 抗性都大肠杆菌基因重组蛋白，分泌的α-因子的分泌信号大肠杆菌和毕赤酵母和C -末端肽含c - myc基因的抗原表位和多聚组氨酸（6xHis）检测和纯化重组蛋白的标记。我们也使用特定的抗 myc - HRP抗体承认父向量的C - myc基因的抗原表位的重组蛋白质免疫印迹分析。

Watch this Scientific Journal Video about Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris at JoVE.com

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

协议描述使用甲醇酵母蛋白的表达<em&gt;毕赤酵母</em&gt;。 electrocompetent酵母细胞的准备，与感兴趣的基因到改造的载体<em&gt; P。酵母</em&gt;和酵母DNA纯化。 Western blot分析和蛋白质纯化，建立在这种蛋白的表达协议的最后步骤。

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

expression-recombinant-proteins-methylotrophic-yeast-pichia

Research

JoVE Journal

Biology

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

45.7K 次观看.  University of British Columbia - UBC. 协议描述使用甲醇酵母蛋白的表达毕赤酵母。 electrocompetent酵母细胞的准备，与感兴趣的基因到改造的载体 P。酵母和酵母DNA纯化。 Western blot分析和蛋白质纯化，建立在这种蛋白的表达协议的最后步骤。

视频: Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

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 ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...