我们感谢安妮 - 玛丽·迪翁 - 科特的讨论导致了该方法的开发。 JK和RB是FQNRT博士学位的学者，米-MG是凡尼尔CIHR学者，和J.-YM是一个FRSQ高级研究员。这项工作是由自然科学和工程研究理事会JY资金支持。 M。

1。生产重组杆状病毒的使用Invitrogen的BAC-到-BAC杆状病毒表达系统主要是根据制造商的协议，只有轻微的修改所产生的杆状病毒: <ol><li>修改后pFastBac1矢量（Gibco公司，生活）是创建一个包含GST和他-标签（ 图2）。一个的pET-52b中（+）载体（Novagen）中的多克隆位点（MCS），含有10xHis-标签，插入一个pFastBac1载体的MCS，生成pFastBac1 - 链霉素-10xHis。在PET-52B（+）的MCS序列进行PCR的XbaI和BLPI限制性位点之间的扩增，增加一个BglⅡ限制性位点的5&#39;末端和一个HindIII限制性位点，在3&#39;末端。将PCR产物然后BamHI和pFastBac1的HindIII限制性位点之间克隆，用BamHⅠ和BglⅡ限制性序列的相容性。因为目的是产生一个pFastBac1允许的表达重组蛋白与链霉亲和10xHis-标签中，PET-52B（+）的BamHI限制性位点中没有使用的MCS序列。此外，XbaI限制性位点没有被使用，以避免已经在pFastBac1的MCS和这些同的pET-52b的MCS插入（+）的位点之间发生的限制性位点的冗余。与断前蛋白酶识别位点（LeuGluValLeuPheGln / GlyPro）的GST编码序列插入到pFastBac1-链球菌，10xHis。从质粒pGEX-6P-2（GE Healthcare公司）的GST cDNA序列的PCR扩增用含有NcoI和KpnI限制性位点的引物，5&#39;和3&#39;末端，分别为。 NcoI位点允许添加一个起始密码子，但也是一个丙氨酸的消费税。然后将PCR产物克隆入pFastBac1 - 链霉素-10xHis的NcoⅠ和KpnⅠ限制位点之间，从而导致删除该链霉亲和标签。由此得到的新的融合载体被命名为pFastBac1-GST-10xHis。 </li><li>克隆的cDNA编码公关关注到GST（N端）和His标签（C端）之间的pFastBac-GST-10xHis矢量otein。小心地取出该cDNA的终止密码子，以允许融合的C-末端His-标签。 </li><li> 转化大肠大肠杆菌 DH10Bac感受态用重组的pFastBac（在步骤1.2中获得），以产生杆状病毒质粒。允许菌落过夜，在37℃和几个小时在含有Bluo-gal和IPTG（10微克/毫升四环素，50微克/毫升卡那霉素，7微克/毫升庆大霉素，100微克/毫升Bluo-选择平板上生长在30℃下加仑，40微克/毫升IPTG）。 </li><li>挑选和重新划2白色菌落（步骤1.3）在选择平板上，以确认白色表型。 </li><li>接种2毫升的LB含10微克/毫升四环素，50微克/毫升卡那霉素，10微克/毫升的庆大霉素与步骤1.4两个白色菌落，并让他们在搅拌下于37℃生长过夜（250转） </li><li>为了净化杆粒DNA，沉淀从步骤1.5的细菌，悬浮细胞在300微升的溶液I并加入300微升的溶液II（自Qiagen大量制备试剂盒）。孵育5分钟，在室温下，加入300微升的3M KOAc pH为5.5，并培育在冰上10分钟。离心样品在4℃，最高速度为10分钟。加入800μl异丙醇的上清，孵育冰​​上10分钟。离心样品15分钟，在4℃，用500μl的70％乙醇洗涤沉淀。让沉淀干燥并在40微升的Tris-EDTA（pH8.0）中在无菌条件下重悬的。该杆粒DNA应保存在4°C。 </li><li>如制造商的方案所述生成杆状病毒。杆状病毒可存放于4℃避光。 </li></ol> 2。重组蛋白的表达<ol><li>为了选择最有效的杆状病毒的净化和监测什么时候到达蛋白表达的高峰期，执行迷你感染测定一个s如下:感染2×10 7的Sf9的感染细胞培养在27℃的Grace的媒体（补充了10％FBS和1％P / S）与133微升杆状病毒（1/150）。收集1.5毫升分装，在0，24，48，和72小时后的感染。沉淀细胞并分析感兴趣使用抗标签抗体的蛋白免疫印迹的表达水平。 </li><li>使用最有效的杆状病毒从步骤2.1感染每毫升含1.0×10 6个Sf9细胞在500毫升的介质与1/150病毒和媒体之间的比例的微调。让感染的细胞生长在悬浮液中在27℃和收获细胞时的最大蛋白表达（如上面步骤2.1中确定的）就达到了。沉淀的细胞可以被储存在-80℃直到进一步的使用。 </li></ol> 3。可溶性细胞裂解液的制备以下所有孵育步骤都在4℃下轻微转动。 <ol><li>裂解S表达的目的蛋白质在约20毫升的GST结合缓冲液（150 mM氯化钠，1mM EDTA中，0.05％的Triton-X-100，1mM的DTT中PBS1X为250mM的NaCl的最终浓度，以及蛋白酶抑制剂F9细胞混合物（Roche），在冰水浴，DOUNCE的20倍溶液用Dounce匀浆，超声处理3次每次30秒（70％产量），并再次DOUNCE 20倍。 </li><li> （可选）。孵育的总细胞裂解物用1mM的MgCl 2和BENZONASE 7核酸酶（2.5单位/毫升）处理30分钟，在4℃以除去DNA或RNA污染。 </li><li>离心机在18,000 rpm离心30分钟，在4°C。保留上清液。 </li><li> （可选）。离心取上清液再30分钟18000转，4℃得到一个明确的可溶性裂解物。通过0.2或0.45微米的过滤器传递上清液，以避免堵塞。 </li></ol> 4。蛋白对GST结合珠<ol><li>孵育可溶性细胞裂解物用1ml GST珠（预洗涤2次10毫升GST的结合缓冲液）1小时，在4℃下轻轻转动。 </li></ol> 评论:培养时间需要根据蛋白质的稳定性和结合效率进行优化。 <ol start="2"><li>以700rpm的转速快速旋转并除去含有未结合蛋白的上清液（这可以保持用于进一步分析）。与GST洗涤缓冲液洗涤的GST-结合蛋白（ 图3B，泳道1）（GST结合缓冲液用350mM的NaCl的终浓度）。 </li><li>重复步骤4.2倍。在最后一次洗涤，去除尽可能多的上清液越好。 </li><li> 1小时孵育用5mM ATP和15mM的MgCl 2的有孔玻璃珠（10毫升GST结合缓冲液）于4℃，以避免热休克蛋白8的非特异性结合。与GST洗涤缓冲液清洗珠三次。 </li></ol> 5。商品及服务税的断前卵裂<ol><li>离心GST珠，除去上清液，用P5 BUF洗GST珠FER（50mM的NaHPO 4 pH值为7.0，500 mM氯化钠，10％甘油，0.05％的Triton-X-100，5mM的咪唑）。 </li><li>从3小时到过夜，4℃孵育在小五缓冲区中的GST-珠断前酶（除以消费税珠在100微升的组分，并添加4-8单位的酶（2-4微升）稀释于100微升的P5缓冲区）。 </li></ol> 评论:需要断前步骤的孵育时间以根据分子量以及该蛋白质的稳定性得到优化。如果降级观察，这个孵化时间可以在一夜之间被减少到最低限度2小时代替。 <ol start="3"><li>快速离心并收集上清液。加入100微升P5后重复此步骤两次 缓冲区的珠子，集中所有分数（ 图3B，泳道2）。 </li></ol> 注释:剩余珠可用于裂解效率分析（ 图3B </stroNG&gt;，泳道3）。通常，蛋白质的70-80％被裂解。 6。蛋白质结合，以TALON金属亲和树脂<ol><li>除以从上面的洗脱分为3个部分1毫升，用100微升爪金属亲和树脂干珠（预洗两次，P5缓冲液）下旋转1小时孵化每个。 </li></ol> 评论:分度洗脱几部分增加结合/洗涤效率。 <ol start="2"><li>用P30缓冲液（含30 mM咪唑的最终浓度P5缓冲液）洗涤树脂5分钟。 </li><li>重复步骤6.2倍，集中在珠在单管。最后一次洗涤之前，除去尽可能多的上清液越好。 </li></ol> 评论:这对应于TALON绑定样品（ 图3B，泳道4）。 7。纯化的蛋白质的洗脱<ol><li>下孵育与P5旋转蛋白结合TALON树脂5分钟00缓冲液（含500mM咪唑的最终浓度P5缓冲液）。使用缓冲液和1:1 V / V的珠之间的比（例如，如果你采取了200微升的干珠，你将不得不加入200μlP500的）。重复此步骤两次，并分别保持各洗脱组分（命名为洗脱1（E1），洗脱2（E2）和洗脱3（E3）， 图3B，泳道5-7）。 </li></ol> 注释:剩余珠可用于洗脱效率（ 图3B，泳道8）的分析。通常情况下，蛋白质的60-80％被洗脱总共。 <ol start="2"><li>通过与在SDS-PAGE和染色用考马斯亮蓝（ 图3B）或SYPRO蛋白染色进行可视化的标准BSA浓度装载约20-40微升的每个级分的分析你纯化的蛋白的数量和质量。 </li></ol> 评论:感兴趣的蛋白质也可以被救援人员到场检测ughout用抗-GST和抗His抗体（ 图3C）的纯化方法。 8。纯化的蛋白质的存储<ol><li>在4℃下透析针对适当的存储缓冲器中的纯化的蛋白质（ 例如，20毫摩尔Tris乙酸盐pH 8.0，200mM的醋酸钾，10％甘油，1mM EDTA，0.5mM的DTT）在透析过程中，检查纯化的蛋白质的沉淀是重要的。我们通常在搅拌下旋转透析2倍1小时，4℃。 </li><li>使纯化的蛋白质（10-20微升）的小等分试样并冷冻在干冰上30分钟。储存分装于-80°C和避免许多解冻/冻结周期。 </li></ol>

<ol>
	<li>Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+affinity+tags+for+protein+purification.">Comparison of affinity tags for protein purification.</a> Protein Expr. Purif. 41, 98-105 (2005).</li><li>Thain, A., Gaston, K., Jenkins, O., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+GST+fusion+proteins+from+co-purifying+GroEL.">A method for the separation of GST fusion proteins from co-purifying GroEL.</a> Trends Genet. 12, 209-210 (1996).</li><li>Carr, 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=Expression+of+a+recombinant+form+of+the+V+antigen+of+Yersinia+pestis,+using+three+different+expression+systems.">Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.</a> Vaccine. 18, 153-159 (1999).</li><li>Armisen, P., 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=Selective+adsorption+of+poly-His+tagged+glutaryl+acylase+on+tailor-made+metal+chelate+supports.">Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.</a> J. Chromatogr. A. 848, 61-70 (1999).</li><li>Kuhn, S., Zipfel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculovirus+expression+vector+pBSV-8His+directs+secretion+of+histidine-tagged+proteins.">The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins.</a> Gene. 162, 225-229 (1995).</li><li>Buisson, R., 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=Cooperation+of+breast+cancer+proteins+PALB2+and+piccolo+BRCA2+in+stimulating+homologous+recombination.">Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.</a> Nat. Struct. Mol. Biol. 17, 1247-1254 (2010).</li><li>Filimonova, M. N., 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=Isoforms+of+Serratia+marcescens+nuclease.+The+role+of+Mg2++in+the+hydrolysis+mechanism.">Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.</a> Biochemistry. 62, 983-988 (1997).</li><li>Thorslund, T., 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=The+breast+cancer+tumor+suppressor+BRCA2+promotes+the+specific+targeting+of+RAD51+to+single-stranded+DNA.">The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.</a> Nat. Struct. Mol. Biol. 17, 1263-1265 (2010).</li><li>Genois, M. M., 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=Interactions+between+BRCA2+and+RAD51+for+promoting+homologous+recombination+in+Leishmania+infantum.">Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.</a> Nucleic Acids Res. 40, 6570-6584 (2012).</li><li>Shrestha, B., Smee, C., Gileadi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+vector+system:+an+emerging+host+for+high-throughput+eukaryotic+protein+expression.">Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.</a> Methods Mol. Biol. 439, 269-289 (2008).</li></ol>

这里提出的GST-他的纯化方案，适用于范围广泛的重组蛋白大小的净化:成功纯化李 RAD51（41kDa），piBRCA2（120kDa），PALB2（130kDa），和一个不稳定的大分子量的蛋白质婴儿利什曼原虫 : 李 BRCA2（125kDa）6,9。此外，与此协议纯化蛋白生化活性6。这种方法的成功完全取决于所需蛋白质的表达，溶解性和稳定性。 在其他宿主中的表达的分子生物学?...

重组蛋白质的纯化是至关重要的，以解决在生物化学的基本问题。蛋白纯化的常规方法类似的离子交换层析和大小排阻层析依赖于目标蛋白的物理性质，例如它的等电点和电荷或大小，分别。后者蛋白质特性是由多种蛋白质，这增加了相当大的常规蛋白质纯化策略污染蛋白的机会共享。这个问题可能会被规避与使用多个纯化柱，这是耗时的。在同一时间，后者的色谱方法需要昂贵的实验装置。亲和标记纯化大力增加目标特异性，因为在大多数的情况下，标签将是唯一的目标蛋白。在最近的研究中，标志或HA-亲和纯化，得到了广泛的使用。 而相比之下，现有的拍摄其中单个标签用于ombinant蛋白质纯化的协议，我们建立两个标签的独特组合。我们的方法是融合GST标签的在N-末端和His-标签在感兴趣的蛋白质的C-末端，用于量和所需蛋白质的纯度之间的最佳比例。的GST是一个长标记（29 kDa）的，这是对纯化的谷胱甘肽琼脂糖珠高效。此外，使用GST保证我们的方法1的成本效益。裂解掉的GST与断前酶（具有识别序列LeuGluValLeuPheGln / GlyPro，导致增加了只有两个氨基酸）的可能性，具有许多优点，例如，这样的策略可避免的生理功能的蛋白质，由于变构阻改变。小的His-tag融合至其它蛋白质末端提供在第二纯化步骤中通过洗涤掉裂解的GST，以及降解的蛋白质和其他增加蛋白质纯度<html>污染物。此外，从GST切换到他的净化步骤柱（TALON树脂）时，协议不需要一个透析步骤。在这样的净化过程常见的污染物是热休克蛋白（HSP）。加入一种培养步骤含MgCl 2和ATP的允许移除这些污染物（ 图1）。 快速蛋白液相色谱（FPLC）和高效液相色谱法（HPLC）是依赖于昂贵的仪器来产生高收益感兴趣的纯化蛋白质的常用技术。我们在座的批次纯化方案，相反，是手动的，不需要昂贵的仪器设置。高产量的蛋白质可以通过扩大的协议达成。在同一时间，用批量纯化方案中，洗脱体积可以提高蛋白质浓度，这是不符合FPLC或HPLC给予调整。 ntent"&gt;此外，在批次GST-他的纯化方案，一个大小范围的〜10-300 kDa的蛋白可被纯化。其中最大的优点是通过以下事实给出，人们可以同时净化几种蛋白质举例来说，两个野生型及其突变体蛋白。所提出的协议的成功完全依赖于目的蛋白质的表达水平和溶解度。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>E.Coli DH10Bac (competent)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Prep Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Solution I and II</td>
		</tr>
		<tr>
			<td>Ultra Pure Bluo-Gal</td>
			<td>Gibco (Life)</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gibco (Life)</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 infected cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>PreScission enzyme</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s infected medium supplemented</td>
			<td>Gibco (Life)</td>
			<td>11605-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum characterized</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (Penicillin-Streptomycin)</td>
			<td>Gibco (Life)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose resin</td>
			<td>GE bioscience</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Talon resin</td>
			<td>Clontech</td>
			<td>635504</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioShop</td>
			<td>288324</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco (Life)</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal GST-antibody</td>
			<td>Production in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 6X-His-antibody</td>
			<td>Clontech</td>
			<td>631212</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dounce homogenizer (tight)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Fisher dismembrator)</td>
			<td>Fisher</td>
			<td>Model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (50 mm)</td>
			<td>Fisher Scientific</td>
			<td>2115217</td>
			<td></td>
		</tr>
	</tbody>
</table>

gst-his purification: a two-step affinity purification protocol yielding full-length purified proteins

在酶学测定法的关键蛋白质的体外生化特性的必要的高浓度的感兴趣的纯化蛋白。蛋白质纯化方案应结合效率，简单性和成本效益1。在这里，我们描述了GST-他的方法作为一种新的小型的亲和纯化系统的重组蛋白，根据N-末端谷胱甘肽琼脂糖标签（GST）的2,3和C-末端10xHis标签4，它们都稠合到感兴趣的蛋白质。后者构建体被用于产生杆状病毒的Sf9对感染细胞中的蛋白质表达5的感染。消费税是用来确保净化效率相当长的标记（29 kDa的）。然而，它可能影响到蛋白质的生理特性。因此，它使用的是断前酶6随后裂解的蛋白。为了确保最大的纯度和去除切割的商品及服务税，我们新增的基础上，比较小His标签的第二亲和纯化步骤。重要的是，我们的技术是基于两个不同的标记侧翼的蛋白质，这是一个有效的工具来除去降解的蛋白质，因此两端，丰富了全长蛋白。这里提出的方法不需要昂贵的仪器设置，诸如FPLC。此外，我们成立氯化镁和ATP清洗以去除热休克蛋白杂质和核酸酶处理，取消污染核酸。总之，两个不同的标记侧翼的组合的N-和C-末端和切割过的标签1的功能，担保感兴趣的高度纯化的和全长蛋白质的回收率。

为了说明GST-HIS纯化方案的效率，我们纯化Rec14，一个S。 32.9 kDa的酵母蛋白质。该Rec14 cDNA克隆在我们修改pFastBac1向量允许GST和他-标签的添加在N-和C-末端，分别为（ 图1A）。重组杆状病毒，然后准备并用于感染SF9感染的细胞用于蛋白质表达。可溶性细胞裂解物与GST珠和束缚蛋白裂解与断前蛋白酶的消费税洗脱。所得Rec14-His蛋白是亲和纯化TALON树脂和束缚的蛋白质用含有500mM...

Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

在本协议中，我们展示了一个高效和具有成本效益的小型蛋白质纯化方法，它允许重组蛋白的纯化结合独特的可裂解GST标签和一个小His标签。

GST-他的净化:一个两步亲和纯化协议屈服全长纯化的蛋白质

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

gst-his-purification-two-step-affinity-purification-protocol-yielding

Research

JoVE Journal

Biology

41.3K 次观看.  Hôtel-Dieu de Québec. 在本协议中，我们展示了一个高效和具有成本效益的小型蛋白质纯化方法，它允许重组蛋白的纯化结合独特的可裂解GST标签和一个小His标签。 

视频: GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

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

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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 ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

GST-他的净化:一个两步亲和纯化协议屈服全长纯化的蛋白质

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