这项工作是由该VENOMICS项目，欧盟项目补助扆278346通过第七框架计划（FP7生2011-2015年）的支持。该VENOMICS项目是在欧洲一些研究机构和公司之间的合作： AFMB，埃克斯 - 马赛UNIVERSITE（法国），东航萨克雷（法国），NZYTech（葡萄牙），SistemasGenomicos（西班牙），大学列日（比利时），VenomeTech（法国），Vitamib（法国），新西兰制药（丹麦） 这项工作是由法国基础设施一体化结构生物学（FRISBI）ANR-10-INSB-05-01支持。 作者还要感谢杰里米Turchetto先生协助筹备影片的拍摄。

<ol>
	<li>Berrow, N. 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=Recombinant+protein+expression+and+solubility+screening+in+Escherichia+coli:+a+comparative+study.">Recombinant protein expression and solubility screening in Escherichia coli: a comparative study.</a> Acta Crystallographica Section D-Biological Crystallography. 62, 1218-1226 (2006).</li><li>Correa, A., Oppezzo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+different+expression+parameters+to+achieve+soluble+recombinant+proteins+in+E.+coli:+advantages+of+high-throughput+screening.">Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening.</a> Biotechnol J. 6, 715-730 (2011).</li><li>Graslund, 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=Protein+production+and+purification.">Protein production and purification.</a> Nat Methods. 5, 135-146 (2008).</li><li>Vera, A., Gonzalez-Montalban, N., Aris, A., Villaverde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conformational+quality+of+insoluble+recombinant+proteins+is+enhanced+at+low+growth+temperatures.">The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures.</a> Biotechnol Bioeng. 96, 1101-1106 (2007).</li><li>Graslund, 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=The+use+of+systematic+N-+and+C-terminal+deletions+to+promote+production+and+structural+studies+of+recombinant+proteins.">The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins.</a> Protein Expr Purif. 58, 210-221 (2008).</li><li>Bird, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+construction+and+small+scale+expression+screening+of+multi-tag+vectors+in+Escherichia+coli.">High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli.</a> Methods. 55, 29-37 (2011).</li><li>Davis, G. D., Elisee, C., Newham, D. M., Harrison, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fusion+protein+systems+designed+to+give+soluble+expression+in+Escherichia+coli.">New fusion protein systems designed to give soluble expression in Escherichia coli.</a> Biotechnol Bioeng. 65, 382-388 (1999).</li><li>Kapust, R. B., Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+maltose-binding+protein+is+uncommonly+effective+at+promoting+the+solubility+of+polypeptides+to+which+it+is+fused.">Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.</a> Protein science: a publication of the Protein Society. 8, 1668-1674 (1999).</li><li>LaVallie, E. R., Lu, Z., Diblasio-Smith, E. A., Collins-Racie, L. A., McCoy, 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=Thioredoxin+as+a+fusion+partner+for+production+of+soluble+recombinant+proteins+in+Escherichia+coli.">Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli.</a> Methods Enzymol. 326, 322-340 (2000).</li><li>Marblestone, J. G., 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=Comparison+of+SUMO+fusion+technology+with+traditional+gene+fusion+systems:+enhanced+expression+and+solubility+with+SUMO.">Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO.</a> Protein science: a publication of the Protein Society. 15, 182-189 (2006).</li><li>Nozach, H., 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=High+throughput+screening+identifies+disulfide+isomerase+DsbC+as+a+very+efficient+partner+for+recombinant+expression+of+small+disulfide-rich+proteins+in+E.+coli.">High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.</a> Microb Cell Fact. 12, 37 (2013).</li><li>Sachdev, D., Chirgwin, 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=Fusions+to+maltose-binding+protein:+control+of+folding+and+solubility+in+protein+purification.">Fusions to maltose-binding protein: control of folding and solubility in protein purification.</a> Methods Enzymol. 326, 312-321 (2000).</li><li>Smith, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+fusions+to+glutathione+S-transferase+for+protein+studies.">Generating fusions to glutathione S-transferase for protein studies.</a> Methods Enzymol. 326, 254-270 (2000).</li><li>de Marco, A., Deuerling, E., Mogk, A., Tomoyasu, T., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone-based+procedure+to+increase+yields+of+soluble+recombinant+proteins+produced+in+E.+coli.">Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.</a> BMC Biotechnol. 7, 32 (2007).</li><li>Hatahet, F., Nguyen, V. D., Salo, K. E., Ruddock, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+reducing+pathways+is+not+essential+for+efficient+disulfide+bond+formation+in+the+cytoplasm+of+E.+coli.">Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli.</a> Microb Cell Fact. 9, 67 (2010).</li><li>de Marco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+contributions+in+the+field+of+the+recombinant+expression+of+disulfide+bonded+proteins+in+bacteria.">Recent contributions in the field of the recombinant expression of disulfide bonded proteins in bacteria.</a> Microb Cell Fact. 11, 129 (2012).</li><li>Katzen, F., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+bond+formation+in+periplasm+of+Escherichia+coli.">Disulfide bond formation in periplasm of Escherichia coli.</a> Methods Enzymol. 348, 54-66 (2002).</li><li>Klint, J. K., 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=Production+of+recombinant+disulfide-rich+venom+peptides+for+structural+and+functional+analysis+via+expression+in+the+periplasm+of+E.+coli.">Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.</a> PloS One. 8, e63865 (2013).</li><li>Braud, 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=Dual+expression+system+suitable+for+high-throughput+fluorescence-based+screening+and+production+of+soluble+proteins.">Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.</a> Journal of Proteome Research. 4, 2137-2147 (2005).</li><li>Douzi, B. G. A., 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=A+new+system+for+expressing+recombinant+animal+toxins+in+E.+coli.+(Collection+Rencontres+en+Toxicologie,+Publications+de+la+SFET,+Chatenay-Malabry).">A new system for expressing recombinant animal toxins in E. coli. (Collection Rencontres en Toxicologie, Publications de la SFET, Chatenay-Malabry).</a> Advances and new technologies in toxinology. , 149-152 (2010).</li><li>Groisillier, A., 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=MARINE-EXPRESS:+taking+advantage+of+high+throughput+cloning+and+expression+strategies+for+the+post-genomic+analysis+of+marine+organisms.">MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms.</a> Microb Cell Fact.. 9, 45 (2010).</li><li>Vincentelli, 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=High-throughput+protein+expression+screening+and+purification+in+Escherichia+coli.">High-throughput protein expression screening and purification in Escherichia coli.</a> Methods. 55, 65-72 .</li><li>Xiao, 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=The+high-throughput+protein+sample+production+platform+of+the+Northeast+Structural+Genomics+Consortium.">The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium.</a> Journal of Structural Biology. 172, 21-33 (2010).</li><li>Saez, N. J., Vincentelli, R., Chen, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch3+High-throughput+expression+screening+and+purification+of+recombinant+proteins+in+E.+coli.">Ch3 High-throughput expression screening and purification of recombinant proteins in E. coli.</a> Structural Genomics: General Applications : Methods in Molecular Biology. 1091, 33-53 (2014).</li><li>Katzen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+(R)+recombinational+cloning:+a+biological+operating+system.">Gateway (R) recombinational cloning: a biological operating system.</a> Expert. Opin. Drug Discov. 2, 571-589 (2007).</li><li>Vincentelli, R., Canaan, S., Offant, J., Cambillau, C., Bignon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+expression+and+solubility+screening+of+His-tagged+proteins+in+96-well+format.">Automated expression and solubility screening of His-tagged proteins in 96-well format.</a> Analytical Biochemistry. 346, 77-84 (2005).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expr Purif. 41, 207-234 (2005).</li><li>Spencer, C. I., 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=Ion+channel+pharmacology+under+flow:+automation+via+well-plate+microfluidics.">Ion channel pharmacology under flow: automation via well-plate microfluidics.</a> Assay Drug Dev Technol. 10, 313-324 (2012).</li><li>Sala, E., de Marco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+optimized+protein+purification+protocols+by+coupling+small-scale+expression+and+mini-size+exclusion+chromatography.">Screening optimized protein purification protocols by coupling small-scale expression and mini-size exclusion chromatography.</a> Protein Expr Purif. 74, 231-235 (2010).</li><li>Moon, A. F., Mueller, G. A., Zhong, X., Pedersen, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synergistic+approach+to+protein+crystallization:+combination+of+a+fixed-arm+carrier+with+surface+entropy+reduction.">A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction.</a> Protein science: a publication of the Protein Society. 19, 901-913 (2010).</li><li>Zanier, K., 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=.">.</a> Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins. 339, 694-698 (2013).</li><li>Kapust, R. B., Tozser, J., Copeland, T. D., Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P1'+specificity+of+tobacco+etch+virus+protease.">The P1' specificity of tobacco etch virus protease.</a> Biochemical and Biophysical Research Communications. 294, 949-955 (2002).</li><li>Vincentelli, R., Romier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+in+Escherichia+coli:+becoming+faster+and+more+complex.">Expression in Escherichia coli: becoming faster and more complex.</a> Current Opinion in Structural Biology. 23, 326-334 (2013).</li><li>Jolma, A., 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=DNA-binding+specificities+of+human+transcription+factors.">DNA-binding specificities of human transcription factors.</a> Cell. 152, 327-339 (2013).</li><li>Aslanidis, C., de Jong, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation-independent+cloning+of+PCR+products+(LIC-PCR).">Ligation-independent cloning of PCR products (LIC-PCR).</a> Nucleic Acids Research. 18, 6069-6074 (1990).</li><li>Haun, R. S., Serventi, I. M., Moss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+reliable+ligation-independent+cloning+of+PCR+products+using+modified+plasmid+vectors.">Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors.</a> BioTechniques. 13, 515-518 (1992).</li><li>van den Ent, F., Lowe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RF+cloning:+a+restriction-free+method+for+inserting+target+genes+into+plasmids.">RF cloning: a restriction-free method for inserting target genes into plasmids.</a> Journal of Biochemical and Biophysical Methods. 67, 67-74 (2006).</li><li>Vincentelli, 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=High-throughput+automated+refolding+screening+of+inclusion+bodies.">High-throughput automated refolding screening of inclusion bodies.</a> Protein science: a publication of the Protein Society. 13, 2782-2792 (2004).</li></ol>

The authors declare that they have no competing financial interests.

有可溶性的，折叠，功能性蛋白的表达没有单一的通用协议。具有成本和时间效益，因此大多数实验室或蛋白质核心设施具有多个目标的工作使用高通量蛋白质表达筛选，找到变量最好的&#39;通用&#39;组合，以获得可溶性活性蛋白，为广大的目标。我们已经确定及DsbC作为以二硫键丰富的肽和蛋白质11的可溶性表达一个普遍适用的融合伴侣。使用及DsbC融合和高通量的方法，在一个星期内的多个目标的可溶性表达，可以观察到11，然后附加变量，比如那些在引言中讨论的，可以在随后的几轮筛选对那些需要进一步优化的目标。本文中所描述的协议的目的是二硫键丰富的蛋白质和肽的表达。然而，对于用户希望为exp以高通量方式RESS非网状的蛋白质，相应的协议已被先前公布的，并且可以在其他地方找到22,24。 关于高吞吐量的设置是理想的许多应用，其中包括了大量不同的蛋白质为可溶性表达或用于在同一时间几个靶基因大量表达构建体（包括各种融合标签）的筛选的筛选（或者，为了提高成功率多个表达构建为一个单一的目标）。该平台还可以用于新的协议上的大量目标的基准测试和验证。其他应用包括的变体的筛选单个困难的目标， 例如 ，所有的直向同源物或同一个家庭的成员，或者测试生产单一靶的突变体在一个实验小组的成功。这个协议也已经在与共同表达载体（机智组合使用高电平单只标记蛋白），使上拉下来的蛋白-蛋白复合物随后更彻底的生物物理分析，以确认正确的复合物的形成和化学计量33初步鉴定。蛋白质纯化的量有时适于微测定法（功能测试，蛋白质-DNA 34或蛋白质-蛋白质相互作用测定法）。有几个优势，以高通量表达筛选策略：（i）向测试大量的目标的能力或大量的变量在单个实验中，（ⅱ）有限批次到批次的变化，（iii）该简单和易于使用的深的井，（ⅳ）可扩展性和重现性在规模较大，（V），用于自动化的潜力，以及跟踪的（ⅵ）简单性在较小规模的加工和处理（无标记单独的管的，更少的错误使用时，板格式较个别管中混合处理或克隆的交换）出台。 <p类=“jove_content”&gt;虽然在协议部分没有讨论，有几个重要的考虑因素是什么下面将简要讨论了实验的准备工作。为更深入的讨论，请参阅我们以前的出版物24。为了获得最大的效率，这是有利于有一个合适的系统，用于高通量克隆中，为了简化亚克隆的大量目标。对于VENOMICS项目的初始阶段，我们利用多功能网关重组系统25，允许亚克隆任何目的载体数百每周克隆的步伐。协议用于Gateway重组克隆可以Invitrogen公司的网站上找到。高通量克隆其他替代方案包括结扎独立克隆（LIC）35,36和限制自由（RF）克隆37。有多种方式来获得目标基因的表达，包括PCR方法从模板DNA，从入门克隆的集合或合成的基因，这是选择用于VENOMICS项目的策略。合成的基因可以与重组位点上的基因和基因合成的每一端责令使靶基因序列的简单的密码子优化（排除罕见的大肠杆菌密码子）。这个建议，但不是必需的。对于包含大量的稀有密码子，罗塞塔2（DE3）pLysS中（它携带的tRNA对于没有高度表达于大肠杆菌稀有密码子）的目标无密码子优化可能更适合比BL21（DE3）pLysS中。虽然有可用的限制二硫键在胞质中的正常的还原环境，减少株，在我们的手中，他们就没有这么成功，因为经常E.大肠杆菌菌株11。 分析型亲和纯化从测试表现文化，以恢复可溶性融合蛋白和量化收益率执行。定量数据可在日获得100 mg / L的文化 - 在0.1之内表达的融合蛋白电子可溶性产量。如果目标不溶于水，不同的融合合作伙伴之前追求另类的表达和诱导温度，压力或媒体可以进行测试。对于二硫键蛋白质丰富的可溶性表达，我们此前排名融合合作伙伴的效果及DsbC&gt; DsbA蛋白&gt; GST&gt; MBP&gt; TRX&gt; His标签的细胞质表达11。周质表达的是，可能有助于二硫键丰富的目标的成功折叠另一种可能性。周质是一个不太还原环境比细胞质和包含有用的氧化还原分子伴侣协助二硫键。及DsbC，DsbA蛋白和MBP蛋白通过其周质信号序列通常定位于周质中。这提供了机会，以引导二硫化物富目标到周质空间中，以协助折叠来利用这些标记。对于顽固性的目标，下一步将是为p从包涵体urify不溶性HIS标签的目标，溶解和复性（这是这项协议的范围，并不会在这里讨论3 9）。这对于只有一个或两个二硫键目标相当简单，但变成为二硫键数目的增加变得越来越困难。或者，特别是对于蛋白质和肽具有四个或更多个二硫键，它可能是有益的尝试较复杂的生产系统，例如酵母，昆虫或哺乳动物表达。 随着微型化和自动化的进步，HTP电生理实验室的单芯片技术28无疑将是未来的功能分析的方法。我们预期，对于大多数用途（或许除了结构研究的），这将否定需要大规模培养。小规模的文化将不仅是筛选表达式条件下非常有用，而且还能够提供萨夫ficient量样品的这些小型化的功能测定法。以足够的数量，以产生并行的多个目标功能特性的能力将降低培养和使用这些种平台的成本，重组蛋白质的表达和表征将变得更加成本和时间效益。 的协议本文已应用于二硫键丰富的肽和蛋白质的表达在FP7欧洲VENOMICS项目的初始阶段。毒液是生物活性肽，往往有有趣的药理潜力的极好来源。然而，它们的生产是由于其复杂的二硫键键合模式和小尺寸有挑战性。使用像这里所描述的高通量平台上，VENOMICS项目旨在产生10,000小说蛇毒肽库再现自然界中观察到的多样性。这个库将被利用为二硫化物-R的表征ICH肽与潜在的药物或治疗应用与开发新药的目标。该平台目前正在使用的基准测试和验证新的协议在VENOMICS项目中使用。

通过基因组学的进步和发现新蛋白质的速度加快推动下，高吞吐量的管道已发展到并行的筛选和鉴定的最佳蛋白生产策略传统方法。潜在变量来进行优化，包括但不限于，不同的表达菌株1,2，3,4的温度，介质2,3，目标变体5，融合伴侣6-13，共表达与蛋白伴侣14,15，细胞质或周质表达16-18，纯化缓冲部件3。通过实现高的吞吐量的方法，许多变量，或者可以在用效率的一个高层次的并行测试多个目标，同时限制批与批之间的变化。根据我们的经验，该战略还给出了在规模化，重复性好使用相同的文化（温度，介质，通风等）和纯化条件（相同的住宅建设N，缓冲器等）。一些高通量平台已被用于在过去十年中，以确定条件为可溶性蛋白表达，即通过改变参数，诸如融合伴侣，表达株或温度19-23。 我们最近使用我们的高通量筛选方法对可溶性二硫化物富含蛋白11的表达。所选择的蛋白质不仅从有毒的来源，而且还包括从各种物种，包括植物，猪，牛和人的二硫化物富酶抑制剂。本实验比较了12个不同的融合配偶体和对28二硫键网状蛋白的溶解度和折叠三种不同的表达菌株的影响。我们证明，使用及DsbC作为生产的菌株BL21融合伙伴（DE3）pLysS中大大outproduced（中获得的水溶性蛋白质的产量和数量）应变和融合的任何其他组合测试11。该该实验的结果所形成的基础为适应我们原来一般高通量管道（其已被用于范围广泛的蛋白质的表达筛选）22,24成一个更适合的二硫键富含目标的表达。从动物毒液二硫键丰富的蛋白质是特别感兴趣的。毒液是生物活性肽和蛋白质的复杂混合物，具有潜在价值的药理学和治疗学。然而，含二硫键蛋白的表达是​​不平凡的。这些蛋白六时五十九二硫键之间通常含有，并且必须以激活与正确的二硫键键合模式被氧化。目前，该平台被用于筛选大量二硫键丰富的动物毒液蛋白的表达作为欧盟第七框架计划的欧洲VENOMICS项目（www.venomics.eu）的一部分和基准新颖的协议为数千个目标的高通量表达。这里，一个自动化的方法提供了一种用于高通量小规模的表达筛选和纯化（参见图1）施加到二硫化物富含动物毒液蛋白。战略二硫化物丰富的肽和蛋白质利用His-标签纯化和氧化还原活性的融合伴侣，及DsbC，创造裂解HIS-及DsbC融合物与靶蛋白（参见图2）。 而该协议的重点是这里使用的自动化液体处理机器人和HTP电泳，这些方法也适用于高吞吐量的手工做法，这意味着即使有基本的设置实验室可以利用协议的优势没有任何先决条件昂贵的设备。为转换到纯化和分析（不特定二硫化物富含蛋白质）手动协议已经在别处24出版并在此将不再重复。吞吐量的手动过程（从表达克隆，通过重组速率产生国家克隆25，以可溶性蛋白水平的分析）为96（用SDS-PAGE检测）或384（4×96;用点印迹和SDS-PAGE 26）每星期培养物（参见图1）。这可以如（使用液体处理机械臂和斑点印迹26或HTP电泳，如用卡尺GXII芯片实验室系统22中的半自动化的方式执行增加 为结果），以高达1152（12×96）培养物在平行超过一周，如本文中所述的分析。培养是在深井24（DW24）格式进行的，这样经常晃动孵化器可以在对比生长在深井96（DW96）格式，这需要使用的短轨道高速晃动孵化器有足够的通风（晃动培养使用在800rpm）。使用自动感应介质27还简化表达，消除了手工归纳步骤。即使在实验室已经在使用预定义的表达和纯化fication条件，这些都可以直接传送到该HTP系统仅仅是为了提高效率。高通量筛选管道为二硫化物富含蛋白质的详细的原理图在图3中提供。在管道中的参数的基础上广泛筛选实验11，22，这使我们能够选择用于蛋白质生产的最有用的条件下被选定。 特性可以直接从在试验性研究，其中几十样品微克的就足够了，或者对于敏感功能测定及结合测定法（例如，低体积HTP膜片钳系统28）的小规模的表达纯化标记的蛋白来进行。相同的，甚至可以在未标记的目标来进行裂解之后，所提供的标记和蛋白酶被除去（例如，通过反相HPLC）。质量控制也可以通过质谱法进行的（以确认预期的大小和氧化STATE）或色谱法（以确认纯度或异质性）29。有时标签切割是不必要的或什至不希望的（特别是对于难溶蛋白30,31），所以在这个协议裂解是任选的。无论如何，在所有构建体有一个TEV蛋白酶切割位点（ENLYFQ / [G] 32）直接与靶基因切割后，以产生天然蛋白质前述（ 见图2和讨论）。如果融合标记的裂解需要，裂解可以被测试（在洗脱级分或“上的列&#39;）在小规模分析效率，优化，如果需要，将获得的产量为随后按比例放大实验的可靠估计的条件。 有两种选择的亲和纯化过程中使用的珠的体积，取决于实验的目的和期望。为了能够捕获尽可能多的蛋白可能（以纯化为导频测定或质谱，或到EXTRAPOL吃了规模化收益率）的200微升树脂的最终体积应使用，使系统的饱和之前在1-100 mg / L的文化范围内检测可溶性蛋白质（见协议（一）第8.1节） 。然而，如果该实验的目的是低量的可溶性蛋白质的检测，然后50μl的树脂的最终体积是合适的，允许在0.1-25毫克/升培养的范围内检测可溶性蛋白质（见协议（乙）第8.2节）。 生产可以按比例增加，如果需要的话，得到的纯化的目标毫克量为，使用鉴定为可溶性表达的条件的其他结构和功能研究。在AFMB使用向上扩展协议的详情已在别处22,24讨论。 有关的实验装置，该协议中的关键步骤的进一步详情，改进和故障排除和技术的限制在盘被提供ussion。在开始实验前，请先阅读讨论。 整个预计的90％的成功率在每个步骤中的协议（例如，至少90％的培养物的生长必须在任何给定工序）。如果在实验中的任何步骤的成功率低于90％的样品被丢弃和重复实验的完整集合构建体。然而，这种成功率不是适用于表达为可溶性蛋白，或具有100％效率的切割结构的比例的结构的数目，因为这将是高度依赖于测试的蛋白质。 提供每个协议的具体细节的设置了机器人工作台（也如图4所示 ），但是它们可以被修改为需要的替代工作台设置窗口。机器人硬件（Tecan公司）由一个96多通道臂（MCA96），机器人机械臂（罗马）和8通道的液体处理头（中LIHA）。也可以使用LIHA如果MCA96不可执行的所有利用MCA96步骤，但是他们会花费更长的时间，因为LIHA需要步骤之间进行洗涤。当机器人在技术上是不是无菌环境，包括抗生素一般可确保不会有问题，污染或不育。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tecan Freedom EVO 200 liquid handling robot</td>
			<td>Tecan Group Ltd.</td>
			<td></td>
			<td>Protocols can be adapted to any liquid handling robot with a vacuum manifold for plates. 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2694&#38;ID=5270&#38;Menu=1&#38;Item=21.1.8</td>
		</tr>
		<tr>
			<td>96-well PCR (PCR96) plates</td>
			<td>Greiner Bio-One</td>
			<td>652270</td>
			<td> 
			http://us.gbo.com/bioscience/detail/2504/</td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td></td>
			<td></td>
			<td>Autoclaved for sterility.</td>
		</tr>
		<tr>
			<td>Antibiotic stocks</td>
			<td></td>
			<td></td>
			<td>Kanamycin (50 mg/ml), Ampicillin (100 mg/ml), Chloramphenicol (34 mg/ml in ethanol), store stocks at -20 &#176;C. Use a 1 in 1000 dilution.</td>
		</tr>
		<tr>
			<td>Deep-well 96 (DW96) plate with 2.42 ml volume capacity</td>
			<td>Greiner Bio-One&#160;</td>
			<td>780270</td>
			<td>Autoclaved for sterility. 
			http://us.gbo.com/bioscience/detail/2462/</td>
		</tr>
		<tr>
			<td>Expression vectors</td>
			<td></td>
			<td></td>
			<td>For the expression of target constructs. Store at &#8722;20 &#176;C.</td>
		</tr>
		<tr>
			<td>BL21 (DE3) pLysS competent cells</td>
			<td></td>
			<td></td>
			<td>Or alternative E. coli expression strain of the user&#39;s choice. Store at &#8722;80 &#176;C.</td>
		</tr>
		<tr>
			<td>Breathseal breathable adhesive film</td>
			<td>Greiner Bio-One</td>
			<td>676050</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine with 96-well plate block</td>
			<td></td>
			<td></td>
			<td>For the transformation heat shock and boiling of SDS-PAGE/Caliper samples.</td>
		</tr>
		<tr>
			<td>24-well sterile tissue culture plates</td>
			<td>Greiner Bio-One</td>
			<td>662165</td>
			<td> 
			http://us.gbo.com/bioscience/detail/2220/</td>
		</tr>
		<tr>
			<td>LB-agar</td>
			<td></td>
			<td></td>
			<td>Autoclaved for sterility.</td>
		</tr>
		<tr>
			<td>200 &#956;l sterile disposable tips</td>
			<td>Tecan Group Ltd.</td>
			<td>30 038 617</td>
			<td> 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2283&#38;ID=4118&#38;Menu=1&#38;Item=21.4.1.2</td>
		</tr>
		<tr>
			<td>Multitron Shaking Incubator, with 3 mm throw</td>
			<td>Infors</td>
			<td>AJ103</td>
			<td>Not essential, a regular shaking incubator can also be used. 
			http://www.infors-ht.com/index.php/en/</td>
		</tr>
		<tr>
			<td>Plate incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td></td>
			<td></td>
			<td>For glycerol stocks and elution using purification protocol B.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td></td>
			<td></td>
			<td>For the preparation of glycerol stocks.</td>
		</tr>
		<tr>
			<td>200 &#956;l disposable tips</td>
			<td>Tecan Group Ltd.</td>
			<td>30 038 616</td>
			<td> 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2283&#38;ID=4118&#38;Menu=1&#38;Item=21.4.1.2</td>
		</tr>
		<tr>
			<td>Adhesive tape pads&#160;</td>
			<td>Qiagen</td>
			<td>19570</td>
			<td> 
			http://www.qiagen.com/Products/Catalog/Lab-Essentials-and-Accessories/Tape-Pads</td>
		</tr>
		<tr>
			<td>Bactinyl</td>
			<td>Orapi Group</td>
			<td></td>
			<td>Or equivalent microbial disinfectant.</td>
		</tr>
		<tr>
			<td>ZYP-5052 medium</td>
			<td></td>
			<td></td>
			<td>See Table 1 for recipes of components.</td>
		</tr>
		<tr>
			<td>Deep well 24 (DW24) plates,10 ml capacity&#160;</td>
			<td>Whatman</td>
			<td>7701-5102</td>
			<td>Autoclaved for sterility. 
			http://www.whatman.com/UNIPLATECollectionandAnalysisMicroplates.aspx#7701-5102</td>
		</tr>
		<tr>
			<td>Flat-bottomed, clear microtiter plate</td>
			<td>Greiner Bio-One</td>
			<td>655101</td>
			<td>For absorbance readings. 
			http://us.gbo.com/bioscience/detail/2029/</td>
		</tr>
		<tr>
			<td>Plate reading spectrophotometer</td>
			<td></td>
			<td></td>
			<td>Optional. For measuring OD600nm of cultures.</td>
		</tr>
		<tr>
			<td>Centrifuge with rotor for deep well plates&#160;</td>
			<td></td>
			<td></td>
			<td>Suitable for 3800 x g.</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td></td>
			<td></td>
			<td>50 mg/ml in water. Store at &#8722;20 &#176;C.</td>
		</tr>
		<tr>
			<td>Imidazole ACS grade</td>
			<td>Merck</td>
			<td>IX0005-1</td>
			<td>A high quality grade of imidazole must be used so that it will not interfere with A280 readings for calculating protein yield.</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 10 mM imidazole pH 8 containing 0.25 mg/ml lysozyme (or your preferred buffer). Add lyzozyme from stocks each time and do not store for extended periods.</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sonicator (Ultrasonic processor XL, adapted for deep well plates)</td>
			<td>Misonix Inc.</td>
			<td></td>
			<td>Not essential. Lysozyme alone is normally sufficient for nearly complete lysis.</td>
		</tr>
		<tr>
			<td>DNase&#160;</td>
			<td></td>
			<td></td>
			<td>2 mg/ml stock in water, filter sterilized. Store aliquots at &#8722;20 &#176;C.</td>
		</tr>
		<tr>
			<td>Magnesium sulphate (MgSO4)</td>
			<td></td>
			<td></td>
			<td>2M stock in water, autoclaved.</td>
		</tr>
		<tr>
			<td>SDS-PAGE or Caliper sample buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Binding buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 10 mM imidazole pH 8 (or your preferred buffer). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 50 mM imidazole pH 8 (or your preferred buffer). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td></td>
			<td></td>
			<td>50 mM Tris, 300 mM NaCl, 250 mM imidazole pH 8 (or your preferred buffer). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>96-well Receiver/Filter Plate 20 &#181;m, 1.8 ml capacity</td>
			<td>Macherey-Nagel</td>
			<td>740686.4</td>
			<td> 
			http://www.mn-net.com/ProductsBioanalysis/Accessories/tabid/10909/language/en-US/Default.aspx</td>
		</tr>
		<tr>
			<td>200 &#956;l disposable wide bore tips</td>
			<td>Tecan Group Ltd.</td>
			<td>30 050 348</td>
			<td> 
			http://www.tecan.com/platform/apps/product/index.asp?MenuID=2283&#38;ID=4118&#38;Menu=1&#38;Item=21.4.1.2</td>
		</tr>
		<tr>
			<td>Ni Sepharose 6 Fast Flow resin</td>
			<td>GE Healthcare</td>
			<td>17-5318-02</td>
			<td>For purification of target fusion proteins. Store at 4 &#176;C. Wash and equilibrate before use. 
			http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/17531802</td>
		</tr>
		<tr>
			<td>Tobacco Etch Virus (TEV) protease</td>
			<td></td>
			<td></td>
			<td>Optional, for cleavage. 2 mg/ml, without reducing agents in storage buffer. Store at &#8722;80 &#176;C.</td>
		</tr>
		<tr>
			<td>96-well 0.22 &#181;m filter plate</td>
			<td>Millipore</td>
			<td>MSGV N22 10</td>
			<td>Optional. To filter the soluble fraction after cleavage. 
			http://www.millipore.com/catalogue/item/msgvn2210</td>
		</tr>
		<tr>
			<td>SDS-PAGE/dot blot equipment or Caliper Labchip GXII equipment</td>
			<td></td>
			<td></td>
			<td>Electrophoresis apparatus and choice of gel type is at the user&#8217;s discretion.</td>
		</tr>
		<tr>
			<td>Spectrophotometer and cuvettes</td>
			<td></td>
			<td></td>
			<td>For measuring absorbance at 280 nm (A280) to calculate yield of soluble proteins. Not required if the analysis is done on the Caliper.</td>
		</tr>
		<tr>
			<td>ZipTip pipette tips</td>
			<td>Millipore</td>
			<td></td>
			<td>Optional. The type of ZipTip is at the user&#39;s discretion. C4 resin should be used for larger proteins, while for peptides C18 may be better. Alternative methods for desalting of samples may also be used to clean up samples before further quality control. 
			http://www.millipore.com/catalogue/module/c5737#0</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials are listed in the order in which they are required in the Protocol section. Reagents can be stored at room temperature unless noted otherwise. Reference numbers for the author&#8217;s preferred choice of materials are provided, however equivalent products may also be suitable. For reagents where the brand will not influence the outcome of the experiment, the company details have been omitted.</td>
		</tr>
	</tbody>
</table>

high throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in e. coli

大肠杆菌（E.coli）是用于生产重组蛋白质的结构和功能研究最广泛使用的表达系统。然而，纯化的蛋白质有时是具有挑战性的，因为许多蛋白质​​被表达的不溶性形式。当有困难或多个目标的工作。因此，建议使用高通量（HTP）在一个小规模的蛋白表达筛选（1​​-4毫升培养），以快速识别的可溶性表达条件。为配合实验室的各种结构基因组学计划，定量（范围为0.1〜100 mg / L的重组蛋白的文化内）和HTP蛋白表达筛查方案实施和验证上千种蛋白质。该协议是自动的使用液体处理机器人的，但也可以不用专门的设备手动执行。 二硫键丰富的蛇毒蛋白的获得日益认识到他们的潜力作为治疗药物的线索。他们可以是非常有力和有选择性，但其复杂的二硫键的网络使他们具有挑战性的生产。由于欧盟FP7项目Venomics（成员<a href="http://www.venomics.eu">www.venomics.eu</a> ），我们的挑战是开发成功的生产策略与生产成千上万的小说蛇毒蛋白质功能特性的目的。通过二硫键异构酶的及DsbC氧化还原性能的帮助下，我们调整了我们生产的HTP管线被氧化，功能毒液肽在大肠杆菌中的表达大肠杆菌细胞质中。该协议也适用于生产多种二硫化物丰富的蛋白质。在这里，我们展示了我们的管道用于生产动物毒液蛋白质。与本文所描述的方案很可能是可溶的二硫键丰富的蛋白质会在短至一周来获得。即使从一个小规模的，还有就是用个潜在的Ë纯化蛋白质通过质谱确认的氧化状态，为表征的试点研究，或敏感微观分析。

代表性的结果示于图6中用于从VENOMICS管道96二硫化物富含蛋白质的表达筛选的蛋白质被配置为通过增加数目的二硫键，然后增加残留的数目。将肽表达的有his标签和及DsbC融合伴侣细胞质中。当使用推荐的培养条件下，12的OD 600通常实现的。将肽用协议8.2-B为50微升镍亲和树脂的终体积进行纯化，从而可以在该实验被检测的最大〜25 mg / L的融合蛋白。 图6A示出了从钳芯片实验室系统（表示切割前的融合）和基于外推法得到的融合蛋白的mg / L的培养的评分系统（在0.1至2毫克/升培养物的水平，2到电泳结果10 mg / L的培养，10〜25 mg / L的培养和无<html>吨检测。）请注意，切割及DsbC标签通常运行在大约32 kDa的，而不是27 kDa的预期。同样，融合及DsbC还奔波5 kDa的高于预期。对于很多的目标，我们不仅看到了完整的融合（上带），而且还融合伙伴单独（低频段）。对其中的一些目标优化培养条件，可以改善完好融合（上频带）相比，单独及DsbC允许增加的最终产率之比。计分是基于仅在完整的融合程度。仅16出96个蛋白质不能在融合水平进行检测。这对应于83％的整体成功程度。可能被检测出的80蛋白，含量大于2毫克/升培养物（56％）中检测到的这些45。取决于靶和融合蛋白产率通常为2-100 mg / L的培养的范围内（尽管在这个例子中使用的协议8.2 B可检测到最大〜25 mg / L的融合蛋白）。 T“&gt;由目前二硫键数目（ 图6B所示）成功的分析显示为测试二硫键所有的数字（1和​​7之间）合理的成功，与成功的最低水平为66％，含二硫化6目标债券。表达成功的基于等电点和残基的数目（在图6C中示出）的分布的分析表明该技术没有特别的偏差，既成功地表达目标而那些没有检测到分散在整个小区的目标。 图6D示出了从电喷雾电离质谱（ESI-MS），用于单个目标获得的，前和DTT还原的样品后的质谱分析结果的一个例子。通常，这样的穷举质谱分析将不需要进行（降低的样品将不需要被分析），但是，对于彻底demonstratio而言n我们已经表明两个结果。所示的目标是5.7 kDa的二硫键丰富的蛇毒蛋白有4个二硫键。在左侧的光谱显示之前DTT还原的结果为蛋白，因为它是裂解和脱盐无需进一步干预后。在右手侧的光谱显示DTT还原随后脱盐以除去任何过量的DTT后的蛋白质。对应于实验质谱中的离子被标记带频谱和指定为每个离子上的箭头显示为绿色。计算出这些离子的实验父块（用于蛋白质之前减少（-DTT）和还原（+ DTT）后）示于表中。群众（5709.6为大前减少和5717.6大的减少了样品的样品）展示的8大的质量差。 2沓的质量差对应的1氧化的二硫键的存在，因此8 Da的质量差表示的4个二硫键的存在下（如预期）中在非还原样品。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig1highres.jpg" width="500" /> 高通量表达筛选方案的图1。示意图。使用该协议，96至384的条件，可以由一个人在一个星期采用手工方式进行测试，或至多1152的条件与所描述的半自动化设备。表达质粒使用的是重组克隆技术让众多的目标可能是亚克隆在同一时间建造。一旦可溶性表达的条件已经确定，并进行切割，如果需要，所述蛋白质可以进行质量控制检查的氧化和纯度，microassays和/或大规模的生产。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig2highres.jpg" wi嗞嗞=“500”/&gt; 图2为示意图通用重组克隆和构建设计，从目标条目克隆，多表达载体可以是子克隆在高通量方式（在一个星期内最多6×96入门克隆）一次实验。表达的蛋白编码镍亲和纯化His标签。及DsbC的（缺少其胞质信号序列的细胞质表达）融合伴侣是用来增加溶解度和/或援助折叠和靶蛋白的正确氧化。请注意，目标编码序列应该包含一个N-末端TEV蛋白酶位点（ENLYFQ）如果标签切割是需要的。在左上角的插图示出了生产中使用的供体载体和靶序列，可以通过PCR或基因合成获得的进入克隆。入门克隆，也可以从商业的入门克隆的集合获得的。多个重组克隆系统可用，但是我们利用网关系统·米，如图中示意。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig3highres.jpg" width="500" /> 图3的高通量筛选管道多个二硫键富含目标。目标最初表示为HIS-及DsbC融合体在大肠杆菌BL21（DE3）pLysS中大肠杆菌的细胞质大肠杆菌在37/18°下使用自动诱导培养基（ZYP-5052）。纯化是在镍树脂随后检测可溶性构建由HTP电泳（或斑点印迹/ SDS-PAGE）中进行。如果第一轮表达式筛选不成功，另类文化条件都试过了。如果构建体产生的可溶性蛋白在足够高的产率，microassays和质量控制可以被执行，如果需要，大规模生产可以追求。对于目标，其中可溶性收益率不够高，表达筛选可以继续使用替代STRA插件和温度那么其他的融合伙伴和周质表达。可选的步骤，由虚线框表示。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig4highres.jpg" width="500" /> 图4。机器人工作台设置为HTP平台。我们的液体处理机器人工作台的布局所示，虽然替代工作台还可以用于提供有可用的等效的网站上。该装置由一个清洗站（WS）为（8通道的液体处理头（LIHA）），两个微孔板运营商有4个位置各（MP4，持仓1-4和5-8），真空站2位置（TE-VACS，位置9和10），与3位置的微载体（MP3，持仓11-13），二板摇床2个位置各（德摇床，持仓14-15和16-17）和载体与3个位置一次性吸头（DITI，持仓18-20）。此外，该再有两个饭店载体深孔板，一个用于微板（未示出）。安装在液体处理机器人的硬件是一个96多通道臂（MCA96）与一次性吸头，一个8通道的液体处理头（LIHA）具有固定的提示和机器人操纵（罗马）的移动板/设备周围使用工作台。该位置的编号是指在整个协议。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig5highres.jpg" width="500" /> 图5原理图，用于从单个96孔板转移到4个24孔板中。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51464/51464fig6highres.jpg" width="500" /> 图6。代表性的结果示于96二硫化物丰富的毒液亲表达屏teins。将蛋白表达为HIS-及DsbC融合体在细胞质中，并使用50μl的镍树脂（协议8.2-B）进行纯化，A）中的表达筛选结果，是表示虚拟凝胶和得分的表达产率。在虚拟凝胶对比度进行了调整车道到车道，以弥补非常微弱的或激烈的乐队。请注意，对于某些目标两个频带中可以看出，上频带相对应的完整的融合蛋白和低频段对应于融合标记单独B）表示在每个表达水平的蛋白质的比例相比，二硫键的数目的蛋白质。蛋白质的各组中的实际数目被叠加在图形上。C）的表达水平的基础上，等电点（pI）和残基的数目的分布。D）的质谱结果为5.7 kDa的二硫键丰富毒液的一个例子蛋白4个二硫键。在频谱左手侧示出了减少前用DTT和在右手侧的频谱的蛋白显示出减少用DTT，然后脱盐的蛋白质。相应的实验群众的离子都标有箭头和他们的任务显示为绿色。前减少和还原后都显示在表中，表现出的8大的质量差，对应氧化蛋白除了还原剂之前存在的实验群众的蛋白质。 <a href="https://www.jove.com/files/ftp_upload/51464/51464fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td>元件</td><td>食谱</td><td>评论</td></tr><tr><td> ZY </td><td> 〜928毫升 10g胰蛋白胨 5g酵母提取物 925毫升水</td><td>混合，然后高压灭菌消毒。 </td></TR&gt; <tr><td> 2M的硫酸镁 </td><td>百毫升 49.3克硫酸镁4·7H 2 O 〜60毫升的水</td><td>搅拌至溶解，然后高压灭菌消毒。 </td></tr><tr><td> 50倍5052 </td><td> 1升 250克甘油 730毫升水 25克葡萄糖 100克α-乳糖</td><td>添加顺序，搅拌过热，直到全部溶解，然后高压灭菌消毒。 </td></tr><tr><td> NPS 20倍</td><td> 1升 900ml去离子水中 66克（NH 4）2 SO 4的 136克KH 2 PO 4 142克的Na 2 HPO 4 </td><td>添加顺序，搅拌至全部溶解，然后高压灭菌消毒。 </td></tr></tbody></table> 表1配方为ZYP-5052培养基的组成部分。

Watch this Scientific Journal Video about High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli at JoVE.com

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

一种协议，用于定量，高通量表达筛选，从小规模的大肠杆菌文化融合蛋白的纯化分析说明，并应用到的二硫键丰富的动物毒液蛋白靶点的表达。

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. ...

high-throughput-quantitative-expression-screening-purification

Research

JoVE Journal

Biology

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

23.9K 次观看.  Aix-Marseille Université. 一种协议，用于定量，高通量表达筛选，从小规模的大肠杆菌文化融合蛋白的纯化分析说明，并应用到的二硫键丰富的动物毒液蛋白靶点的表达。 

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

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

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

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 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 ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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 ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

纯化及复性的淀粉样纤维（他）<sub&gt; 6</sub&gt; -tagged重组Shadoo蛋白表达的包涵体<em&gt;大肠杆菌</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...