This research was supported by Cleveland State University Faculty Development Funds.

1.准备寄生虫细胞培养，收集寄生虫微丸<ol><li>加入10克的RPMI-1640粉末，66毫克硫酸庆大霉素的，2克碳酸氢钠，5.9克HEPES缓冲液，50毫克次黄嘌呤和3.8g的葡萄糖在1L制备的RPMI-1640-HEPES培养基的无菌卫生署2 O.混合使用表磁力搅拌器，直到1升蒸馏水中所有的粉末溶解。执行使用0.22微米的过滤介质微过滤。存储介质中的无菌瓶中。 </li><li>未感染的洗净（鲜）型使用RPMI A +红细胞。制备红细胞的5％血细胞比容（0.5毫升洗涤新鲜红细胞的9.5毫升10％的RPMI +人血清）。 </li><li>让15％的人血清+ RPMI（15毫升的人血清和85ml的RPMI）发起寄生虫的培养。使10％的人血清和RPMI（10毫升人血清和90ml的RPMI），以继续寄生虫培养中的红血细胞。 </li><li>文化P.恶性疟原虫 （3D7和FCR-3株）型A +人红细胞在5％血细胞比容和20％原虫在培养皿或75 cm 3的培养瓶。根据Trager和詹森烛缸26的方法， 在体外保持养殖。交替，保持培养物在孵化器在37℃保持9.5％的CO 2和3.4％的N 2气。文化P.疟原虫的RPMI 1640-HEPES培养基补充有10％的人血清。 </li><li>同步P.疟原虫培养通过添加65％的Percoll（65毫升Percoll密度+35毫升的RPMI-1640的没有人类血清）的培养物浓缩物27。 </li><li>离心培养物浓缩物以Percoll密度在2500×g离心5分钟，这导致3层。仔细，用移液管隔离无菌条件27下的中间层转移到单独的管裂殖。 </li><li>洗所述分离培养物，用10％人血清+ RPMI-1640两次通过在7500×g离心离心5分钟以除去过量的Percoll。迪在无菌条件27下每次SCARD上清液。 </li><li>继续培养同步P.疟原虫裂殖在10％人血清+ RPMI-1640 27。 </li><li>通过处理疟原虫 -感染的红细胞用10mM的Tris pH值8.8，并在15000×g离心离心15分钟23收集裂殖。在-70℃以下加入5微升蛋白酶抑制剂（抑肽酶）的粒料商店寄生虫粒料。使用寄生虫粒料用于蛋白质提取，基因组DNA分离和RNA分离。 </li></ol> 2.准备质粒载体<ol><li>隔离P.基因组DNA从约20％寄生虫血症的培养物制备疟原虫裂殖粒料。 </li><li>添加600微升的10mM的Tris-HCl的pH值7.6，50毫摩尔EDTA pH为8.0，0.1％SDS和1mg / ml蛋白酶K，来粒料在微量​​离心管中，用1ml注射器附接至＆G针均质沉淀。交替填充注射器配合颗粒混合物和排空到一个离心管中的20倍。含孵育匀浆管O / N在50℃水浴。 </li><li>加入600微升P +的C（300微升酚和300微升氯仿）均质颗粒氯仿（P + C）提取 - 执行苯酚。帽筒颠倒管端至端紧紧地摇晃混合物剧烈。使用微量到一个新的离心管在14000×g离心2分钟，并离心管收集上层水溶液。重复此步骤两次。 </li><li>从步骤2.1.2添加600微升氯仿到水层中，在新管中。涡管10秒和离心机在14000×g离心2分钟。测量上层的水层用微量并将其转移到一个新的离心管中。 </li><li>加入0.1体积的5M乙酸钠pH5.5的和1体积的异丙醇（如果水层为300微升加入30微升的5M乙酸钠pH5.5加上330微升异丙醇）到水捻呃从步骤2.1.3。孵育管中，在室温进行15分钟。 </li><li>离心反应管在14000×g离心10分钟以沉淀DNA沉淀。弃上清，洗涤沉淀用100μl的70％乙醇（70微升乙醇和30微升的dh 2 O），以从DNA除去过量的盐。存储在DNA的50微升的dh 2 O在-20℃。 </li><li>设计正向和使用软件反向引物或手动为P.扩增疟原虫基因PF3D7_1361800，PF3D7_0925900，PF3D7_1436300和PF3D7_0114100先前在蛋白质组研究16,18用适当的限制性位点，以允许该基因序列插入表达质粒pT7CFE1-CHIS的多克隆位点的克隆鉴定为示于表2。 </li><li>请加入50微升的反应混合物; 5微升隔离P的疟原虫的基因组DNA，5微升的10x缓冲液，MgCl 2的的各5μl，1.5微升正向引物，1.5微升反向引物，0.5微升的T7 DNA聚合酶和31.5微升的dh 2 O，并执行聚合酶链反应（PCR），在94℃下进行8分钟变性，50℃进行1分30秒为延伸和72℃9分的退火和复性，用于放大在表1所示的基因。 </li><li>分离PCR产物在1％琼脂糖凝胶（1克琼脂糖和100ml的Tris乙酸EDTA（TAE）缓冲液中）32。 </li><li>从琼脂糖凝胶上切的PCR扩增的DNA条带，将含有DNA的凝胶切片中的DNA凝胶提取旋转柱并冻结在-20℃下5分钟。加入100微升异丙醇，以冷冻凝胶切片和离心机在14000×g离心5分钟。 </li><li>测量水流量通过从步骤2.5过滤在收集管中，使用微量并且转移至新管。加入0.1体积的5M乙酸钠和离心机在14000×g离心5分钟。弃去上清液，加入卫生署2的10微升O操作THËDNA沉淀。 </li><li>通过加入3微升质粒和5μl的PCR基因产物向每个管消化在两个独立的管中的pT7CFE1-CHIS表达载体和纯化的DNA片段（从2.6）。添加到每个管中，1微升的特定限制性内切酶，2微升反应缓冲液和14微升的dh 2 O的</li><li>加入3微升消化的质粒，5微升消化的DNA片段，2微升10×连接酶缓冲液，1微升连接酶和9微升的dh 2 O为在一个离心管中。孵育12°CO / N。 </li><li>加入0.1体积的3M乙酸钠，pH为5.5和2倍体积的乙醇​​（如果你的结扎管体积是20微升加入2微升3M乙酸钠+ 44微升乙醇）于管从步骤2.14。拌匀，并孵化在-20℃下15分钟。 </li><li>离心反应管在14000×g离心15分钟。小心取出用微型吸管上清，而不会干扰颗粒。 </li><li>加入100微升70％乙醇牛逼Ø从步骤2.10沉淀。离心反应管在14000×g离心5分钟。取出并弃去上清液。添加10微升不含核酸酶的水至DNA沉淀。 </li></ol> 3.蛋白表达利用体外哺乳动物细胞表达自由制度<ol><li>通过测量2微升重组pT7CFE1-CHIS质粒DNA，4微升的5×转录缓冲液，4μl的NTP混合物，2微升T7 RNA聚合酶和8微升无核酸酶水两步骤制备20微升转录混合物的人类使用 DNA模板体外蛋白质的表达。混合组件在一个离心管中，并在水浴中孵育75分钟，在30℃。 </li><li>取2微升的转录混合物，并把它添加到23微升含12.5微升的HeLa细胞裂解物，2.5微升辅助蛋白，1微升的盐溶液A，0.5微升氨基酸-Met，0.5微升的氨基酸翻译混合物​​的-Leu，181，L RNA酶抑制剂，1.25微升能源结构和3.75微升无核酸酶水。 </li><li>孵育来自步骤3.2的翻译混合物​​在30℃下90分钟。商店翻译产物在-20℃下。 </li><li>通过加入3微升重组pT7CFE1-CHIS质粒DNA，2微升无核酸酶水，5微升反应混合物，12.5微升的HeLa细胞裂解液和2.5微升辅助蛋白的1步制备25μl的转录和翻译混合物​​的人类再加IVT蛋白表达的DNA模板。 </li><li>孵育来自步骤3.4的90分钟至6小时，将反应混合物在30℃。存储的翻译产物在-20 C。 </li></ol> 4.纯化表达的重组蛋白的<ol><li>添加75微升1×纯化缓冲至25微升翻译产物，使100微升纯化的混合物。 </li><li>加入100微升镍螯合树脂，以100微升净化mixt的中URE。洗镍树脂两次通过加入300微升的dh 2 O和300微升的结合缓冲液中。重悬树脂和离心机在14000×g离心1分钟，以去除过量的乙醇。 </li><li>从步骤加入100微升纯化混合物的4.2至100μl镍螯合树脂并在室温60分钟孵育的混合物。 </li><li>在14000 XG 1 min离心混合物并收集上清到一个新的管标记为“流过”。 </li><li>洗涤树脂用100μl1×洗涤缓冲液（20 mM咪唑，250mM的的NaH 2 PO 4在pH 8，2.5M NaCl和卫生署2 O）。离心在14000×g离心1分钟，并在新的管中标记为“洗涤”收集上清液。重复步骤4.5的两倍。 </li><li>加入100微升1×洗脱缓冲液（100mM咪唑，250mM的的Na 2 PO 4在pH 8.0，2.5M NaCl和卫生署2 O）与该树脂并孵育15分钟。离心在14000×g离心1分钟。做洗脱步骤两次。每时间收集上清到新的离心管和标签为“洗脱”。 </li><li>洗脱后，如在步骤4.5中所述两次洗涤树脂。商店流经，洗涤和洗脱样品在-20℃或更低。树脂必须储存在4℃。使用30微升各样品进行SDS-PAGE和免疫印迹分析。 </li></ol> 5.考马斯（布拉德福德）蛋白质含量34 <ol><li>与浓度制备标准的牛血清白蛋白（BSA）溶液20微克/毫升（混合10微升BSA（2毫克/毫升）和90微升的dh 2 O的），40微克/毫升（混合20微升BSA（2毫克/ ml）和80微升的dh 2 O）的60微克/毫升（混合30微升BSA（2毫克/毫升）和70微升的dh 2 O）的80微克/毫升（混合40微升的BSA（2毫克/ ml）和60微升的dh 2 O），100微克/毫升（混合50微升BSA（2毫克/毫升）和50微升的dh 2 O）的，160微克/毫升（混合80微升BSA（2毫克/毫升）和20微升卫生署2 O）和200微克/毫升（100微升BSA（2毫克/毫升）。 </li><li>措施5微升流过，洗涤和洗脱物样品从步骤4.7。加入95微升的dh 2 O的</li><li>添加5毫升考马斯亮蓝试剂从步骤5.1和5.2的混合物。覆盖用封口膜，涡旋10的管子秒然后孵育20分钟，在RT。 </li><li>使用分光光度计，测量光密度（OD）在650nm的蛋白管从步骤5.3波长。积浓度与 OD值，以确定该蛋白浓度34。 </li></ol> 6.免疫印迹<ol><li>加入5微升裂殖提取物P.疟原虫 ，2微升大肠杆菌表达的重组RHOP-3蛋白31，5微升的重组蛋白的方法，用2-步骤和1-步骤在体外人表达系统和10μl的蛋白产物纯化用亲和法来分离管表达。 </li&gt; <li>添加含巯基乙醇电泳样品缓冲液中向每个管的20微升的最终体积，以使增溶的蛋白质样品。煮沸管2分钟。 </li><li>加载溶解蛋白质样品到10％SDS-PAGE凝胶分离蛋白质。 </li><li>通过电泳在半干印迹腔室转移从SDS-PAGE凝胶到硝化纤维素纸（NCP）的分离的蛋白在每凝胶35毫安电流2小时。 </li><li>块的NCP以下转印用2％脱脂牛奶。 </li><li>孵育阻断的NCP用稀释1以下多克隆抗体：100在2％牛奶进行蛋白质印迹;兔抗体＃676 21特异性P.疟原虫裂殖子棒状体，鼠抗体20项具体为P.疟原虫和P.疟原虫裂殖子棒状体，抗血清＃685具体为P.疟原虫寄生泡蛋白，血清（丝氨酸丰富的抗原）22和抗体特异性的毛雷尔217; S裂蛋白PFMC-2TM 28。 </li><li>孵育的NCP也以1：100稀释的正常小鼠和兔血清和从SP2骨髓瘤细胞耗尽培养上清液（SCS）作为阴性对照。 </li><li>国家联络点孵育在与4ºCO / N抗体和控制。 </li><li>洗涤的NCP 4倍带印迹缓冲液孵育的NCP用缀合辣根过氧化物酶（HRP）物种特异性的二抗稀释1：1000在2％的牛奶。 </li><li>国家联络点洗净用4倍缓冲印迹。洗孵育国家联络点与显色液A + B（溶液A：加入50ml 1X印迹缓冲液和30微升H 2 O 2;溶液B：加入10毫升甲醇和30毫克的3-氯萘酚）30分在黑暗中。色度分析Western blot结果。 </li></ol>

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Y., 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+Plasmodium.+gene+family+encoding+Maurer&#8217;s+cleft+membrane+proteins:+Structural+properties+and+expression+profiling.">A Plasmodium. gene family encoding Maurer&#8217;s cleft membrane proteins: Structural properties and expression profiling.</a> Genome Res. 14 (6), 1052-1059 (2004).</li><li>Sam-Yellowe, T. Y., Banks, T. L., Fujioka, H., Drazba, J. A., Yadav, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+yoelii.:+Novel+rhoptry+proteins+identified+within+the+body+of+merozoite+rhoptries+in+rodent+Plasmodium+malaria.">Plasmodium yoelii.: Novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria.</a> Exp Parasitol. 120 (1), 113-117 (2008).</li><li>Sam-Yellowe, T. Y., Rio, D., Fujioka, H., Aikawa, M., Yang, J. C., Yakubu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+merozoite+rhoptries,+identification+of+novel+rhoptry+associated+proteins+from+P.+yoelii.,+P.+chabaudi.,+P.+berghei.+and+conserved+interspecies+reactivity+of+organelles+and+proteins+with+P.+falciparum.+rhoptry-specific+antibodies.">Isolation of merozoite rhoptries, identification of novel rhoptry associated proteins from P. yoelii., P. chabaudi., P. berghei. and conserved interspecies reactivity of organelles and proteins with P. falciparum. rhoptry-specific antibodies.</a> Exp Parasitol. 89 (3), 271-284 (1998).</li><li>Sam-Yellowe, T. Y., Fujioka, H., Masamichi, A., Messineo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum.+rhoptry+proteins+of+140/130/110kd+(Rhop-H)+are+located+in+an+electron+lucent+compartment+in+the+neck+of+the+rhoptries.">Plasmodium falciparum. rhoptry proteins of 140/130/110kd (Rhop-H) are located in an electron lucent compartment in the neck of the rhoptries.</a> J Euk Microbial. 42 (3), 224-231 (1995).</li><li>Sam-Yellowe, T. Y., Hishashi, F., Masamichi, A., Messineo, D. G., Leash, A. M., Hall, T., Drazba, J. A., Ndengele, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Organization+and+cross+linking+analysis+of+the+Plasmodium+falciparum+erythrocyte+binding+proteins+Rhop-H+and+SERA.">Molecular Organization and cross linking analysis of the Plasmodium falciparum erythrocyte binding proteins Rhop-H and SERA.</a> J Protozool. Res. 10 (3), 128-154 (2000).</li><li>Sam-Yellowe, T. Y., Shio, H., Perkins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+of+Plasmodium+falciparum.+rhoptry+protein+into+plasma+membrane+of+host+erythrocytes.">Secretion of Plasmodium falciparum. rhoptry protein into plasma membrane of host erythrocytes.</a> J Cell Biol. 106 (5), 1507-1513 (1988).</li><li>Sundaresh, 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=Identification+of+humoral+immune+responses+in+protein+microarrays+using+DNA+microarray+data+analysis+techniques.">Identification of humoral immune responses in protein microarrays using DNA microarray data analysis techniques.</a> Bioinformatics. 22 (14), 1760-1766 (2006).</li><li>Terpe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+bacterial+expression+systems+for+heterologous+protein+production:+from+molecular+and+biochemical+fundamentals+to+commercial+systems.">Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.</a> Appl. Microbiol. Biotechnol. 72 (2), 211-222 (2006).</li><li>Trager, W., Jensen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+malaria+parasites+in+continuous+culture.">Human malaria parasites in continuous culture.</a> Science. 193 (4254), 673-675 (1976).</li><li>Tsarukyanova, I., Drazba, J. A., Fujioka, H., Yadav, S., Sam-Yellowe, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+of+the+Plasmodium+falciparum.+two+transmembrane+Maurer&#8217;s+cleft+protein+family,+PfMC-2TM+and+the+130+kDa+Maurer&#8217;s+cleft+protein+define+different+domains+of+the+infected+erythrocyte+intramembranous+network.">Proteins of the Plasmodium falciparum. two transmembrane Maurer&#8217;s cleft protein family, PfMC-2TM and the 130 kDa Maurer&#8217;s cleft protein define different domains of the infected erythrocyte intramembranous network.</a> Parasitol Res. 104 (4), 875-891 (2009).</li><li>Tsuboi, 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=Wheat+germ+cell-free+system-based+production+of+malaria+proteins+for+discovery+of+novel+vaccine+candidates.">Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates.</a> Infect. Immun. 76 (4), 1702-1708 (2008).</li><li>Tsuboi, T., Takeo, S., Sawasaki, T., Torii, M., Endo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+approach+to+the+production+of+vaccines+against+the+malaria+parasite.">An efficient approach to the production of vaccines against the malaria parasite.</a> Methods in Mol. Biol. 607, 73-83 (2010).</li><li>Wang, J., 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+versatile+protein+microarray+platform+enabling+antibody+profiling+against+denatured+proteins.">A versatile protein microarray platform enabling antibody profiling against denatured proteins.</a> Proteomics Clin. Appl. 7 (5-6), 378-383 (2013).</li><li>Wang, T., Fujioka, H., Drazba, J. A., Sam-Yellowe, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhop-3+protein+conservation+among+Plasmodium.">Rhop-3 protein conservation among Plasmodium.</a> species and induced protection against lethal. 99 (3), 238-252 (2006).</li><li>Yadavalli, R., Ledger, C., Sam Yellowe, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+human+cell+free+expression+for+synthesis+of+malaria+proteins.">In vitro human cell free expression for synthesis of malaria proteins.</a> Parasitol Res. 111 (6), 2461-2465 (2012).</li><li>Machida, K., Masutan, M., Imataka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Synthesis+in+vitro.:+Cell-Free+Systems+Derived+from+Human+Cells.">Protein Synthesis in vitro.: Cell-Free Systems Derived from Human Cells.</a> Intech. , (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantification+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

We do not have any competing financial interests in this study.

使用两个步骤在体外人细胞表达系统和纯化蛋白的成功表达的重要步骤是：1）成功的扩增和基因引入pT7CFE-CHIS质粒载体的克隆; 2）转录的RNA在30℃;蛋白3）翻译在30℃在RNA酶抑制剂的存在; 4）开发使用涂覆有镍和5树脂珠）上免疫印迹分析的结果使用多克隆和单克隆抗体的亲和纯化方案。用于使用一个步骤的IVT偶联蛋白翻译系统蛋白质的成功表达的重要步骤是：1）成功的扩增和基因引入pT7CFE...

在疟疾研究，使用原核或真核细胞的系统免疫原性蛋白的表达仍然是一个挑战。 疟原虫的基因组的和未知的翻译后机制14,7，的AT丰富有助于与获得正确折叠的和免疫原性蛋白产生抗体和疫苗研究的困难。原核系统如大肠杆菌已被用于重组蛋白质表达。原核系统是低成本，有效，产生重组蛋白的高产率 并有多个克隆载体。 E.主机大肠杆菌细胞很容易变换和细胞生长迅速。然而，原核系统有例如缺乏氨基酸取代，翻译后修饰，污染，异构产品和重组蛋白的积累的包涵体24内危险的限制。 在一个研究由Mehlin 等人（2006年），1000的开放阅读框（ORF）表达。约7％的表达的蛋白质的可溶于14。所观察到的不溶性是由于基因的偏压性质和密码子的高频率理想地使用由疟原虫富含AT的基因组14。克服该问题的另一种策略已开发通过使用质粒或含有的tRNA识别稀有密码子或匹配频率3中的密码子的宿主细胞。即使在执行这些优化后，蛋白质的非常小的部分被表示为可溶的，活性和免疫原性14。无细胞表达系统包含了所有必需的转录和翻译元件，如核糖体，起始因子，延伸因子（翻译因子），tRNA和氨酰基tRNA合成酶。转录和翻译反应被耦合在一步法11,29。该transcription反应在一个试管中进行前mRNA的适当量的孵育翻译机器在不同的管11,29。虽然这些方法是成功的疟原虫属蛋白的表达，一个主要缺点是时间为蛋白质的表达，这是约22小时29的长度。此外，耗材列入成本高的协议29，细胞裂解液的劳动密集型筹备和不一致成分制剂，使这些系统无法实现的。研究人员对无细胞表达系统发展的主要焦点取决于多种因素，如快速的遗传修饰，具有较高的浓度和直线前进裂解液制剂1快产量。 真核系统例如酵母，哺乳动物细胞系，杆状病毒介导的表达系统， 四膜虫thermophilia，粘菌和寄生表达系统ス通道作为利什曼已经用于重组蛋白质表达7。真核系统中共享的亲缘关系，因此也共享特性，如糖基化，酰化，二硫键的形成，分子伴侣相互作用，蛋白水解和亚细胞区室。在真核生物中蛋白分泌事件预防的积累和降低毒性表达系统13。利用酵母系统是优选的，因为它们是适合于在大规模蛋白质表达和获得高的产率4。两个P.疟原虫蛋白PfCP-29和Pvs25用酵母系统4产生。然而，在大多数的蛋白质的合成的主要缺点是不规则N和O-糖基化模式，不当折叠和从它们的天然形式4蛋白的截断。 使用哺乳动物细胞合成重组蛋白质的是劳动密集型的并且它expen西伯来维持稳定的重组细胞系4。因此，哺乳动物细胞被限制为蛋白信号，蛋白质相互作用和寄生虫-宿主相互作用的分析，而且还用于测试DNA疫苗4。人类DNA和mRNA 体外蛋白表达的无细胞系统本文中所描述的HeLa细胞来源的哺乳动物为基础的系统表达的蛋白质中3小时。蛋白表达使用pT7CFE1-CHIS表达载体已C-末端标签促进鉴定和纯化。的HeLa细胞为基础的系统中补充有翻译起始因子和翻译调节器，从而提高翻译系统的效率。蛋白质可迅速转化，筛选，验证和在各种免疫学和结构应用15处理以供使用。 目前用于变种的表达的真核细胞表达系统，例如小麦胚芽，兔网织红细胞白条真核蛋白，包括恶性蛋白质11,29。另外，E。大肠杆菌基于无细胞系统也用于真核蛋白表达11。 表1通过比较系统的功能，以及易于使用的系统用于蛋白质表达的总结了在不同的分类细胞表达系统。相较于其他人无细胞系统必须执行后并共翻译修饰的能力，并且是不太昂贵。密码子优化是可能的，并且所有的反应可以在3小时来进行。的HeLa细胞系统是一种理想的翻译系统蛋白质合成在实验室设置。 在其它细胞系统人类自由细胞表达系统的一个主要优点是，从不同的器官和组织来源的多种不同的细胞系的可用性。几个品种无细胞系统可根据不同的细胞系进行设计。提取物从哺乳动物细胞衍生s具有更高的效率来合成大量的蛋白质，比任何其他的分类细胞表达系统。这些无细胞表达系统也可用于诊断和蛋白质表征的应用，如微阵列30。流行的HeLa细胞系是最广泛地用于进行蛋白33的表达。在HeLa细胞系内质网不发达，导致缺乏翻译后糖基化修饰的活性33。然而，该系统被认为是有利的疟原虫蛋白合成疟原虫也缺乏糖基化交翻译修改29。 HeLa细胞游离转录 - 翻​​译协议是容易执行的，廉价和蛋白质可表示在90分钟，准备用于纯化和进一步的下游应用。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Two-step in vitro human cell free expression system</td>
			<td>Thermo fischer</td>
			<td>88856</td>
			<td>Discontinued. Always store at -80oC. Do not thaw in warm water</td>
		</tr>
		<tr>
			<td>One-step in vitro coupled translation system</td>
			<td>Thermo fischer</td>
			<td>88860</td>
			<td>Always store at -80oC. Do not thaw in warm water</td>
		</tr>
		<tr>
			<td>ProBond Purification system</td>
			<td>Invitrogen</td>
			<td>K850-01</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Probond Nickel-Chelating Resin</td>
			<td>Invitrogen</td>
			<td>R801-01</td>
			<td>Store at 4oC.</td>
		</tr>
		<tr>
			<td>&#160;A+ Human Blood</td>
			<td>Interstate Blood Bank</td>
			<td></td>
			<td>Store at 4oC.</td>
		</tr>
	</tbody>
</table>

hela based cell free expression systems for expression of plasmodium rhoptry proteins

疟疾导致显著的全球发病率和死亡率。没有常规疫苗是当前可用。其中一个主要的原因，缺乏疫苗是确定合适的候选疫苗的挑战。疟疾蛋白使用原核和真核基于细胞的表达系统很差糖基化，通常不溶性且经受不当折叠导致降低的免疫原性表达。小麦胚芽，兔网织红细胞裂解物和大肠杆菌裂解物无细胞表达系统目前用于疟疾蛋白的表达。然而，表达时间和不适当的糖基化蛋白质的长度仍然是一项挑战。我们使用的HeLa基于无细胞表达系统，被称为“ 在体外人的分类细胞表达系统”表明在体外恶性疟原虫蛋白质的表达。 2.基于海拉细胞自由表达系统表达转录在75分钟和3微升transcrib的编的mRNA足以翻译的蛋白质在90分钟。的1步表达系统是一个转录和翻译偶联表达系统;转录和共翻译发生在3小时。该方法也可以通过提供额外的能量扩展6小时。在2步表达系统，表达被第一转录，然后添加至翻译组合用于蛋白质表达。我们描述了如何表达疟疾蛋白;疏水PF3D7_0114100毛雷尔的唇腭裂 - 2跨膜（PFMC-2TM）的蛋白质，亲水PF3D7_0925900蛋白质和含蛋白质PF3D7_1361800犰狳重复使用的HeLa细胞基于自由表达系统。的蛋白质表达于微体积采用2步和1-步骤表达的策略。亲和纯化方法纯化25μl的使用也被描述的体外人细胞表达系统表达的蛋白质。蛋白质产量是由布拉德福德测定法测定和表达和纯化的蛋白质可以通过蛋​​白质印迹分析来证实。表达的重组蛋白可用于免疫接种，免疫测定和蛋白质测序。

通过反应性的特异性抗体表达的重组蛋白的验证是在确认表达的蛋白质的正确折叠的重要的第一步。重组疟疾蛋白采用一步法和两步体外培养的人无细胞表达系统表达。重组蛋白是纯化的镍螯合亲和方法。然后，我们使用抗血清在SDS-PAGE分离的重组蛋白的Western印迹整个裂殖子棒状体。 图1示出PY17X_1139200，PY17X_1402200，PY17X_1366000，PY17X_0830000和PF3D7_1436300在1％琼脂糖?...

Watch this Scientific Journal Video about HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins at JoVE.com

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

基于细胞系统疟疾蛋白的表达仍然具有挑战性。我们证明两步并一步IVT（ 体外翻译）无细胞表达系统从HeLa细胞表达的重组疟疾蛋白质棒状体。我们使用的Ni树脂基于亲和性的纯化系统纯化棒状体蛋白质。

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

hela-based-cell-free-expression-systems-for-expression-plasmodium

Research

JoVE Journal

Biology

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

12.0K 次观看.  Cleveland State University. 基于细胞系统疟疾蛋白的表达仍然具有挑战性。我们证明两步并一步IVT（ 体外翻译）无细胞表达系统从HeLa细胞表达的重组疟疾蛋白质棒状体。我们使用的Ni树脂基于亲和性的纯化系统纯化棒状体蛋白质。 

视频: HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

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

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

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

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

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

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...