Name: hela based cell free expression systems for expression of plasmodium rhoptry proteins
Uploaded: 2015-06-10T14:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins at JoVE.com

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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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-CHIS质粒载体的克隆; 2）转录和蛋白质的翻译在30°C; 3）开发使用涂有Ni和4的树脂珠）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。 当前LY，无细胞表达系统用于表达重组疟疾蛋白。无细胞表达系统的关键特征来表达疟疾蛋白是解码富含AT的基因的能力，缺乏长平移暂停，重复氨基酸序列的成功表达，缺少糖基化机制和质粒来控制基因的截断的能力。在这项研究中，我们展示使用一个两步骤在体外人细胞表达系统32和一个单步IVT耦合翻译系统为P的表达P.恶性疟原虫的基因同源基因如表1所示，一个纯化方案被开发和用于纯化的微量从1步和第2步在体外人细胞游离表达系统获得的重组蛋白的优化疟原虫裂殖子棒状体的基因。 4 P.疟原虫基因，PF3D7_0114100跨膜蛋白疏水性，预测假设ARM重复蛋白PF3D7_1361800和亲水蛋白质PF3D7_0925900，使用2步和1-步骤在体外人细胞表达系统中表达。纯化的蛋白质的产量进行了测定。蛋白质PFMC-2TM被包括在这些研究中，因为它是一个完整的膜蛋白具有两个跨膜结构域，我们感兴趣的是确定在体外表达系统是否能正确表达跨膜蛋白，并允许易于纯化。 同时在一个步骤和两步表达系统的主要区别在于，两个步骤的表达系统是mRNA变体系，其中一个稳定的mRNA转录，并加入到翻译反应中。与此相反，在体外偶联转录的一个步骤/翻译系统包括一个连续的转录和供应的mRNA随后蛋白质的反应中的共翻译。两个1步IVT转录/翻译加上ëXPRESSION系统和2-步骤在体外人细胞表达系统进行了测试高分子量蛋白质的表达。这两种系统都成功地表达的低分子量的重组蛋白质。在1步表达系统，并在翻译反应混合物蛋白抗体的交叉反应性进行了观察。具体的蛋白质与交叉的抗体反应是未知的，需要进一步调查。同时使用1步和第2步系统高分子量蛋白质的表达是不成功的。一个纯化的表达同时使用1步和第2步在体外培养的人无细胞表达系统制定了不同的溶解特性的重组蛋白纯化microvolumes。蛋白被成功纯化，将得到的产率为每25微升3.5微克表达时使用2-步骤体外蛋白质翻译系统和每25微升1.5微克当与1步翻译我VT蛋白表达系统。低分子量蛋白质改变从18 1kDa至37kDa的表达是可重复的和直线前进。然而，高分子量的表达导致蛋白截断。由于对克隆的DNA获得的microvolumes，PCR扩增被用来验证克隆产物。潜在的障碍表达使用无细胞表达系统可能是的eIF2α亚基磷酸化不变，由于反应高浓度的ATP高分子量蛋白。这损害了翻译起始的系统，该系统可能会影响蛋白质合成的伸长合成大蛋白在无细胞表达系统10中。 这里所描述的协议由优化条件为蛋白质的表达不受影响。既无基因密码子也不质粒为蛋白质的表达进行了优化，并且是不需要的蛋白质的重折叠。该系统表示p在3小时和蛋白质的纯化roteins可以在2小时而获得，从而允许进一步的下游应用，如重组蛋白用于免疫接种或肽测序的处理。多个重组蛋白可以与海拉细胞系统表达，使用微阵列格式免疫血清筛选和鉴定潜在的疫苗候选分子。此外，潜在的免疫原性蛋白可以通过屏幕进行识别和选择通过放大表达所得的蛋白质。

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><tbody><tr><td>2-step &lt;em&gt;in vitro&lt;/em&gt; human cell free expression system</td><td>Thermo Fisher</td><td>88856</td><td>Discontinued. Always store at -80 &amp;deg;C. Do not thaw in warm water</td></tr><tr><td>1-step &lt;em&gt;in vitro&lt;/em&gt; coupled translation system</td><td>Thermo fischer</td><td>88860</td><td>Always store at -80 &amp;deg;C. Do not thaw in warm water</td></tr><tr><td>ProBond Purification system</td><td>Invitrogen</td><td>K850-01</td><td>Store at RT</td></tr><tr><td>Probond Nickel-Chelating Resin</td><td>Invitrogen</td><td>R801-01</td><td>Store at 4&lt;sup&gt;o&lt;/sup&gt;C.</td></tr><tr><td>A+ Human Blood</td><td>Interstate Blood Bank</td><td></td><td>Store at 4 &amp;deg;C.</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％琼脂糖凝胶的PCR扩增的基因片段。 DNA的条带切出和DNA通过在DNA提取离心柱冷冻纯化。 疟疾基因成功地扩增来自基因组DNA（在表2中示出）被克隆到pT7CFE-CHIS表达载体。再放大从RECO的基因mbinant质粒证实克隆基因片段的载体中的存在。用于蛋白质翻译表达系统的流程图如图2所示。 图2A和2B示出了两步骤在体外人细胞表达系统和1步IVT耦合翻译系统的分步过程。成功利用体外人类细胞自由表达系统疟原虫蛋白的表达，棒状体特异性兔抗血清证实。 如图3A所示，重组蛋白是由棒状体特异性兔抗血清＃676识别。如前所示，表达的重组蛋白不被抗血清从P.虫空泡蛋白识别疟原虫和P.约氏疟原虫表明兔抗血清676的特异性对表达的蛋白32。正常兔血清（NRS）不与重组蛋白反应翻译使用2-步骤在体外人细胞表达系统和1-步骤IVT耦合翻译系统（ 图3B）。 疟原虫的重组蛋白的成功表达，如在凝胶组氨酸染色和镍的HRP染色32不同的技术确认。表达的蛋白通过亲和纯化系统进行纯化。净化以批处理方式进行（没有列使用）。该技术是通过改变咪唑的浓度从60至100mM优化。的最佳浓度为洗脱是250mM的。 图4示出了从25微升翻译的蛋白产物被翻译的蛋白质的成功纯化。 图4A示出毛雷尔的裂跨膜蛋白18的成功的纯化。 图4B示出PF3D7_0925900，P的成功纯化约氏疟原虫基因PY17X_0830000，一个恶性的同源基因假定蛋白32。 图4C示出含有蛋白质PY17X_1139200，用Ni树脂珠和100mM咪唑的1×洗脱缓冲液中的假定蛋白犰狳重复成功纯化。蛋白质的纯化后的产率是3.5微克/ 25微升。 <img alt="图1" src="/files/ftp_upload/52772/52772fig1.jpg" /> 图1.扩增P的疟原虫和P.约氏疟原虫的基因。PFc14_0344，PF3D7_0925900，PF3D7_1361800，PY07482和PFA0680cw从P.扩增的基因片段的1％琼脂糖凝胶，显示溴化乙锭染色的PCR产物疟原虫的基因组DNA。 <img alt="图2" src="/files/ftp_upload/52772/52772fig2highres.jpg" width="700" /> 海拉BAS图2.流程图<html> ED无细胞表达系统。两个2步和1步在体外培养的人无细胞表达系统的示意图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52772/52772fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52772/52772fig3highres.jpg" width="650" /> 图3.免疫印迹确认蛋白质的表达。使用全棒状体特异性兔抗血清＃676和正常兔血清表达的重组蛋白的Western印迹分析。抗血清676（A）与反应表达的重组蛋白。 （B）的正常兔血清不与表达的重组蛋白反应。虫空泡蛋白不与重组蛋白32发生反应。hres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/52772/52772fig4highres.jpg" width="700" /> 图从翻译产品的微卷4纯化的蛋白质。使用镍螯合树脂亲和方法表达的重组蛋白的纯化。表达的蛋白质（翻译）（A） 的PF3D7_0114100，（B）的PY17X_0830000，和（C）PY17X_1139200蛋白孵育镍-螯合树脂纯化，用镍基螯合树脂在不存在咪唑的结合缓冲液和纯化缓冲器。孵育后，珠子离心，未结合的蛋白分离，并且将珠粒在洗涤缓冲液洗涤两次。洗脱，接着结合的重组蛋白两次通过的珠子洗涤。翻译蛋白质，洗，洗出液珠溶解在电泳样品缓冲液中。咪唑，使用在洗涤中（20mM咪唑）和洗脱缓冲液（的咪唑100毫摩尔）。抗血清＃676成功识别表达并纯化的重组蛋白。正常兔血清不与表达蛋白作为秀图3B反应。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52772/52772fig4highres.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"><tbody><tr><td>特征</td><td>人无细胞表达系统</td><td>小麦胚芽无细胞表达系统</td><td>兔网织红细胞表达系统</td><td>大肠杆菌裂解物蛋白质表达系统</td></tr><tr><td>时间</td><td>完成转录和翻译在3小时</td><td>转录6小时和翻译为10 - 20小时</td><td>的RNA已被分离的翻译需要90分钟</td><td>唐E在1小时</td></tr><tr><td>合适的载体</td><td> T7载体可以是</td><td> SP6载体</td><td> T7可以使用</td><td> T7可以使用</td></tr><tr><td>合适的规模</td><td>适用于实验室合成</td><td>用于大规模蛋白质的合成</td><td>适用于实验室和免疫研究</td><td>适用于实验室和免疫研究</td></tr><tr><td>摘录</td><td> HeLa细胞提取免费</td><td>小麦提取物的胚胎</td><td>网织细胞提取物</td><td> 大肠杆菌细胞裂解物中提取</td></tr><tr><td>修改</td><td>共翻译和翻译后修饰</td><td>翻译后修饰</td><td>共翻译修饰</td><td>翻译后修饰</td></tr><tr><td>蛋白质的大小翻译</td><td> 8千道尔顿至250千道尔顿</td><td> 220千道尔顿</td><td> 250千道尔顿</td> &lt;TD&gt; 200千道尔顿</td></tr><tr><td>密码子优化</td><td>紧</td><td>紧</td><td>松</td><td>紧</td></tr><tr><td>二硫键的形成</td><td>是的</td><td>是的</td><td>没有</td><td>是的</td></tr><tr><td>产量</td><td>产量低</td><td>高产</td><td>产量低</td><td>极丰产</td></tr><tr><td>费用</td><td> 130.00美元10反应</td><td> 173.00美元10反应</td><td> 136.00美元5反应</td><td> 159.00美元8反应</td></tr><tr><td>蛋白浓度</td><td>每个反应3至5微克</td><td> 100微克/毫升</td><td> 100微克/毫升</td><td> 500微克/毫升</td></tr><tr><td>参考</td><td> 32，30 </td><td> 7，28，29 </td><td> 7 </td><td> 7，24，25 </td></tr></tbody></table> 表1.无细胞表达系统在疟疾研究，细胞的比较目前在疟疾研究中使用自由表达系统。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td fo:width="25mm">基因ID </td><td fo:width="25mm">同源基因标识</td><td>蛋白质</td><td>引物序列进行PCR </td></tr><tr><td rowspan="2"> PF3D7_1436300 </td><td rowspan="2"></td><td rowspan="2">位子组件</td><td> CTCGAGAATAATAACAATCATAATAATAAG </td></tr><tr><td> GAATTCATTATCATCAGGTTTAGCTAATTTTC </td></tr><tr><td rowspan="2"> PF3D7_0114100 </td><td rowspan="2"></td><td rowspan="2"> PFMC-2TM毛雷尔的裂缝蛋白</td><td> GGATCCATGTTAGGTCAAAAAAACACAAATA </td></tr><tr><td> CTCGAGTGTTATTTGCTTTTTGTTTTGAAAA </td></tr><tr><td rowspan="2"> PY17X_0830000 </td><td rowspan="2"> PF3D7_0925900 </td><td rowspan="2">假设蛋白</td><td> GGATCCATGAAATTTTTTAATATTCTCGCA </td></tr><tr><td> CTCGAGTCCTTGGACAACATATATACT60; </td></tr><tr><td rowspan="2"> PY17X_1139200 </td><td rowspan="2"> PF3D7_1361800 </td><td rowspan="2">假设蛋白</td><td> GGATCCATGGGGTGCGACCCTGGGGTCAGCA </td></tr><tr><td> CTCGAGGCTCTTCAGATACTAAGCTACTAAT </td></tr></tbody></table> 表2棒状体的基因在研究中。表达本研究19个不同基因的引物。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>材料名称</td><td>公司</td><td>目录编号</td><td>描述</td></tr><tr><td> 2步在体外培养的人细胞表达系统32 </td><td>赛默飞世</td><td> 88856 </td><td>停产。一直存放于-80℃。不要解冻在温水中。 </td></tr><tr><td> 1步体外耦合翻译系统</td><td>赛默飞世</td><td> 88860 </td><td>一直存放于-80℃。不要THAW在温水中。 </td></tr></tbody></table> 表3的Hela基于无细胞表达系统。用于蛋白质的表达试剂盒。

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

Universidad San Sebastian

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

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

科研

生物化学

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

免疫与感染

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

生物学

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

生成转基因 伯氏疟原 虫孢子

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

重组蛋白表达的结构生物学在HEK 293F悬浮细胞:一种新的和可访问方法

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

<meta charset="utf8"></head><body>利 什曼原虫 翻译提取物 （LTE） 生产的简化方案

Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.

<meta charset="utf8"></head><body>作者聚焦：使用 LTE 真核生物无细胞蛋白表达系统生产蛋白质

Leishmania Translational Extract (LTE) is a eukaryotic cell-free protein expression system derived from the single-celled parasite, Leishmania tarentolae. This optimized protocol makes LTE simple and cost-effective to manufacture. It is suitable for various applications focused on the multiparallel expression and study of complex eukaryotic proteins and their interactions.

LTE 的生产和优化，一种用于重组蛋白生产的利 什曼原虫 衍生的无细胞蛋白表达系统

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate ...

基于海拉细胞自由表达系统中的表达

摘要

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此视频中的章节

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

高产净化

方法调查小RNA和核糖体占用的调控作用

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

单细胞基因转录的RNA荧光分析

生成转基因伯氏疟原虫孢子

重组蛋白表达的结构生物学在HEK 293F悬浮细胞:一种新的和可访问方法

LTE 的生产和优化，一种用于重组蛋白生产的利什曼原虫衍生的无细胞蛋白表达系统

LTE 的生产和优化，一种用于重组蛋白生产的利什曼原虫衍生的无细胞蛋白表达系统

LTE 的生产和优化，一种用于重组蛋白生产的利什曼原虫衍生的无细胞蛋白表达系统

基于海拉细胞自由表达系统中的表达

摘要

探索更多视频

此视频中的章节

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

高产净化

方法调查小RNA和核糖体占用的调控作用

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

单细胞基因转录的RNA荧光分析

生成转基因 伯氏疟原 虫孢子

重组蛋白表达的结构生物学在HEK 293F悬浮细胞:一种新的和可访问方法

LTE 的生产和优化，一种用于重组蛋白生产的利 什曼原虫 衍生的无细胞蛋白表达系统

LTE 的生产和优化，一种用于重组蛋白生产的利 什曼原虫 衍生的无细胞蛋白表达系统

LTE 的生产和优化，一种用于重组蛋白生产的利 什曼原虫 衍生的无细胞蛋白表达系统

生成转基因伯氏疟原虫孢子

LTE 的生产和优化，一种用于重组蛋白生产的利什曼原虫衍生的无细胞蛋白表达系统

LTE 的生产和优化，一种用于重组蛋白生产的利什曼原虫衍生的无细胞蛋白表达系统

LTE 的生产和优化，一种用于重组蛋白生产的利什曼原虫衍生的无细胞蛋白表达系统