The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/&#954;.

注:合适的哺乳动物表达载体，必须使用该协议。这里，使用含有两个表达盒的单个构建体（ 即重链和轻链表达由单独的启动子驱动）。曲妥单抗重和轻链先前克隆到载体中。这个载体是从安德鲁Beavil的礼物，通过一个不以营利为目的质粒库13获得。 1.恢复和载体DNA的规模化发展注:载体DNA收到大肠杆菌 XL-1蓝色株软琼脂穿刺培养;载体携带潮霉素抗性。 <ol><li>将无菌接种刺伤或循环进入穿刺培养，然后连胜75微克准备了卢里亚BERTANI（LB）琼脂平板上的软琼脂/ ml潮霉素的单菌落。孵育在37℃的板18-24小时。 </li><li>接种单个菌落到5ml了不起肉汤（TB）的含有75微克/毫升潮霉素。 INCU大怒的文化，在37℃，18〜24小时，225转摇晃。 </li><li>使用过夜培养轻轻用200μl80％甘油混和并冷冻在-80℃下混合800微升培养以制备载体DNA的甘油。 <ol><li>将100μl过夜培养物加至100毫升结核病含75微克/一个挡板摇瓶中（ 即 1/1000稀释）ml潮霉素。孵育在37℃下培养18至24小时，以225 rpm振摇。 </li></ol></li><li>过夜培养后，提取和纯化根据与以下异常的midi / MAXI制备试剂盒的制造商的说明将DNA;期间与水（pH 7.0-8.5）中的最后一步，洗脱或重悬的DNA。 </li><li>检查浓度与由吸光度读数的DNA纯度在260和280nm，然后储存的DNA在-20℃。 注:可选的（强烈推荐）:DNA序列使用载体特异性引物，以确认身份。 </li></ol> 2。 HEK-293细胞的稳定转染<ol><li>生长并保持根据在补充有在锥形瓶中的0.1％的非离子表面活性剂的无血清培养基的标准协议HEK-293细胞（悬浮细胞）。保持在2×10 5个细胞/ ml传代培养每第四天。培养细胞在37℃，5％的CO 2和120rpm的旋转。 注:细胞应已在培养不少于4天，不超过4周前，转染。生长在含有血清的培养基单层的HEK-293细胞也可以用于此过程。 </li><li>在转染前一天，种子的HEK-293细胞以3×10 5个细胞/在12孔平板的孔中毫升2-毫升Dulbecco氏改良的Eagle培养基（DMEM）中的补充有10％热灭活的胎牛血清（ FBS）。在转染日，检查细胞达到80-90％汇合</li><li>稀释转染媒体DNA和聚乙烯亚胺（PEI）分别然后混合在一起。<ol><li>稀释300微升转染媒体每孔1.25微克载体DNA（15微克的12口井）。在室温下孵育5分钟。 </li><li>稀释的1mg / ml的PEI溶液2.5微升（30μl的12口井， 即 1:PEI的2比的DNA）在300微升转染培养基。在室温下孵育5分钟。 </li><li>稀释的DNA添加至稀释的PEI，轻轻混合并在室温下孵育15分钟。 </li></ol></li><li>加入50μl的DNA / PEI混合物滴加至板的各孔中。轻轻摇动平板分发然后转染混合物放置板在37℃，5％CO 2的24小时。 </li><li>加50微克/ ml潮霉素，每孔B和返回板37℃，5％CO 2的10天。 注意:通过第10天，未转染的细胞将死亡和拆卸从板的表面上，而稳定地转染的细胞将是可行的，并附着到板上。 </li><li>在水井更换介质1/4体积的无血清培养基和3/4体积的DMEM补充有10％FBS（ 注:0.5 ml无血清培养基+ 1.5的10ml DMEM = 7.5％的最终血清浓度），并孵育4天。 </li><li>用补充有10％FBS（ 即 1毫升无血清培养基+ 1的10ml DMEM = 5％的最终血清浓度）1/2体积的无血清培养基和1/2体积的DMEM替换在孔中的媒体并孵育4天。 </li><li>用补充有10％FBS（ 即 1.5毫升无血清培养基+ 0.5的10ml DMEM = 2.5％的最终血清浓度）3/4体积的无血清培养基和1/4体积的DMEM替换在孔中的媒体并孵育4天。 </li><li>用2ml补充有0.1％的非离子表面活性剂（即0％的最终血清浓度）的无血清培养基的更换在孔中的媒体并孵育4天。 注:保持无血清适应过程中50μg/ ml的潮霉素B的选择性细胞上的压力。适应于无血清培养基的细胞从孔表面分离，并且可以第一组ñ停牌。参见步骤2.1培养条件与50微克的额外补充/ ml潮霉素B. </li><li>使用移液管，轻轻在12孔板和池细胞悬浮培养物混合到一个单独的管中。 <ol><li>使用血细胞计数器，枚举汇集细胞和种子在30毫升无血清培养基中一个锥形烧瓶中，在2×10 5个细胞/ ml。培养细胞在37℃，5％的CO 2和120rpm的旋转。亚文化，每第四天扩大。 </li></ol></li><li>继续扩大培养规模到所需细胞密度为1×10 7个细胞，以获得10小瓶/小瓶在液氮冷冻保存。冻结细胞在含有10％二甲基亚砜（DMSO）的无血清培养基。 注:空调含有抗体媒体可以在每个传代培养收获确认蛋白质生产或用于优化纯化条件。收获条件培养基和维护传代细胞，离心在文化300 XG 5分钟，然后用0.22微米的过滤器过滤消毒上清液。过滤的上清液可保持在4℃下1-2周或-20℃下长期储存。 </li></ol> 3.大规模抗体生产批量从长满文化注:抗体的生产可以从步骤2.11，其中现有的单元格扩展到基于批处理长满培养体积上所需的细胞密度是设置应遵循的。否则，已经从冷冻解冻细胞开始在该阶段，一旦扩大到合适的细胞密度和体积。各种培养补充剂可用于优化抗体生产;利用蛋白胨，增加抗体产量是表现在这个协议。 <ol><li>种子细胞在两个Erlenmeyer烧瓶的100ml无血清培养基2×10 5个细胞/ ml（以比较从非补充和营养补充培养抗体的产量）。文化在37 ℃，5％CO 2的</sUB&gt;和120转。 </li><li> 24小时后，加入胰蛋白胨为0.5％的终浓度向营养补充培养。均衡与无血清培养基未补充的文化音量。 </li><li>从第8天，进行使用血球监测细胞生存力每日培养物的细胞计数。收获培养上清一次细胞存活率小于80％。 <ol><li>收获上清液通过培养物离心以3000 xg离心15分钟，然后通过0.22微米的过滤器过滤，消毒上清液（含有抗体）。存储上清在4℃（短期），或在-20°C（长期）冻结。 </li></ol></li></ol> 4.抗体纯化通过亲和层析法使用自动化快速蛋白质液相色谱（FPLC）系统注:以下步骤一般可以适用于大多数的自动化系统。纯化可在室温下或在4℃下（如果FPLC系统被保持在阴凉室）中进行。可以进行一系列的侦察测试，以找出最佳纯化条件包括适当的列矩阵，结合缓冲液，洗脱缓冲液和pH值，以确保从条件培养基纯化的抗体的最大恢复（参见结果部分）。最优条件取决于被纯化的抗体或蛋白质。纯化了一个自动化FPLC系统上进行。纯化用5ml的蛋白A柱在室温下进行。 <ol><li>使用超纯水制备下列缓冲液和调整到推荐pH值，然后通过0.22微米的过滤器进行过滤。 <ol><li> 0.14 M氯化钠，0.0027米氯化钾，0.01M 的 Na 2 HPO 4和0.001摩尔KH 2 PO 4:通过混合下列制备1升磷酸盐缓冲盐水（PBS）pH 7.4的（结合缓冲液）的。过滤缓冲区之前必要时进行调整pH值。 </li><li>制成500毫升之0.1M甘氨酸 - 盐酸pH值2.7（洗脱缓冲液）的。过滤的buff前调节pH值呃。 </li><li>准备50ml的摩尔Tris pH值9.0（和缓冲液）的。 </li></ol></li><li>确保计算机是由系统电源和通讯连接。设备应在软件"系统控制"模块可见。确保UV单元被设定在280nm波长。可选的:如果连接，也可以使用校准pH计。 </li><li>沉浸A，B和采样泵的入口管超纯水冲洗系统。清除泵用注射器是否有空气管道或空气的系统中的一个怀疑。通过系统控制器或经由自动化的方法，通过手工操作用水冲洗该系统。观察到压力，导电性，UV280跟踪和pH保持一致，并且该列中的压力限制不超过根据制造商的规范。 注:异常可能表明空气或堵塞应该在继续之前加以解决系统。 </li><li>在系统控制器模块，从1开始的流速毫升/分钟通过手动在任何负载泵A（绕过样品环）或注射（通过样品环）的位置，开始连接列。在列位置入口断开系统管道和删除连接到柱入口塞子。 <ol><li>允许水从系统管滴流在塔的顶部则系统管道连接到柱上。具有柱的入口和与水滴入口配件溢流确保无气泡的连接。 </li></ol></li><li>连接柱子出口的下游出口，并确认所有的配件都紧紧系住。与列现在连接至系统，如在列规范概述不超过最大压力极限和流量限制。 </li><li>用的水5倍柱体积（CV）的洗涤塔。如果有必要，继续洗柱，直到UV280跟踪已趋于稳定。 </li><li>沉浸A的入口管结合缓冲液，B在洗脱缓冲液和结合缓冲器T采样泵Ø平衡系统在正确的缓冲区。使用PumpWash通过手动操作缓冲填充进气管。 </li><li>在软件的"方法编辑器"模块中，使用该方法的向导设置为，目的是要使用的柱的亲和色谱法。 注:自动化系统FPLC配方法预先填充推荐的设置（ 即流量和压力限制），并运行基于选定净化柱（制造商，矩阵和大小）的步骤。 <ol><li>使用蛋白质A亲和层析以下运行步骤: <ol><li>平衡系统用5CV结合缓冲液，并收集到流出液废物容器。 </li><li>负载样品上柱（体积要加载手动指定的方法），并收集流过到一个单独的容器中。 </li><li>洗系统用5CV的结合缓冲液，并收集流过到一个单独的容器中。 </li><li>用5CV isocrat柱洗脱IC分离使用洗脱缓冲液，并收集纯化抗体样品由馏分收集器部分。 </li><li>平衡系统用5CV结合缓冲液，并收集到流出液废物容器。 </li></ol></li></ol></li><li>一旦该方法已经设置，指定的条件培养基的体积要施加到列和保存方法。 注:在该方法中指定的体积应比实际体积，以避免样品运行期间引入空气少5-10毫升。在这个协议中指定的卷是90-120毫升。 </li><li>浸没样品泵管到含有经调节的培养基的容器中。 </li><li>准备一个单独的容器通过指定的出口管收集样品流出液。 注:收集在运行或柱的结合能力过程中发生错误的情况下的流过液是很重要的超过需要的流过液被重新应用到列或外净化epeated。 </li><li>在馏分收集器准备收集管。加入100微升每毫升体积分数和缓冲的。在FPLC系统将自动洗脱馏分进入收集管中。 </li><li>在软件的"系统控制"模块，打开方法来运行。 注:该方法运行在一系列的包括检查的方法中，馏分收集器安装程序的变量和定义的结果的文件名和存储位置的页面启动。 </li><li>点击START开始运行。运行可以在"系统控制"模块中进行监控。 </li><li>在净化运行完成后，检查所生成的色谱系统软件的"评估"模块中。 </li><li>将所有含蛋白质的分数到一个单独的管，缓冲交换和使用离心过滤装置与切断30 kDa的分子量在PBS集中。根据制造商的执行离心步骤说明。 </li><li>使用根据制造商的说明双金鸡宁酸测定法（BCA）测定抗体浓度。 </li><li>如果另一个净化运行是要执行，主要在结合缓冲液，并重复采样泵管步骤4.9-4.14。 </li><li>在纯化轮次的完成，沉浸A，B和样品泵的入口管在超纯水中，并如在步骤3中进行清洗系统和柱。 </li><li>淹没A，B和泵管在20％乙醇，重复洗涤程序对系统和列存储。 </li><li>断开从下游出口列和替换柱塞，然后在入口断开柱和更换塞子，重新连接管连接到该系统。储存在4℃下的列。 </li></ol>

<ol>
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L., 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=Transient+gene+expression+in+HEK293+cells:+peptone+addition+posttransfection+improves+recombinant+protein+synthesis.">Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.</a> Biotechnol Bioeng. 90 (3), 332-344 (2005).</li><li>Pham, P. L., 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=Large-scale+transient+transfection+of+serum-free+suspension-growing+HEK293+EBNA1+cells:+peptone+additives+improve+cell+growth+and+transfection+efficiency.">Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency.</a> Biotechnol Bioeng. 84 (3), 332-342 (2003).</li><li>Yang, W. C., 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=Addition+of+valproic+acid+to+CHO+cell+fed-batch+cultures+improves+monoclonal+antibody+titers.">Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers.</a> Molecular Biotechnology. 56 (5), 421-428 (2014).</li><li>Backliwal, 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=Valproic+acid:+a+viable+alternative+to+sodium+butyrate+for+enhancing+protein+expression+in+mammalian+cell+cultures.">Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures.</a> Biotechnol Bioeng. 101 (1), 182-189 (2008).</li><li>Jiang, Z., Sharfstein, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+butyrate+stimulates+monoclonal+antibody+over-expression+in+CHO+cells+by+improving+gene+accessibility.">Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility.</a> Biotechnol Bioeng. 100 (1), 189-194 (2008).</li><li>Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+adherent+and+suspended+cells+by+calcium+phosphate.">Transfection of adherent and suspended cells by calcium phosphate.</a> Methods. 33 (2), 136-143 (2004).</li></ol>

The authors declare that they have no competing financial interests.

该协议细节的转染，稳定表达和治疗性抗体的纯化在HEK-293细胞。抗体基因的稳定表达是在产生用于治疗性抗体的开发和制造的抗体产生细胞系的第一步。而中国仓鼠卵巢（CHO）细胞仍然是治疗性蛋白质的选择的表达平台，HEK-293细胞系获得突出的实现，在这些细胞中产生的蛋白质是天然存在的人类蛋白质更匹配，在后方面-translational修改和功能14,15。 ...

治疗性抗体的成功将继续推动大量投资进入的抗体开发为下一代疗法的浪潮开始。抗体市场预期由抗体片段1，抗体-药物偶联物2，双特异性抗体3和工程化抗体具有有利性质4重塑。另一类获得医药感兴趣的是生物仿制药。生物仿制药抗体是"高度相似"复制这种已经获得监管部门的批准治疗性抗体产品。拟议的生物仿制药必须是就其结构，功能，动物毒性，临床安全性和有效性，人体药代动力学（PK），药效学（PD）和免疫原性5,6鼻祖抗体相媲美。 批准ř生物仿制药的抗体的茨一直缓慢由于在产品的最终质量的严格限制。确切的制造过程，如通过对最终的处理步骤的特定细胞系和培养条件可以保持专有的。更重要的是，抗体的制造固有涉及程度可变性的可加至生产高度类似产品的挑战。全面理化和生物物理表征和比较是相当困难的，但许多研究表明生物仿制药的抗体的特性出现在文献7，8，9。 产生治疗性抗体便从哺乳动物宿主细胞用携带的基因的各抗体的载体的转染。载体设计，细胞系和培养条件是建立前的关键考虑因素PRESSION系统。 抗体的DNA序列可以从药物银行（www.drugbank.ca），IMGT（www.igmt.org）或研究的出版物，包括专利中采购。例如，曲妥单抗的序列，可通过药物银行（DB编号:DB00072）。可变区的氨基酸序列可经历基因设计和优化用于合成在所需宿主物种。它是没有修饰的氨基酸序列制成的生物仿制药抗体重要。一旦合成，抗体基因可亚克隆到所选择的合适的载体。 人IgG抗体由两条相同的重链和两个相同的轻链组成。两条链的紧密调节表达是用于在哺乳动物细胞10的异源抗体蛋白的最佳生产的关键。帧内以及链间二硫键必须形成和一些翻译后修饰的需要成为在蛋白质的生物合成过程中troduced。许多载体是已专为表达抗体基因（参考材料表）可用。这些特定抗体 - 载体通常表示为重链和轻链，以便仅在每个链的可变区所需要克隆的恒定区。 有两个独立的构建体（共转染）细胞的转染是用于输送重和轻链编码基因的最常见的方法。也就是说，每个基因通过其自身的启动子被组装的内质网前驱动和转录为单独的抗体链。另一方面，多顺反子载体具有掺入内部核糖体进入位点（IRES）元件，使多个基因作为与由mRNA 11的内部区域允许翻译单一的mRNA转录物的表达。在这种情况下，重链和轻链编码基因被连接在一个arrangEMENT同时实现抗体链10,12的共表达。 而瞬时转染细胞产生足够的蛋白来执行的实验的数量有限，已经经过选择基因组整合稳定转染的细胞系可以提供更高的产量。较高的蛋白质的量允许为涉及体外表征检测开发，并能提供在考虑抗体质量为下游应用的指示，如克隆细胞系和铅候选选择。 本文的目的是描述在哺乳动物表达系统中产生的治疗性抗体的稳定表达和纯化。事实上，这种方法可以适用于一生物仿制药抗体的表达。该方法可用于抗体的初始特性继续到关键的，尽管是费时STE之前确定一个理想的克隆用于大规模生产的PS。此外，这种方法可用于表达其它蛋白质，而不只是抗体。 下面的详细方案描述的治疗性抗体曲妥单抗的表达。这包括制备载体DNA随后在HEK-293细胞系和抗体蛋白质的纯化稳定转染通过自动化色谱法的。

<table border="0" cellpadding="0" cellspacing="0" width="1730">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">pFUSE vector series</td>
			<td style="width:189px;">N/A</td>
			<td style="width:191px;">InvivoGen</td>
			<td style="width:1003px;">Heavy and light antibody genes expressed in separate vectors that require co-transfection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAbXpress vector series</td>
			<td>N/A</td>
			<td>ACYTE Biotech Pty Ltd.</td>
			<td>Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pVITRO1 vector</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Heavy and light antibody genes are each driven by a separate promoter in a single vector.&#160; Refer to: Dodev, T. S. et al. A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GS vector series</td>
			<td>N/A</td>
			<td>Lonza</td>
			<td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-cistronic vector series 1</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. et al. A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-cistronic vector series 2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. et al. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pVITRO1-Trastuzumab-IgG1/&#954;</td>
			<td>61883</td>
			<td>Addgene</td>
			<td>Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast-Media Hygro Agar</td>
			<td>fas-hg-s</td>
			<td>Jomar Life Research</td>
			<td>Used to prepare low salt LB agar containing 75 &#181;g/ml hygromycin.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast-Media Hygro TB</td>
			<td>fas-hg-l</td>
			<td>Jomar Life Research</td>
			<td>Used to prepare low salt TB broth containing 75 &#181;g/ml hygromycin.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol, BioXtra, &#8805;99%</td>
			<td>G6279</td>
			<td>Sigma-Aldrich</td>
			<td>Prepare to 80% with water and autoclave. Store at room temperature.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Jestar 2.0/LFU Plasmid Maxi Kit</td>
			<td>G221020</td>
			<td>Astral Scientific</td>
			<td>Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FreeStyle 293-F Cells</td>
			<td>R790-07</td>
			<td>Life Technologies</td>
			<td>HEK-293 cell line adapted to suspension culture in serum-free media.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FreeStyle 293 Expression Medium</td>
			<td>12338-018</td>
			<td>Life Technologies</td>
			<td>Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Kolliphor P188</td>
			<td>K4894</td>
			<td>Sigma-Aldrich</td>
			<td>Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 &#956;m filter. Store at 4oC.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose&#160;</td>
			<td>11995-065</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat-Inactivated Foetal Bovine Serum</td>
			<td>10082-147</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:348px;">Polyethylenimine, Linear, MW 25,000&#160;</td>
			<td>23966</td>
			<td>Polysciences, Inc.</td>
			<td>Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 &#956;m filter. Store at -80oC until use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OptiPro SFM</td>
			<td>12309-050</td>
			<td>Life Technologies</td>
			<td>Transfection formulated serum-free media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin B Solution</td>
			<td>ant-hg-1</td>
			<td>Jomar Life Research</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethylsulphoxide (DMSO)</td>
			<td>AJA2225</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tryptone (casein peptone)</td>
			<td>LP0042B</td>
			<td>Thermo Fisher Scientific</td>
			<td>Prepare to 20% in PBS and filter-sterilize using 0.22 &#956;m filter. Store at 4oC.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml</td>
			<td>09-2051-100</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HiTrap Protein A High Performance, 1 x 5 ml column</td>
			<td>GE17-0403-01</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AKTApurifier 100</td>
			<td>28406266</td>
			<td>GE Healthcare</td>
			<td>Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine-HCl</td>
			<td>G2879</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citric Acid, monohydrate</td>
			<td>BIOC2123</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Citrate, trisodium salt dihydrate&#160;</td>
			<td>BIOCB0035</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M Tris-HCl solution pH 9.0</td>
			<td>BIOSD8146</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra Centrifugal Filters (30 MWCO)</td>
			<td>UFC803008/UFC903008</td>
			<td>Merck Millipore</td>
			<td>Used to buffer exchange and concentrate purified protein.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Bicinchoninic Acid (BCA) Assay Kit</td>
			<td>23227</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BLItz System</td>
			<td>45-5000</td>
			<td>fort&#233;BIO</td>
			<td>Instrument used for bio-layer interferometry (BLI) measurements.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein A biosensors</td>
			<td>18-5010</td>
			<td>fort&#233;BIO</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylamide/Bisacrylamide (37.5:1), 40% solution</td>
			<td>786-502</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Persulfate (APS)</td>
			<td>AM0486</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEMED</td>
			<td>AM0761</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomassie Brilliant Blue R-250</td>
			<td>786-498</td>
			<td>Astral Scientific</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Precision Plus Dual-Color Protein Standard</td>
			<td>1610374</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory scale production and purification of a therapeutic antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

使用生物层干涉（BLI）转染HEK-293细胞的稳定生产单抗的结果确认， 如图1中。通过测量IgG抗体标准和蛋白质的生物传感器（ 图1A）之间的结合率产生的IgG的标准曲线。粗制上清液样品类似地测量，那么其浓度从标准曲线（ 图1B）内插。上清液的浓度测得为大约25微克/毫升双周（从传代培养收集50毫升取样）的无血清适应之后和之前为?...

Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

Laboratory Scale Production and Purification of a Therapeutic Antibody

这个协议描述在哺乳动物表达系统中生产的治疗性抗体的。所描述的方法包括制备载体DNA，稳定转染和人胚肾293细胞系的无血清适应的，设置大规模培养和纯化使用亲和层析组成。

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

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Biochemistry

17.2K 次观看.  University of Sydney. 这个协议描述在哺乳动物表达系统中生产的治疗性抗体的。所描述的方法包括制备载体DNA，稳定转染和人胚肾293细胞系的无血清适应的，设置大规模培养和纯化使用亲和层析组成。 

视频: Laboratory Scale Production and Purification of a Therapeutic Antibody

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

生产，结晶和结构测定<em&gt;℃。难辨</em&gt;通过Microseeding和锌SAD PPEP-1

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

烟草 Benthamiana叶在制备鳞片上的瞬态表达

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...