Name: high yield expression of recombinant human proteins with the transient transfection of hek293 cells in suspension
Uploaded: 2015-12-28T15:00:00.000+00:00
Duration: 11 min 42 s
Description: Watch this Scientific Journal Video about High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension at JoVE.com

这项工作是由财政拨款K22AI099165（AWB）的支持，P41GM103390（KWM）和P01GM107012（KWM）由美国国立卫生研究院，以及来自生物化学，生物物理学和分子生物学的罗伊J.卡佛系美国爱荷华州立大学基金。这项工作的内容​​完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

该协议是充分利用两种HEK293F或HEK293S细胞中的表达。 1.细胞建立<ol><li>培接种 注:所有培养操作过程必须在BSL-2设施内进行和每个项目带入生物安全柜必须通过在水溶液中的70％的乙醇喷雾进行灭菌。 <ol><li>操作培养摇床中于135转，湿度80％，并在8.0％的CO 2和37℃。打开"的"生物安全柜中至少1小时前工作的紫外灯。 Prewarm密封培养基A和培养基B瓶在水浴在37℃进行1小时。 </li><li>通过在水溶液中70％乙醇消毒喷洒125毫升锥形瓶中生长具有通风帽，移液器，移液器和预热媒体瓶。发生在生物安全柜这些项目。注意:消毒只有外塑料覆盖125毫升锥形烧瓶中培养，然后，只有当它我删除S中的生物安全柜中。 </li><li>为30ml的培养，撤回和26 ml的培养基A 3毫升培养基B的（最终培养的10％）和转移到125毫升的锥形烧瓶中培养，并轻轻振荡混合（以下称为新鲜培养基）。 </li><li>传递一个小瓶含有HEK293F细胞，冷冻在-80℃，在冰上，并移动到培养室的细胞。注意:HEK293F细胞以1×10 7个细胞/ ml的密度提供的。 </li><li>轻轻暖小瓶在水 - 浴中于37℃，以部分解冻细胞（大约需要1分钟，细胞不应该被完全解冻）。消毒小瓶的外部与在水溶液中的70％的乙醇，并将其移动到生物安全柜。 </li><li>使用1毫升吸管，撤回细胞悬浮液（约0.7毫升）并转移到含有29日毫升新鲜培养基125毫升锥形瓶中。关闭培养瓶的盖子，并将其移动到培养摇床。 </li></o升&gt; </li><li>电池保养:检查细胞密度，活力和细胞传代<ol><li>按照下面的协议检查文化解冻24小时后的细胞密度。注意:将细胞解冻，以确保必要的细胞生长&gt; 80％的存活率和后生长24小时。 </li><li>消毒培养瓶中，在水溶液中的70％的乙醇，并把它移动到了生物安全柜。 </li><li>使用1ml吸管和移液器，慢慢撤回100微升悬浮培养并转移到无菌的0.5毫升的Eppendorf管中。关闭并尽快培养瓶中转移放回摇床越好。 </li><li>混合7.5微升台盼蓝溶液与7.5微升细胞。与7.5微升的细胞/台盼蓝的混合物剧烈混合，并将其转移到计数幻灯片。 </li><li>打开自动细胞计数器，然后将计数滑入装载室。自动读取器被激活和细胞中的单元自动计数每毫升和百分比生存力的细胞数的</li><li>确定所需的转移到新鲜的培养基中以0.3×10 6个活细胞/ ml的细胞密度的供体培养物体积。 注:查看示例计算补充资料。 </li><li>重复步骤1.1.2和1.1.3以上的"1.1培接种"协议的准备所需的新鲜培养基材料。 </li><li>转移培养基A（23毫升）和培养基B（3毫升）中，以一个新的125个毫升培养瓶中。从生物安全柜中取出瓶子股票中A和培养基B的。 注意:介质瓶窗饰不应被保持在生物安全柜，同时作为HEK 293F细胞。这限制污染库存介质瓶的可能性。 </li><li>从培养箱中取出HEK293细胞培养和喷洒在水溶液中的70％的乙醇。使用新的移液管，撤细胞的等分（4毫升，这个量是确定的，根据牛逼从培养Ò步骤1.2.6），并将其转移到含有新鲜培养基的烧瓶中。 </li><li>从生物安全柜转让双方文化的培养摇床根据1.1.1设置。生长细胞2-3×10 6个活细胞/ ml的密度近似。 注:细胞的近似倍增时间为32小时。这将需要3-4天达到这一密度。孵育细胞3-4代以具有先于所述第一转染细胞的稳定生长模式。 </li></ol></li></ol> 2.准备材料转<ol><li>准备HEK293细胞（第1天） <ol><li>根据传代培养的细胞，以1×10 6个细胞/ ml，在新鲜培养基B 24 hr的密度在转染之前步骤1.2。 注意:转染培养的体积将取决于细胞的数目。较大的转染体积（&gt; 20毫升）将要求在此阶段有较大体积的培养。 </li></ol></li><li>准备圣材料ocks <ol><li>在含有25mM 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）和150毫摩尔NaCl（pH7.5）中的缓冲液制备的线性聚乙烯亚胺（PEI）原液以1mg / ml的浓度。完全溶解PEI;这可能需要30-60分钟，在室温。通过0.22微米注射器式滤器并储存消毒在-20℃下长期使用。 </li><li> （按步骤2.2.2.1-2.2.2.4）准备无菌质粒DNA。该质粒的构建过程，其他地区42描述。一个简短的程序进行DNA准备在此说明: <ol><li>生长在1升LB培养基（Tryptone- 10克/升，酵母extract- 5克/升和钠氯化物10克/升），补充了100微克/的转化的pGEn2矢量化学感受态大肠杆菌细胞的培养ml氨苄青霉素。E.大肠杆菌生长在37℃下在非加湿，振荡培养箱。 </li><li>根据马努净化pGEn2矢量编码对象质粒DNA厂家的DNA纯化的过程。 </li><li>以确保质粒DNA完全无菌，执行最终异丙醇沉淀和再悬浮步骤的生物安全柜内将DNA提取程序。 </li><li>异丙醇（在质粒制备试剂盒中指定）的体积添加到洗脱的DNA和离心机以10,000× 离心10分钟。消毒的DNA的容器，用70％的乙醇的外侧水溶液和生物安全柜内传送。丢弃用吸管和异丙醇上清液风干DNA沉淀。一旦干燥，重悬沉淀在无菌的10mM Tris缓冲液，pH 8.0。 </li></ol></li><li>制备的220毫米丙（VPA）酸储备溶液中的水，通过无菌0.22微米的过滤器通过通道消毒。存储所述溶液在-20℃直至进一步使用。 </li></ol></li></ol> （三）建立瞬时转染<ol><li>转（第0天） <ol><li>通过下面的步骤1.2.2通过"1.2。电池维护"上面的1.2.5检查细胞密度。确定用于转染（2.5-3.0×10 6个活细胞/ ml的存活率&gt; 95％）细胞的量。查看示例计算补充资料。 </li><li>转移到250毫升的离心管中的前一步骤（此处67毫升）计算悬浮培养细胞使用无菌血清吸管的体积。收集细胞离心5分钟，在100×g下。 </li><li>在此期间，准备根据"1.1。培养接种"上方的新鲜染培养基以下步骤1.1.3。见补充材料的计算示例。此外，新鲜的培养基转移5 ml到无菌的15毫升管，备用。 </li><li>含有沉淀的细胞到生物安全柜灭菌管的外面与70％乙醇在水中的溶液，并使用无菌移液管，以滗次后传输管Ë上清包括废培养基。 </li><li>大力通过上下抽吸用10ml新鲜培养基，并转移到新鲜的转染培养基烧瓶悬浮沉淀的细胞（在上述步骤3.1.3制备）。拧上排出细胞培养烧瓶中的帽并与摇动烧瓶培养箱（如在1.1.1中设置）为15分钟-1小时。 </li><li>在此期间，稀释质粒DNA，并使用新鲜的培养基（使用5 ml的体积来自上述步骤3.1.3撤销）为0.5微克/微升的最终浓度的PEI的股票。见补充材料样品计算，以确定DNA和PEI的量。转移与分散的细胞进入生物安全柜的培养瓶中。 </li><li>加入质粒DNA（300微升0.5微克/微升）用微量移液器的文化和温和的人工摇动拌匀。加入PEI（900微升0.5微克/微升），以文化，轻轻晃动混​​匀。加入3.8毫升新鲜的小路TURE介质，从步骤3.1.3，上方和培养摇床24小时转移烧瓶中。 </li><li>清洁按照步骤1.1生物安全柜。 </li></ol></li><li>稀释（1天）（适用于50毫升的转染量） <ol><li>孵育转染的培养24小时。 </li><li>撤回1 ml的预热220毫丙戊酸（VPA;根据步骤2.2.3制备。），使用无菌的1 ml的血清吸液管，并将其转移到无菌的50ml管中。 </li><li>撤回5.0毫升预热培养基B和44毫升预热培养基A和用无菌血清吸管以制备50ml新鲜培养基与丙戊酸钠4.4 1mM的终浓度将其添加到VPA溶液。 </li><li>移动转染培养到生物安全柜灭菌烧瓶的外观与在水溶液中的70％的乙醇后，添加所制备的稀释介质将烧瓶转移回温育摇床。 </li></ol></li><li>表达和收获（2-6天） <ol><li>孵育细胞额外4-5天（5-6天以来的总转）。 </li><li>撤回培养基的以下步骤1.2.2等分试样。到1.2.5。中所述"1.2。细胞维持，"上方用无菌1 ml的血清吸液管，以监测细胞生存力和保存等分试样来分析蛋白质表达在每天通过SDS-PAGE（见第5，下文）。 </li><li>后5-6天通过离心细胞加介质在1000克5分钟收获细胞。此外，收获上清，如果细胞生存力低于50％（见步骤1.2.2-1.2.5）。 </li><li>倒出将包含分泌蛋白质的上清液。加入5毫升10％的漂白剂的HEK细胞沉淀并丢弃作为生物危害。 </li></ol></li></ol> 4.蛋白质纯化<ol><li>制备柱用5ml蛋白A琼脂糖。平衡柱用缓冲液A（20mM的3-（5倍柱体积的N-吗啉代）丙磺酸（MOPS），pH值7.4，100mM的NAC升）。 </li><li>离心收集的上清液以表达的蛋白质以高速（14,000个克，10分钟）以去除任何剩余的细胞碎片。通过滗入新的试管收集。弃沉淀为一个生物危害。 </li><li>收集上清液的10微升等分试样并另外收集在每个蛋白质纯化步骤来分析样品通过SDS-PAGE的小样本（第5部分）。加载澄清的上清液，并收集通过的流动。 </li><li>洗涤用缓冲液A的3-柱体积的柱，洗脱蛋白与100mM的甘氨酸，pH值3.0 5柱体积。收集在含有二分之一体积的1M TRIS缓冲液，pH值为8.0到迅速中和pH管中的洗出液。 </li><li>用缓冲液A和存储的10个柱体积为将来使用洗柱。 </li><li>缓冲交换洗脱的蛋白具有至少10倍过量的缓冲液A，用10 kDa的分子量截止离心过滤器。注意:不要集中洗脱样品一个非常低的体积（&lt;2毫升）中的洗脱缓冲液中。使用离心过滤器以用缓冲液A交换缓冲</li><li>商店纯化的蛋白质在4°C，直到下次使用。 注:蛋白质表达在上清液可通过蛋白的属性的基础上或在使用亲和标签的选定亲和柱进行纯化。对于一个典型的纯化方法，蛋白A柱可用于抗体或Fc片段或镍柱可以用于表达与聚组氨酸标签的蛋白质。这里，是的纯化步骤的IgG1-Fc的使用蛋白A柱的描述。 </li></ol>蛋白通过SDS-PAGE 5.分析<ol><li>稀释各样品的等分试样（7.5微升）与2×上样缓冲液（0.125摩尔Tris，pH8.0中，4％SDS，10％β巯基乙醇，20％甘油，和0.01％溴酚蓝）等体积。 </li><li>在90℃下煮沸混合物用于使用热块或水浴5分钟。离心样品以10000g FO的R 1分钟。加载与5微升标记蛋白和14微升样品用于使用20微升吸管一次性尖端分析所制备的凝胶。 </li><li>运行一个凝胶在25毫安60分钟。一旦电泳步骤完成后，将凝胶转移到容器用1L水和微波5分钟。 </li><li>染色用染色溶液（40％乙醇，10％乙酸和0.1％考马斯亮蓝）2小时，脱色在水凝胶中。 </li></ol> 6.聚糖分析质谱<ol><li>如前面所述44分析聚糖。简言之，75微克的IgG1-Fc的被胰蛋白酶处理，然后，处理通过PNGaseF为聚糖45的释放。释放聚糖全甲基化，并通过使用飞行时间的基质辅助激光解吸电离质谱（MALDI-TOFMS）分析。 </li></ol> 7.协议调整为特殊场景<ol><li>标记蛋白15 N-标记的氨基酸（对于50毫升的转染体积） <ol><li>分配5毫克每种氨基酸为单个的无菌玻璃小瓶中。悬浮用4ml蒸压水（注意酪氨酸不会完全溶解，不像Lys和Phe的生产）。消毒在121℃将溶液高压灭菌15分钟。允许溶液冷却至约50-60℃。 </li><li>对于50毫升的转染量，按照"3.1建立瞬时转染"按照步骤3.1.3。见有适当数量的一个例子补充材料部分。 </li><li>对于转染，按照步骤的"（3）建立一个瞬时转染的"使用取代有标记的氨基酸残基如上制备新鲜培养基。 </li><li>转染后的24小时，在培养稀释的天，提取5.0毫升预热培养基B中加入40ml预热培养基A和新鲜蒸压4毫升一份的氨基酸混合物（其制备如步骤7.1.1。段）并添加1毫升预热的VPA的溶液原液，制备50ml新鲜稀释培养基与丙戊酸钠4.4 10mM的终浓度。添加使用无菌血清吸管上述培养基转染培养。 </li><li>传送转染培养到生物安全柜灭菌烧瓶的外观与在水溶液中的70％的乙醇后，添加所制备的稀释介质将烧瓶转移回温育摇床。 注意:培养基A是化学成分确定的，无血清的培养基。因此，有可能制备不同的制剂。联系供应商以获取自定义中的制剂，缺乏某些氨基酸。有可能有所有氨基酸冷落，但是，我们优选使用介质缺乏只有一些特定的残基，因为完全失落培养基将补充后需要调整渗透压。我们选择了可补充，并且不需要摩尔渗透压调整一个赖氨酸酪氨酸 - 苯丙氨酸漏失介质。福rthermore，中等提供者不自由分享培养基成分的浓度;我们用100毫克/升的每个为赖氨酸，酪氨酸和苯丙氨酸与成功。转染和表达培养基的体积必须被调整以考虑所添加的氨基酸。下面是调整上述的协议同位素标记</li></ol></li><li>补充转染与小分子<ol><li>准备的使用高压灭菌水2-脱氧-2-氟-1-岩藻糖的100mM储备溶液，并用0.22微米过滤灭菌该溶液。 </li><li>为50毫升的培养，添加125微升的岩藻糖类似物溶液的转染和稀释介质（250μM）。注意:我们忽略了执行任何进一步的音量调整，因为增加取代基在此浓度只占0.25％的总培养体积。它是可能的修改使用聚糖处理的小分子抑制剂的表达。这里，我们描述的条件，包括2-脱氧2-氟-1-岩藻糖，岩藻糖基聚糖的抑制剂。我们发现在岩藻糖基&gt;减少90％的添加岩藻糖类似物在250μM，在转染和稀释介质的浓度。 </li></ol></li></ol>

All authors declare no competing financial interests in this manuscript.

这个协议说明通过HEK293F或S细胞的瞬时转染的蛋白表达。建立的倒钩和Moremen实验室的最佳转染条件采用细胞密度和试剂浓度的临界组合，以实现高效率的转染。实施这项协议时，关键的考虑因素包括:保持在转染前一个稳定的文化（具有一致的文化倍增时间）;活跃生长的细胞的转染细胞存活率大于95％（按稀释细胞至1×10 6个细胞/ ml 24小时在转染之前实现）;在转染细胞密度应在含90％培养?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/53568">High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension</a>. The Authors section was updated. from:
Ganesh P. Subedi1 
Roy W. Johnson2 
Heather A. Moniz2 
Kelley W. Moremen2 
Adam Barb1 
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 
2Complex Carbohydrate Research Center, University of Georgia
to
Ganesh P. Subedi1 
Roy W. Johnson2 
Heather A. Moniz2 
Kelley W. Moremen2 
Adam W. Barb1 
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 
2Complex Carbohydrate Research Center, University of Georgia

An erratum was issued for:&#160;<a href="https://www.jove.com/t/53568">High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension</a>. The Authors section was updated.

Erratum: High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

制备适当折叠和翻译后修饰的人蛋白质的高产量的结构和功能的详细的分析仍然是一个显著挑战。可产生重组蛋白具有天然样的功能和行为的大量表达系统。细菌表达系统，主要是大肠杆菌菌株，代表了最方便和常用的工具，在研究领域，由于这些表达系统的简单性，尽管酵母，植物，昆虫和哺乳动物系统也描述1-4。然而，大多数这些系统是不能的靶蛋白的适当的翻译后修饰。锋芒毕露和Moremen实验室的根本利益，是生产真核蛋白与适当的糖基化。许多人类蛋白质需要适当的糖基化的功能是否正常（见图5）。 真核糖基化机制是广泛的，能够进行修改多种多样，包括天冬酰胺（N）的-和丝氨酸/苏氨酸（O） -连接的复合聚糖6。据估计，人蛋白质的&gt; 50％是N-糖基化的7。聚糖是许多蛋白质​​包括治疗性单克隆抗体，促红细胞生成素，和凝血因子样因子IX的必要成分，仅举几例。虽然多种方法可用来制备适当N-糖基化蛋白和范围从纯合成8-10，以酶法11月14日或恢复从工程改造的重组系统15-20，这并不奇怪，人表达系统迄今被证明是最稳健的方法产生的人类蛋白质。 许多治疗人糖蛋白产生在使用哺乳动物细胞的重组系统。值得注意的系统是中国仓鼠卵巢（CHO），小鼠骨髓瘤（NS0），幼仓鼠KidneY（BHK），人胚肾（HEK-293），并且被用在粘附或悬浮培养生产蛋白质4,21,22人视网膜细胞系。然而，哺乳动物蛋白表达系统需要的稳定细胞株，昂贵的生长培养基和基质辅助转程序23的生成。 哺乳动物细胞的转染与众多试剂，包括磷酸钙24,25，阳离子聚合物（DEAE-葡聚糖，聚凝胺，聚赖氨酸，聚乙酰亚胺（PEI））或带正电荷的阳离子脂质体26-29的帮助来实现。 PEI是聚阳离子，充电，直链或支链的聚合物（25 kDa）的26，其形成稳定的络合物与DNA和被内吞。在核内体的酸化，PEI被认为是膨胀，导致内体和DNA的释放的破裂进入细胞质26,30。 直到最近，在suspensi瞬时转染上培养进行事先的DNA / PEI复合物的形成，随后加入到细胞培养物29。然而，亚龙和同事报道了生产重组蛋白的HEK293细胞在原位形成31,32的DNA / PEI复合物进行了优化的高效的协议。这避免了准备，复杂的灭菌，并进入培养基缓冲液交换。通过包括导致显著的产量增加33表达增强质粒进一步优化。这里是建立在这些进展和广泛适用的方法。表达的条件也可以被改变以影响N-聚糖组合物。 所述HEK293S细胞系，与基因缺失，其停止N-聚糖处理在中间级，导致与均匀N-聚糖的蛋白质组成的2 N-乙酰葡糖胺残基的加五甘露糖残基（曼5的GlcNAc 2）34中的表达， 35。这些细胞缺乏N-乙酰转移酶I（GntI），这是需要的下游N-聚糖处理36,37基因。使用糖基转移酶抑制剂，包括kifunensine，唾液酸类似物和岩藻糖类似物和2-脱氧-2-氟-岩藻糖也有类似的影响和限制N-聚糖处理38-41。 协议报这里使用pGEn2矢量如图1 42,43，PEI辅助瞬时转染到哺乳动物细胞系（HEK293F或HEK293S细胞），并适当地糖基化蛋白的高产率的恢复。这个系统是稳健，可容纳各种因素，包括同位素标记和聚糖工程用于生产重组蛋白的大效价。

<table><tbody><tr><td>Biosafety cabinet&amp;nbsp;</td><td>NuAire, Inc.</td><td>CellGard ES NU-S475-400</td><td>Class II, Type A2 Biological Safety Cabinet</td></tr><tr><td>Incubation shaker</td><td>INFORS HT</td><td></td><td>Multitron Cell&amp;nbsp;</td></tr><tr><td>Medium A: FreeStyle Expression Medium&amp;nbsp;</td><td>Life Technologies</td><td>12338-018</td><td></td></tr><tr><td>Medium B: ExCell 293 Serum-Free Medium&amp;nbsp;</td><td>SIGMA</td><td>14571C&amp;nbsp;</td><td></td></tr><tr><td>125 Erlenmeyer Flask with Vented Cap</td><td>Corning Incorporated/Life Sciences</td><td>431143</td><td></td></tr><tr><td>250 Erlenmeyer Flask with Vented Cap</td><td>Corning Incorporated/Life Sciences</td><td>431144</td><td></td></tr><tr><td>FreeStyle HEK 293F Cells</td><td>Life Technologies</td><td>R790-07</td><td></td></tr><tr><td>1 ml Disposable serological pipette</td><td>Fisher Scietific</td><td>13-676-10B</td><td></td></tr><tr><td>10 ml Disposable serological pipette</td><td>Fisher Scietific</td><td>13-676-10J</td><td></td></tr><tr><td>25 ml Disposable serological pipette</td><td>Fisher Scietific</td><td>13-676-10K</td><td></td></tr><tr><td>Pipettor (Pipet-Aid XP)</td><td>Drummond Scientific</td><td>161263</td><td></td></tr><tr><td>Trypan Blue Solution&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>SV30084.01</td><td></td></tr><tr><td>Counting slides&amp;nbsp;</td><td>Bio-Rad</td><td>145-0011</td><td></td></tr><tr><td>TC20 Automated Cell Counter&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>145-0102</td><td></td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences Inc.</td><td>23966</td><td>Prepare stock solution at a concentration of 1 mg/ml in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mM NaCl (pH 7.5). Dissolve PEI completely; sterilize through 0.22 &amp;mu;M syringe filter and store at -20 &amp;deg;C.</td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>EMD Chemicals Inc.</td><td>7365-45-9</td><td></td></tr><tr><td>25 mm Syringe Filter, 0.22 &amp;mu;M</td><td>Fisher Scietific</td><td>09-719A</td><td></td></tr><tr><td>Trisaminomethane (Tris base)</td><td>Fisher Scietific</td><td>BP152-1</td><td></td></tr><tr><td>XL1-Blue</td><td>Stratagene</td><td>222249</td><td></td></tr><tr><td>Trypton</td><td>Fisher Scietific</td><td>BP1421-2</td><td></td></tr><tr><td>Yeast extract</td><td>Fluka Analytical&amp;nbsp;</td><td>92144</td><td></td></tr><tr><td>Sodium chloride</td><td>BDH chemicals</td><td>BDH8014</td><td></td></tr><tr><td>Plasmid Purification Kit</td><td>QIAGEN</td><td>12145</td><td></td></tr><tr><td>Valproic (VPA)</td><td>SIGMA</td><td>P4543</td><td>Prepare stock solution of 220 mM&amp;nbsp; in water, sterilize by passage through a sterile 0.22 mm filter and store at -20 &amp;deg;C</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific&amp;nbsp;</td><td>EW-17707-65</td><td></td></tr><tr><td>Protein A-Sepharose column</td><td>SIGMA</td><td>P9424</td><td></td></tr><tr><td>Ni-NTA superflow</td><td>QIAGEN</td><td>30430</td><td></td></tr><tr><td>3-(N-morpholino)propanesulfonic acid (MOPS)</td><td>Fisher Scietific</td><td>BP308-500</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scietific</td><td>BP381-500</td><td></td></tr><tr><td>10 kDa molecular weight cut-off Amicon&amp;reg; Ultra centrifugal filters&amp;nbsp;</td><td>Millipore</td><td>UFC901096</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>ALDRICH</td><td>L3771</td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>ALDRICH</td><td>M6250</td><td></td></tr><tr><td>Glycerol</td><td>SIGMA</td><td>G5516</td><td></td></tr><tr><td>Precision Plus Protein All Blue Standards</td><td>Bio-Rad</td><td>161-0373</td><td></td></tr><tr><td>Acetic Acid, Glacial&amp;nbsp;</td><td>Fisher Scietific</td><td>64-19-7</td><td></td></tr><tr><td>coomassie brilliant blue&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>161-0406</td><td></td></tr><tr><td>MALDI-TOFMS Voyager-DE PRO&amp;nbsp;</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;N labeled L-Tyrosine</td><td>ALDRICH</td><td>332151</td><td></td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;N labeled L-Lysine</td><td>ALDRICH</td><td>592900</td><td></td></tr><tr><td>Unlabeled L-Phenylalanine</td><td>SIGMA-ALDRICH</td><td>P2126</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C6-Glucose</td><td>ALDRICH</td><td>389374</td><td></td></tr><tr><td>2-deoxy-2-fluoro-l-fucose&amp;nbsp;</td><td>SANTA CRUZ Biotechnology</td><td>sc-283123</td><td></td></tr></tbody></table>

high yield expression of recombinant human proteins with the transient transfection of hek293 cells in suspension

产生重组蛋白的复杂的翻译后修饰的技术代表的结构和功能研究的主要挑战。迅速建立和高回收率从瞬时转染的哺乳动物细胞系解决了这一障碍并为表达该通过ER和高尔基体介导的分泌途径自然引导蛋白质的一种有效手段。这里是使用人类的HEK293F和转染的设计的高蛋白质产量的哺乳动物表达载体HEK293S细胞系表达一个协议。这个系统的适用性用与之间95-120毫克每升培养物中回收纯化的蛋白的产量表达三个具有代表性的糖蛋白是证明。这些蛋白质是人类和FcγRIIIa的大鼠α2-6唾液酸转移酶，ST6GalI，既表达具有N-末端GFP融合，以及未经修饰的人免疫​​球蛋白G1的Fc。这个强大的系统UTIlizes一个无血清培养基是能适应于同位素富集蛋白质和碳水化合物用于使用质谱和核磁共振谱结构研究的表达。此外，所述N-聚糖的组合物可以通过添加一个小分子，以防止某些聚糖修饰的方式，并没有降低产量来调节。

高水平表达和纯度这种优化的表达系统产生的高收率糖基化蛋白。典型的图案显示在IgG1的-Fc的（图1）的表达。在这种情况下，第0天是转染一天随后1天（稀释）和随后的培养天至第5天的蛋白质表达使用在粗培养基中的可溶性表达馏分进行分析。观察到在第1天的一个非常小的量的蛋白质表达作为培养等分试样取出?...

Watch this Scientific Journal Video about High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension at JoVE.com

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

high-yield-expression-recombinant-human-proteins-with-transient

Research

JoVE Journal

Biology

29.9K 次观看.  Iowa State University. Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

视频: High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

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.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

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 ...

科研

生物化学

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

遗传学

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

使用瞬时表达系统高效、可扩展地生产哺乳动物细胞中的全长人类亨廷顿蛋白变异

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

生物学

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. ...

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 ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

人类颗粒酶在哺乳动物细胞中高产量和经济高效表达系统

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

使用 HEK293 悬浮培养物简化高效的 AAV 生产

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

作者聚焦：通过 HEK293 悬浮细胞培养推进高产量 AAV 载体的基因治疗

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

使用 HEK293 悬浮细胞生产高产腺相关载体批次

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

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此视频中的章节

实验室规模的生产和治疗性抗体的纯化

通过诱导瞬时转染可控离子通道表达

使用瞬时表达系统高效、可扩展地生产哺乳动物细胞中的全长人类亨廷顿蛋白变异

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

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

人类颗粒酶在哺乳动物细胞中高产量和经济高效表达系统

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

使用 HEK293 悬浮细胞生产高产腺相关载体批次

使用 HEK293 悬浮细胞生产高产腺相关载体批次