我们要感谢小雯杨博士（PROTEX克隆服务）准备用于这项工作，莱斯特核心生物技术服务大学的表达结构。这项工作是由BBSRC助学金支持，威康信托计划及高级调查员补助WT085408＆WT100237和BBSRC授予RM31G0224。

注:该协议是适用于任何规模的表达，因此，试剂的体积和数量应当按比例进行缩放。合适的哺乳动物表达载体必须用于本协议。在这里，我们使用的改性pcDNA 3.1表达载体便利地允许根据在克隆（ 图1）中使用的限制性酶的选择的亲和标签的包含或排除。以下协议描述了一个大规模的转染。 1，大型文化/转1升转瓶生长并根据标准方法保持HEK293F悬浮适应细胞。通常的30至100毫升的起始培养生长于250ml锥形细胞培养瓶中。 <ol><li>种子细胞以0.5×10 6个细胞/ ml至300 ml，在每1 ​​升转瓶的终体积。 注:摇瓶最少150毫升，以容纳最多300毫升悬浮培养。对于较大规模的转染使用多个瓶子。 </li><li>孵育24小时，在轨道摇床培养箱中，在37℃，120rpm下，和5％的CO 2，直至细胞达到1.0×10 6个细胞/ ml的密度（细胞应划分约每24小时）。 </li><li>吸取总共300微克过滤灭菌的DNA（参见补充方法）到30的PBS中的溶液，并涡旋混合3秒。 注:使用共有1微克每百万转染细胞的DNA。如果两个或更多个质粒进行共转染减少每个的量，使得DNA的细胞的比率保持恒定。 </li><li>补充加入1.2ml 0.5毫克/毫升过滤灭菌的PEI与PBS / DNA溶液和涡旋混合3秒。 </li><li>孵育混合物在室温下20分钟。 </li><li>加入该DNA / PEI混合的细胞-这应当是在1×10 6个细胞/ ml（步骤1.3）的密度。 </li><li>以下共转染，培养的细胞在轨道摇床培养箱中进一步的48小时，在37℃，120rpm下，和5％的CO 2。 </li><li>收获细胞内蛋白质通过在3000×g离心5分钟，离心细胞，将片状沉淀储存于-80℃。 注意:可替换地，可使用标准（非CO 2）的振荡培养箱中，如果培养上述的气氛置换为5％的CO 2和瓶盖密封。大气中会需要在每个通道（通常每2天）进行更换。 </li></ol>从全细胞提取2蛋白复合物的分离纯化这个协议被优化用于使用标记标签蛋白核配合物的纯化。 <ol><li>解冻的细胞沉淀进入〜40预冷的裂解缓冲液（100mM的醋酸钾，的50mM Tris pH值7.5，5％甘油，0.3％的Triton X-100，蛋白酶抑制剂），每升培养物。 </li><li>重悬沉淀吹打向上和向下几次（避免起泡，不要旋涡）。 </lI&gt; <li>彻底重新暂停使用玻璃匀浆细胞。超声清洗3次（15秒时，15秒关闭）。离心30000×g离心25分钟，在4℃，并保留上清液。 </li><li>平衡1.25抗旗洗涤三次，用树脂的平衡缓冲液（100mM乙酸钾，50毫摩尔Tris pH7.5）中填充每升培养物的琼脂糖树脂的溶液中。 </li><li>培养从步骤2.3中一个或多个的50ml离心管中的亲和树脂的全细胞提取物，并在4℃下轻轻地旋转样品为30-120分钟。 </li><li>离心机在3000 XG为1分钟，在4℃下，弃上清。 </li><li>用45ml预冷的缓冲液1中洗涤树脂（100mM的醋酸钾，的50mM Tris pH值7.5，5％甘油，0.3％的Triton X-100）。离心机在3000×g离心1分钟，弃上清。 </li><li>重复步骤2.7用高盐缓冲液（300mM的醋酸钾，的50mM Tris pH值7.5，5％甘油）中，然后用低盐缓冲液（50mM醋酸钾，50毫摩尔Tris pH 7的0.5，5％甘油）和TEV裂解缓冲液（50mM醋酸钾，的50mM Tris pH值7.5，0.5 mM的TCEP）。 注:确保每次洗涤是短暂即足以充分重悬浮树脂和不再。 </li><li>收集树脂的10微升样品，并稀释至1体积的分析2×蛋白加样缓冲液（结合蛋白对照）。不使用的还原剂，以避免从树脂释放的抗体。 </li><li>重悬的树脂注入8-10毫升预冷TEV裂解缓冲液和转移至15ml离心管中。 </li><li>添加〜40微克（1毫克/毫升的股票）每升原生态文化的TEV蛋白酶和移液器向上和向下几次拌匀。 </li><li>更换管的氛围，100％ 氮气 ，以防止蛋白质氧化。 </li><li>轻轻转动在o / n 4℃。 </li><li>离心机在3000×g离心10分钟。将上清液转移到一个超离心过滤器具有合适的分子量截断和集中下降到500微升。 </li><li>收集浓缩的蛋白质的10微升样品，并稀释至1倍体积的2×蛋白加样缓冲液进行分析。 （TEV洗脱液控制）。 </li><li>收集该树脂的10微升样品，并稀释成1倍体积的2×蛋白加样缓冲液进行分析。不使用的还原剂，以避免从树脂释放出大量的抗体。 （后TEV树脂控制）。 </li><li>平衡用凝胶过滤缓冲液（50mM醋酸钾，的50mM Tris pH值7.5，0.5 mM的TCEP）的大小排阻层析柱。 </li><li>通过0.22微米的过滤器过滤的蛋白质</li><li>加载样品进入柱，并收集级分。 </li><li>运行样品从步骤2.9，2.15和2.16和从上的SDS-PAGE和考马斯亮蓝染色分析步骤2.19的凝胶过滤级分。 注:最好是从步骤2.9，2.15和2.16之前的凝胶过滤运行的SDS-PAGE样品以确认靶蛋白的表达。</li>
</ol>

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The authors declare that they have no competing financial interests.

我们已经开发出一种简单和成本有效的方法（PEI成本比市售的亲脂转染试剂少得多）用于表达和纯化大量的重组蛋白，并从哺乳动物细胞多亚基复合物。最佳的转染和表达效率可如高纯度质粒DNA（二百八分之二百六十〇在1.8和2.0）中的协议部分中所述的组合使用的PEI到达。细胞必须在无血清和无抗生素的培养基中培养，因此，无菌技术要求严格的传代和转染细胞，以避免昂贵的和费时的感染。细胞生存力应为90％或更高和文化不应该发展到密度大于2.5×10 6个细胞/毫升紧接在转染前，因为这样做会降低蛋白质产率。对于一个成功的转染，文化应该只包含单个或分裂的细胞。集群可以通过活力被打破了项涡旋20到30秒（ 图5）。在共转染中使用的每种质粒的比例可以由用户根据复杂的被研究的化学计量可以变化。表达效率可以针对感兴趣的蛋白进行优化，以使适当的表达时，可以建立。纯化应执行保持在所有时间该蛋白质样品冷以减少不需要的蛋白水解降解的风险。的标记和纯化缓冲器的选择是至关重要的，因为它们可能干扰结构元件或某些蛋白质的活性位点，通常导致降低的溶解度和/或酶活性的损失。小规模的转染是用于大型的蛋白质复合物的纯化测试不同的构建体是特别有用的。 今天，大量不同的替代方法是可用的重组真核蛋白质（ 表2）的表达。例如，Baculovi在昆虫细胞中表达RUS被广泛使用，由于其高转染效率和其相比于其它病毒物种6缺乏细胞毒性的。然而，使病毒的方法是费时和它的不稳定性不允许病毒可以长时间保存。另一方面，酵母表达系统提供了在发酵罐中培养生长的细胞以非常高的密度的可能性，从 ​​而导致高蛋白质产率7。但它们仍然缺乏充分各种正确折叠和与其蛋白伴侣相互作用所需的真核生物蛋白质的翻译后修饰。 HDAC3，例如，被表达，但不折叠在大肠杆菌大肠杆菌 。然而，当在293F细胞中表达，我们能够净化在复杂的活性酶，其共抑制（SMRT）和肌醇四磷酸（IP4）8，它没有在原核细胞中发现的分子。这个方法也使我们以纯化并解决晶体STructure的HDAC1与MTA1在NURD复杂的，这是同样由IP4 9活化的相互作用。理想情况下，我们总是要表示在其自然的，生理环境的真核蛋白质。用人的HEK 293-EBNA1细胞在生物反应器10-12哺乳动物表达系统已经描述和产生非常高水平的蛋白质，但这些可能是复杂的使用。 我们的方法是一种简单的和可访问的替代方法在细菌，酵母和生物反应器系统中的表达。表达的方法是快速的，并且不需要使用昂贵的设备，尤其是当该滚瓶或烧瓶中的气氛替换为5％的CO 2和标准摇动孵化器中使用。我们使用这些协议来共转多种质粒和纯化物与最多五个蛋白质随着培养的&gt; 1 mg / L的产率。有趣的是，该系统有助于稳定性能良好配合物的鉴定。例如，前HDAC1和Sin3A的二元配合物的pression，得到蛋白质的有限产量，但除了SDS3的导致了5倍以上的量，因此可指导选择合适的构建体和稳定的复合物结晶的结构生物学家。

分子细胞生物学的迅速发展和提高药物在医学上需要不断创造了一个需要进行结构生物学家看着越来越复杂的蛋白质结构。这些通常可能需要特定的翻译后修饰，分子伴侣和辅因子，以支持他们的精心折叠和酶活性。虽然目前在用细菌表达系统已获得的蛋白质数据库结构的绝大部分，原核生物无法执行大量的这些修改和缺乏许多基本的真核合作的因素。这可能是一个问题对于大型多亚基复合物，是由小信号分子，以及用于核，细胞表面和需要复杂的折叠机器的分泌蛋白活性的研究。一些E的大肠杆菌菌株已被工程化以克服这些局限性1。近年来，然而，使用米ammalian表达系统一直在增加，因为它们可靠地生产的真核蛋白质，是有问题的，否则在其他系统2来表示。今天它是可能的，通过各种范围从病毒转导到化学介导的转染技术，以获得稳定的和短暂的哺乳动物表达细胞系，物理的基因转移，如电穿孔和直接注射3。虽然所有这些方法都具有其自己的优点和缺点，仅其中的一些适合的结构研究是不是太昂贵和/或耗时的。 在这里，我们描述了用于在悬浮液中生长的哺乳动物细胞表达的蛋白质复合物的结构生物学非常简单，快速，廉价而高效的方法。该方法采用瞬时共转染人胚肾（HEK）细胞系（ 如自由式HEK 293F细胞）。这些细胞已被来自HEK 293 CELL线，适合于悬浮培养物生长，达到了使用无血清培养基的高密度（如自由式293表达培养基）。然后将细胞瞬时使用的聚乙烯亚胺的支链版本（PEI），已报道的功能对于大范围的哺乳动物细胞4通过形成DNA / PEI复合物进入宿主细胞通过内吞作用5廉价的聚合物试剂进行转染。此方法是同时适合小规模（30毫升）和大规模（最多300毫升）中的实验，可以产生高水平的纯化蛋白质复合物。它是研究需要复杂的折叠机器，辅因子或不能由细菌，酵母和昆虫细胞中进行特定的翻译后修饰的蛋白是特别有用的。 在这个协议中，我们提出从Sin3A转录抑制复合物的三个蛋白中央支架的表达和纯化。这是由组蛋白的去乙酰化酶1（HDAC1）的缺陷沉默3（SDS3）抑制器和开关独立3（Sin3A）。纯化的复合物被用于高通量结晶试验。

<table><tbody><tr><td>FreeStyle HEK 293F cells&amp;nbsp;</td><td>LifeTechnologies</td><td>R790-07</td><td></td></tr><tr><td>FreeStyle 293 Expression Medium</td><td>LifeTechnologies</td><td>12338-018</td><td></td></tr><tr><td>Anti-FLAG M2 Affinity Gel</td><td>SIGMA</td><td>A220</td><td></td></tr><tr><td>250 ml Erlenmeyer Flask with Vented Cap</td><td>Corning</td><td>431144</td><td></td></tr><tr><td>Roller bottles with vented caps</td><td>Corning</td><td>01836-02</td><td></td></tr><tr><td>Polyethylenimine, 25 kDa, branched</td><td>Sigma-Aldrich</td><td>408727</td><td>Make 0.5 mg/ml stocks in H&lt;sub&gt;2&lt;/sub&gt;O, adjust pH to 7.0 with dilute HCl and store at -20 - 4&amp;ordm;C</td></tr><tr><td>Mammalian expression vector</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>0.22 &amp;micro;m Centrifugal Filter Units</td><td>Amicon</td><td>UFC30GV00</td><td></td></tr><tr><td>15 ml Ultra centrifugal filters (10 kDa cut-off)</td><td>Amicon</td><td>UFC901008</td><td></td></tr><tr><td>Superdex&amp;nbsp; 10/300 GL</td><td>GE Healthcare Life Sciences</td><td>17-5175-01</td><td></td></tr></tbody></table>

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.

在这里，我们展示一个2升（8×250毫升培养）瞬时共转染和净化HDAC1，SDS3，Sin3A三元复合物。通过Sin3A的HDAC-相互作用结构域（HID）与Sin3A HDAC1和SDS3交互。典型的纯化产量可达1毫克每升培养物络合物。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig1highres.jpg" width="500" /> 图1原理图的改性pcDNA 3.1表达载体的构建。的质粒用EcoRV消化和EcoRI消化，以包括关于Sin3A的氨基末端亲和标签和TEV切割位点。克隆HDAC1和SDS3的未标记的版本中，该载体用KpnI消化和EcoRI消化。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig2highres.jpg" width="500" /> 图2 SDS-PAGë表示纯化的第一步，"结合蛋白"行结合到亲和树脂的复合物。以下TEV消化，上Sin3A-HID标记本被裂解掉，复合物从树脂中洗脱所示凝胶的第二和第三通道。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig3highres.jpg" width="500" /> 图3的色谱的蛋白质复合体的纯化使用尺寸排阻色谱法。注意，纯配合物中洗脱的空隙体积，因为它形成了一个二聚物在溶液中。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig4highres.jpg" width="500" /> 图4的SDS-PAGE显示在图3中示出的凝胶过滤的级分。 图5台盼蓝染色的HEK 293F细胞在2.3×10 6个细胞/ ml准备待转染。细胞应仅存在作为单个或分裂的细胞，而大型集群可以被打破，剧烈涡旋振荡约25秒。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> 问题 </td><td> 可能的原因 </td><td> 动作 </td></tr><tr><td rowspan="4">低蛋白产量。 </td><td>细胞数量低转。 </td><td>确定细胞密度是约1.0×10 6之前加入转染反应混合物至培养。 </td></tr><tr><td>细胞可能已传代次数太多。 </td><td>使用新鲜的股价细胞后约90通年龄。 </td></tr><tr><td> DNA可以被降解或具有高的量的杂质。 </td><td>确保所使用的质粒DNA具有260/280比1.8和2.0之间。在琼脂糖凝胶上运行的DNA是可取的，以评估其质量。 </td></tr><tr><td>蛋白（S）被表达并不稳定，或溶于足够了。 </td><td>测试小规模向上调整的转染前不同的结构。确保标签不与感兴趣的蛋白（S）的结构干扰。 </td></tr><tr><td>细胞看起来浑浊并有一个不寻常的颜色和/或气味。 </td><td>细胞被感染的细菌或酵母。 </td><td>良好的无菌技术，必须使用在任何时候。熏蒸的层流净化罩和UV消毒的细胞培养室中的情况下感染有助于含有问题。 </td></tr><tr><td rowspan="2">细胞具有较低的生存能力。 </td><td>错误的pH值在媒体上。 </td><td>确保文化是在5-8％的CO 2增长在任何时候。 </td></tr><tr><td>细胞被培养至密度超过3.0×10 6个细胞/ ml。 </td><td>细胞不应该长大密度高于2.5×10 6个细胞/毫升，立即转染之前，从不高于3.0×10 6个细胞/ ml。 </td></tr><tr><td rowspan="2">亲和纯化没有工作</td><td>蛋白（次）不被表达。 </td><td>见上面。 </td></tr><tr><td>纯化条件可能是错误的。 </td><td>调整缓冲条件（ 如高盐，低盐，pH值。） </td></tr></tbody></table> 表1故障排除。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> 系统 </td><td> 优点 </td><td> 缺点 </td></tr><tr><td> 大肠杆菌 </td><td><ul><li>快表达系统</li><li>高蛋白质产量</li><li>便宜</li><li>适合经济实惠的生产同位素标记的蛋白质</li></ul></td><td><ul><li>缺少的重要伴侣分子可能需要对蛋白质折叠</li><li>缺乏许多翻译后修饰</li><li>可能缺乏的辅助因子，这对于蛋白的功能和/或复杂的装配很重要的。 </li></ul></td></tr><tr><td> 毕赤酵母 </td><td><ul><li>可以执行大部分的翻译后修饰</li><li>便宜</li></ul></td><td><ul><li>缺乏一些翻译后修饰</li><li>可能缺乏的辅助因子，这对于蛋白的功能和/或复杂的装配很重要的。 </li></ul></td></tr><tr><td> 杆状病毒表达在昆虫细胞中 </td><td><ul><li>可以执行所有的翻译后修饰&lt;/ LI&gt; <li>杆状病毒比其他病毒株的细胞毒性更小</li></ul></td><td><ul><li>制备病毒的转导是耗时</li><li>该病毒是不稳定的，不能保存很长一段时间</li><li>昆虫细胞可能缺乏的辅助因子，这对于蛋白的功能和/或复杂的装配很重要的。 </li></ul></td></tr><tr><td> 生物反应器与哺乳动物系统 </td><td><ul><li>可以执行所有的翻译后修饰</li><li>可以提供所需的蛋白质功能和复杂的装配所有共同因素</li><li>文化能细胞在非常高的密度</li><li>可以表达需要复杂的折叠机械所有哺乳动物的蛋白质</li><li>非常高的蛋白产量</li></ul></td><td><ul><li>需要专门的工作人员使用的生物反应器。 </li><li>昂贵的设备是必需的。 </li><li>优化表达条件S可以是耗时的。 </li></ul></td></tr><tr><td> HEK 293F悬浮细胞 </td><td><ul><li>可以执行所有的翻译后修饰</li><li>可以提供所需的蛋白质功能和复杂的装配所有共同因素</li><li>可以表达需要复杂的折叠机械所有哺乳动物的蛋白质</li><li>快速，直观的转协议</li><li>相对便宜的相比的生物反应器和/或其它市售的转染试剂。 </li></ul></td><td><ul><li>虽然价格实惠，不便宜，因为在细菌中表达</li></ul></td></tr></tbody></table> 表2的优势和主要表达系统的优缺点。

Watch this Scientific Journal Video about Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach at JoVE.com

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

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 ...

recombinant-protein-expression-for-structural-biology-hek-293f

Research

JoVE Journal

Biology

53.7K 次观看.  University of Leicester. 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.

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

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

科研

遗传学

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

生物工程

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

生物学

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) ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

免疫与感染

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. ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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 ...

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

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

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.