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>

<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 border="0" cellpadding="0" cellspacing="0" width="865">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Reagent/ Equipment</td>
			<td style="width:140px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FreeStyle HEK 293F cells&#160;</td>
			<td style="width:140px;">LifeTechnologies</td>
			<td>R790-07</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:315px;">FreeStyle 293 Expression Medium</td>
			<td style="width:140px;">LifeTechnologies</td>
			<td style="width:119px;">12338-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Anti-FLAG M2 Affinity Gel</td>
			<td style="width:140px;">SIGMA</td>
			<td style="width:119px;">A220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">250 ml Erlenmeyer Flask with Vented Cap</td>
			<td style="width:140px;">Corning</td>
			<td style="width:119px;">431144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Roller bottles with vented caps</td>
			<td style="width:140px;">Corning</td>
			<td style="width:119px;">01836-02</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Polyethylinamine, 25 kDa, branched</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">408727</td>
			<td style="width:292px;">Make 0.5 mg/ml stocks in H2O, adjust pH to 7.0 with dilute HCl and store at -20 - 4&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mammalian expression vector</td>
			<td style="width:140px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">0.22 &#181;m Centrifugal Filter Units</td>
			<td style="width:140px;">Amicon</td>
			<td style="width:119px;">UFC30GV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">15 ml Ultra centrifugal filters (10 kDa cut-off)</td>
			<td style="width:140px;">Amicon</td>
			<td style="width:119px;">UFC901008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Superdex&#160; 10/300 GL</td>
			<td style="width:140px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-5175-01</td>
			<td></td>
		</tr>
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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

53.3K 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

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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

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

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

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

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

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) ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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 ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

细菌，昆虫细胞和植物系统：重组蛋白表达在不同的生物工厂的比较分析

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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.

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 ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...