该项目由 NIH 赠款 EY025290、GM127652 和罗切斯特大学开办资金资助。

1. 产生 BacMam 表达杆状
<ol><li>将所需哺乳动物 bestrophin 蛋白的编码序列插入 pEG BacMam 向量18中, 用烟草蚀刻病毒 (TEV) 蛋白酶识别序列, 然后在蛋白质的 C 末端 GFP-10x 他的标记。</li>
	<li>瞬时染表达质粒成胶粘剂 HEK293 细胞19,20,21,22,23。用10X 或20X 放大倍数、488 nm 激发源和 510 nm 发射过滤器 (图 1A) 检查荧光显微镜下的 GFP 融合蛋白的表达式。 注: 以聚合物为基础的转染通常以1微克的质粒 DNA 为 HEK293 细胞的35毫米皿进行。</li>
	<li>在 Sf9 昆虫细胞中产生杆状, 如前所述16、17。 注: 通过 3 (P3) 病毒, 将用于感染 HEK293-F 细胞蛋白质生产, 可以储存在4摄氏度1月。</li>
	<li>通过斑块形成试验确定 P3 病毒的效价 (传染性单位)16,17。 注: 由于感兴趣的蛋白质是用 GFP 标记的, 快速检查感染单位的方法是通过病毒的序列稀释感染 Sf9 细胞, 并计算48小时后荧光细胞的数量。</li>
</ol>2. 蛋白质表达
<ol><li>保持悬浮培养中的 HEK293-F 细胞与 HEK293-F 表达培养基在37°c 湿润的孵化器与 8% CO2。使用一次性培养瓶的 2-2. 5 倍大于文化体积和旋转的文化在轨道平台上, 在 135 rpm。 注意: 建议在通道5和30之间对细胞进行感染, 并在细胞培养罩下执行这些操作。</li>
	<li>24小时前病毒感染, 增加 15 ul 的台盼蓝到 15 ul 整除细胞培养, 并检查细胞密度和可行性使用一个 hemocytometer 显微镜下。稀释细胞到 0.6 x 106细胞/毫升在 2 x 2 L 瓶每, 与500毫升中等预热在37°c。 注意: 死细胞被台盼蓝染成蓝色。所需的细胞生存能力为 > 95%。</li>
	<li>在感染日, 检查细胞密度和生存能力 (步骤 2.2)。 注意: 高细胞活力 > 95% 对于有效的感染和蛋白质表达是必不可少的。预期细胞密度为 ~ 1.0 x 106细胞/毫升 (从 500 x 0.610细胞/毫升细胞培养的 2 x 6 毫升一夜之间生长)。预期的单元格总数是 ~ 1.0 x 109。</li>
	<li>添加 P3 杆状的细胞在多种感染 (语言) 的5。要确定接种病毒的数量, 请使用以下公式: <img alt="Equation" src="/files/ftp_upload/57832/57832eq1.jpg">
</li>
	<li>摇晃的轨道平台上的细胞在 135 rpm 在一个湿润的37°c 孵化器与 8% CO2为 24 h。</li>
	<li>在细胞培养中加入10毫米丁酸钠, 继续孵化48小时。 注: 对于某些蛋白质, 在丁酸钠添加后, 将细胞培养温度降低到30摄氏度可以改善表达和稳定性。</li>
	<li>在具有10X 或20X 放大倍数的荧光显微镜下, 488 毫微米激发源和 510 nm 发射过滤器, 检查绿色细胞的百分比和亮度, 直接代表蛋白质 (bestrophin-GFP-10xHis) 产量 (图 1B)。 注: 绿色细胞的百分比计算为: 100 x (绿色细胞/总细胞)。显微镜下强绿色荧光表示良好的蛋白表达水平。</li>
	<li>通过离心在 1000 x g 在4°c 收获细胞20分钟. 取出上清液, 再用磷酸盐缓冲生理盐水 (PBS) 将细胞颗粒重新悬浮至80毫升的最终体积。分裂细胞悬浮成 2 x 50 毫升圆锥管和离心机在 1000 x g 在4°c 为20分钟. 将细胞小球储存在-80 摄氏度。</li>
</ol>3. 蛋白质纯化
<ol><li>在室温搅拌水浴中, 将含有细胞小球的锥形管孵化10-15 分钟, 使其解冻。在 2x (w/v) 缓冲区容量中重新挂起单元格 (表 1) (例如, 20 毫升的缓冲器中的10克细胞颗粒) 补充了蛋白酶抑制剂 (抑肽酶、Leupeptin、Pepstatin A 和 phenylmethylsulfonyl 氟), 其 0.1-1.0 毫米。吸管广泛的向上和向下, 以获得一个均匀的细胞悬浮。</li>
	<li>用高压均质机溶解细胞。通过 7-10 MPa 的均质机进行细胞悬浮, 共3-4 次, 达到完全均匀化。把细胞裂解液放在冰上2-3 分钟。</li>
	<li>添加洗涤剂 [例如, 2% 瓦特/v 溶胶级的 n-月桂-Maltopyranoside ()) 的细胞裂解。在20摄氏度孵育1小时以提取膜蛋白。 注: 洗涤剂的类型需要为每个蛋白质目标18,24进行优化。</li>
	<li>离心机在 15万 x g 在 ultracentrifuge 在4°c 为 1 h。 注意: 以下所有步骤都在4摄氏度执行。</li>
	<li>收集清楚的细胞裂解液, 并通过一个5毫升他的陷阱镍2 +NTA 亲和柱预平衡与缓冲 a (表 1)。 注: 离心后, 透明细胞裂解液将夹在底部的颗粒和顶部的混浊层之间。使用10毫升转移吸管仔细收集清楚的裂解, 然后切换到1毫升吸管的最后几毫升裂解。避免颗粒, 可堵塞镍2 +柱;然而, 一小部分的顶层是罚款或可以过滤出与1.5 微米的过滤器。</li>
	<li>用25毫升的缓冲器 B 和40毫升的缓冲 C (分别为表 2 和 3) 冲洗柱。 注意: 这是一个休息的好地方, 因为总纯化可能需要多达12小时, 蛋白质可以保持稳定, 而附加到专栏一夜之间。</li>
	<li>将该柱连接到快速蛋白液相色谱 (FPLC) 系统, 并洗脱13毫升的缓冲 D (表 3) 与分馏的蛋白质。根据紫外吸光度读数, 收集富含蛋白质的分数。 注: FPLC 条件如下: 1.0 毫升分数大小, 前柱压力报警设置为0.3 兆帕, 流量0.5 毫升/分钟。</li>
	<li>测定 microvolume 分光光度计中洗脱蛋白产品浓度。读280纳米的吸光度。</li>
	<li>选要去除 GFP-10xHis 标签, 添加 TEV 蛋白酶在1:1 质量比和孵化在4°c 30 分钟。 注: 该标签可能会或不影响纯化通道的功能或结构。计算从 3.7-3.8 步骤中收集到的蛋白质的体积和浓度的 TEV 量。例如, 如果10毫升的洗脱产品是收集和测量0.1 毫克/毫升, 总蛋白质量是 10 x 0.1 = 1 毫克, 和1毫克的 TEV 将用于消化。</li>
	<li>将蛋白质与15毫升离心 (50 或 100 kDa) 过滤单元, 通过旋转在 4000 x g 在4°c 到最后容量 400-500 ul (为2毫升样品装载循环在 FPLC)。 注意: 离心时间的变化取决于蛋白质的浓度和大小。为了避免过度集中, 这可能导致蛋白质沉淀, 首先旋转10分钟, 然后观察剩余的体积, 并估计随后的旋转 (如2-5 分钟) 的时间, 以获得最终的体积。吸管之间的旋转, 以分散的蛋白质, 这是拉到底部的过滤器。</li>
	<li>将浓缩蛋白转移到新的1.5 毫升管中, 从浓缩产物中除去任何沉淀或气泡。旋转在 > 1.2万 x g 5-10 分钟在4°c 和收集上清在一个新的1.5 毫升管。</li>
	<li>用1毫升注射器和一个圆尖针在 FPLC 系统上装载最终产品, 尺寸排除色谱与缓冲器 E (表 5) 预平衡。 注: FPLC 条件如下: 0.5 毫升分数大小, 前柱压力报警设置为2.50 兆帕, 流量设置为30毫升的缓冲 E (表 5), 流量设置为0.5 毫升/分钟。</li>
	<li>注意, 表现良好的蛋白质作为一个单一的峰值运行 (图 2A), 并收集与该峰值对应的蛋白质分数 (图 2A)。</li>
	<li>浓缩蛋白质与4毫升或0.5 毫升离心过滤单元 (与步骤3.10 相同的分子量切断) 和旋转在 4000 x g 在4°c 到最后浓度5-10 微克/ul。使用分光光度计检查最终产品浓度 (步骤 3.8)。 注: 离心时间不同, 因为最终的产品体积可以不同的纯化试验。通常, 离心需要10-20 分钟。建议吸管之间的旋转, 以分散的蛋白质, 这是拉到底部的过滤器。</li>
	<li>将浓缩蛋白转移到新的1.5 毫升管中。用离心在 > 1.2万 x g 处去除任何沉淀, 5-10 分钟4摄氏度。将上清液转移到新的1.5 毫升管。使 10 ul 整除数存储在-80 摄氏度, 并保存一个 2-5 ul 小整除运行在4-15% 梯度 SDS 页凝胶在9伏/厘米 (图 2B)。</li>
	<li>用质谱法确定纯化蛋白的鉴定8。 注: 对纯化蛋白的功能和结构进行体外分析可以通过平面脂质双层和 x-射线晶体学分别进行5、8。</li>
</ol>

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<li>Sun, W., Zheng, W., Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+discovery+and+development+for+rare+genetic+disorders%5BTitle%5D)+AND+%22American+Journal+of+Medical+Genetics+Part+A%22%5BJournal%5D)">Drug discovery and development for rare genetic disorders.</a> American Journal of Medical Genetics Part A. 173 (9), 2307-2322 (2017).</li>
</ol>

作者没有什么可透露的。

该协议描述了一种有用的管道, 用于表达和纯化哺乳动物 bestrophin 离子通道, 用于未来的体外分析。虽然 FPLC 设备是需要的大小排除色谱, 注射器泵是足够的所有步骤的亲和层析, 包括捆绑, 洗涤和洗脱。当使用注射器泵来推动溶液 (在注射器中) 通过一列, 这是必不可少的垫弹簧一侧, 以避免推动气泡进入柱。如果蛋白质的纯度在亲和层析后已经足够的后续实验, 大小排除色谱可以省略, 以便 FP...

Bestrophins 是一个通过从细菌到人类的不同物种而保存的离子通道的家族1。在人类中, BEST1基因, 位于染色体 11q12.3, 编码的膜蛋白 Bestrophin-1 (BEST1), 主要表现在基底膜的视网膜色素上皮 (RPE) 细胞的眼睛2,3 ,4。由585种氨基酸组成, 其中第一 ~ 350 在物种间高度保守, 含有其跨膜区域, BEST1 在人类1、5、6中起 CaCC 作用。此外, 鸡和肺炎克雷伯杆菌的 BEST1 同系物的作用为 homopentamers7,8, 表明整个进化过程中的保护水平很高。
在人类中, BEST1基因的200多个突变已经临床上与一组称为 bestrophinopathies1,9的视网膜变性疾病有关。报告了五项具体 bestrophinopathies, 包括最佳疾病、成人发病 vitelliform 营养不良、常染色体显性 vitreoretinochoroidopathy、常染色体隐性 bestrophinopathy 和视网膜色素变性3、4 ,10,11,12,13,14。这些疾病, 导致视力下降, 甚至失明, 目前无法治愈。为了发展治疗治疗和潜在的个性化医学, 重要的是要了解这些BEST1疾病引起的突变如何影响 BEST1 通道15的功能和结构。为了这些目的, 研究人员需要获得纯化的 bestrophin (野生类型和/或突变) 通道, 并进行体外分析5,8。
第一个关键步骤是哺乳动物细胞中较高物种的 bestrophin 通道的表达。由于 HEK293-F 细胞的杆状病毒转导 (BacMam 系统) 是 heterologously 表达膜蛋白16、17的一种强有力的方法, 该协议利用优化的 BacMam 向量 (pEG BacMam) 进行鲁棒表达。靶蛋白18, 在这种情况下是哺乳动物 bestrophin 同源。该载体用于各种膜蛋白的表达, 包括 G 蛋白偶联受体、核受体和其他离子通道18。还有证据表明, 所生产的蛋白质适合晶体学18。在 HEK293-F 细胞中具有较高的表达水平, 可以用色谱法纯化蛋白质;具体地说, 在 bestrophins 的情况下, 可以使用亲和性和大小排除色谱。
一旦该协议对 bestrophin 通道进行微调, 则可以通过平面脂质双层和 X 射线晶体学分别对纯化后的蛋白质进行分析, 以5、8为其功能和结构。总之, 这些技术为 bestrophins 和其他离子通道的功能和结构研究提供了强有力的管道。

<table><tbody><tr><td>HEPES</td><td>Fisher Scientific</td><td>AC327265000&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>AC446212500</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33-500</td><td></td></tr><tr><td>Imidazole</td><td>Fisher Scientific</td><td>AC301870010</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>AC197530010&amp;nbsp;</td><td></td></tr><tr><td>TCEP</td><td>Fisher Scientific</td><td>AA4058704&amp;nbsp;</td><td></td></tr><tr><td>Aprotinin</td><td>Fisher Scientific</td><td>AAJ63039MA</td><td></td></tr><tr><td>Leupeptin</td><td>Fisher Scientific</td><td>AAJ61188MB&amp;nbsp;</td><td></td></tr><tr><td>Pepstatin A</td><td>Fisher Scientific</td><td>AAJ20037MB</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride</td><td>Fisher Scientific</td><td>AC215740050</td><td></td></tr><tr><td>DDM sol-grade</td><td>Anatrace</td><td>D310S</td><td></td></tr><tr><td>DDM anagrade</td><td>Anatrace</td><td>D310</td><td></td></tr><tr><td>Sf-900 II SFM</td><td>ThermoFisher</td><td>10902179</td><td></td></tr><tr><td>FreeStyle medium</td><td>ThermoFisher</td><td>12338018</td><td></td></tr><tr><td>NanoDrop spectrophotometer</td><td>ThermoFisher</td><td>ND-2000</td><td></td></tr><tr><td>High pressure homogenizer</td><td>Avestin</td><td>Emulsiflex-C5</td><td></td></tr><tr><td>HisTrap column</td><td>GE</td><td>17-5248-01</td><td></td></tr><tr><td>Superdex-200 column</td><td>GE</td><td>28990944</td><td></td></tr><tr><td>AKTA Pure</td><td>GE</td><td>29018224</td><td></td></tr><tr><td>Ultra-15 centrifugal filter units</td><td>Millipore</td><td>UFC910024</td><td></td></tr><tr><td>Ultra-4 centrifugal filter units</td><td>Millipore</td><td>UFC810024</td><td></td></tr><tr><td>Ultra-0.5 centrifugal filter units</td><td>Millipore</td><td>UFC505024</td><td></td></tr><tr><td>Optima XE-90 Ultracentrifuge</td><td>Beckman Coulter</td><td>A94471</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>1658004</td><td></td></tr><tr><td>Mini-PROTEAN precast gel</td><td>Bio-Rad</td><td>4561084</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>PolyJet transfection reagent</td><td>SignaGen</td><td>SL100688</td><td></td></tr><tr><td>pEG BacMam vector</td><td></td><td></td><td>Obtained from the Gouaux lab at Vollum Institute</td></tr></tbody></table>

expression and purification of mammalian bestrophin ion channels

人类基因组编码四 bestrophin paralogs, 即 BEST1、BEST2、BEST3 和 BEST4。BEST1, 编码的BEST1基因, 是一个 Ca2 +激活 Cl 通道 ( CaCC) 主要表现在视网膜色素上皮 (RPE)。BEST1 的生理和病理意义被强调的事实是, 在BEST1基因的200多个不同的突变已经基因链接到至少五视网膜退行性疾病的频谱, 如最佳 vitelliform黄斑营养不良 (最佳疾病)。因此, 了解 bestrophin 通道在单分子水平上的生物物理学具有重要的意义。然而, 获得纯化的哺乳动物离子通道往往是一项具有挑战性的任务。在这里, 我们报告了哺乳动物 bestrophin 蛋白的表达与 BacMam 杆状病毒基因转移系统和他们的亲和性和大小排斥色谱纯化的协议。纯化蛋白在随后的功能和结构分析中有潜在的应用前景, 如脂质双层和晶体学中的电生理记录。重要的是, 这条管道可以适应研究其他离子通道的功能和结构。

瞬态转染胶粘剂 HEK293 细胞的荧光强度 (图 1A) 是悬浮 HEK293-F 细胞中预测蛋白表达水平的良好指标 (图 1B)。如果靶蛋白在瞬态转染后在 HEK293 细胞中表达不良好或被错误地定位, 建议考虑修改表达结构 (例如, 改变 GFP 标记的位置或做出突变/截断目标蛋白)。小 HEK293-F 悬浮培养 (例如, 25 毫升培养) 用于优化蛋白质表达?...

Watch this Scientific Journal Video about Expression and Purification of Mammalian Bestrophin Ion Channels at JoVE.com

Expression and Purification of Mammalian Bestrophin Ion Channels

哺乳动物 Bestrophin 离子通道的表达与纯化

离子通道的纯化往往具有挑战性, 但一旦达到, 它就有可能允许对通道的功能和结构进行体外调查。在这里, 我们描述的步骤, 以表达和纯化哺乳动物 bestrophin 蛋白, 一个家庭的 Ca2 +激活 Cl 通道.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

expression-and-purification-of-mammalian-bestrophin-ion-channels

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.3K 次观看.  University of Rochester, School of Medicine and Dentistry. 离子通道的纯化往往具有挑战性, 但一旦达到, 它就有可能允许对通道的功能和结构进行体外调查。在这里, 我们描述的步骤, 以表达和纯化哺乳动物 bestrophin 蛋白, 一个家庭的 Ca2 +激活 Cl 通道.

视频: Expression and Purification of Mammalian Bestrophin Ion Channels

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

科研

生物化学

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

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

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

表达和纯化溶质载体转运蛋白的策略

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

作者聚焦:使用密码子优化基因表达和纯化人溶质载体转运蛋白 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

用于结构和生化研究的人溶质载体的高通量表达和纯化

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

哺乳动物 Bestrophin 离子通道的表达与纯化

摘要

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