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>

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="680">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:119px;">AC327265000&#160;</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC446212500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">G33-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imidazole</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC301870010</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgCl2</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC197530010&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AA4058704&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aprotinin</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ63039MA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ61188MB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin A</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ20037MB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenylmethylsulfonyl fluoride</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC215740050</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DDM sol-grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DDM anagrade</td>
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			<td style="width:119px;">D310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf-900 II SFM</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:119px;">10902179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FreeStyle medium</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">12338018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop spectrophotometer</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High pressure homogenizer</td>
			<td style="width:148px;">Avestin</td>
			<td style="width:119px;">Emulsiflex-C5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HisTrap column</td>
			<td style="width:148px;">GE</td>
			<td>17-5248-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superdex-200 column</td>
			<td style="width:148px;">GE</td>
			<td style="width:119px;">28990944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AKTA Pure</td>
			<td style="width:148px;">GE</td>
			<td style="width:119px;">29018224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-15 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-4 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC810024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-0.5 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC505024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optima XE-90 Ultracentrifuge</td>
			<td style="width:148px;">Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN Tetra Cell</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN precast gel</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>4561084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T100 Thermal Cycler</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PolyJet transfection reagent</td>
			<td style="width:148px;">SignaGen</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pEG BacMam vector</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></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

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

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Expression, Purification, and Antimicrobial Activity of S100A12

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

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

S100A12的表达，纯化和抗菌活性

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

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Mechanosensitive ion channels are often studied in terms of fluid flow/shear force sensitivity with patch-clamp recording. However, depending on the experimental protocol, the outcome on fluid flow-regulations of ion channels can be erroneous. Here, we provide methods for preventing and correcting such errors with a theoretical basis.

开放补丁夹具移液器测量未搅拌边界层中的离子浓度: 流体流动对离子通道控制的启示

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

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

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

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

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

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

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

哺乳动物组织和异种卵母细胞与胆固醇的丰富

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

PCR 突变体、克隆、表达、快速蛋白质纯化协议和色氨酸合成酶野生类型和突变形式的结晶

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...