Name: water in oil emulsions: a new system for assembling water-soluble chlorophyll-binding proteins with hydrophobic pigments
Uploaded: 2016-03-21T14:00:00
Description: Watch this Scientific Journal Video about Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments at JoVE.com

DN承认欧盟FP7项目PEPDIODE（GA 256672）和REGPOT-2012-2013-1（GA 316157），并从以色列科学基金会个人研究资助（编号十分之二百六十八 ）的支持。我们感谢什穆埃尔·鲁宾斯坦教授，工程学院和应用科学，哈佛大学，马萨诸塞州剑桥，美国采取的共聚焦显微镜图像。

1.准备叶绿素a股票方案<ol><li>关键步骤：执行叶绿素准备的所有步骤在化学罩，在绿光（520纳米），或在黑暗中，以尽量减少光损伤。始终冻结颜料存放前添加氮气或氩气。确保所有的溶剂均为分析纯。 </li><li>称取约5mg仅含有叶绿素a在类囊体膜冻干螺旋藻细胞或其它蓝藻细胞，并使用研钵和杵粉碎它。 </li><li>加载破碎细胞到玻璃柱以除去类胡萝卜素用约50-100毫升100％丙酮洗涤。?...

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Botrys binds various chlorophyll derivatives.</a> Biochemistry. 42, 7427-7433 (2003).</li><li>Takahashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+characterization+and+analysis+of+the+intracellular+localization+of+a+water-soluble+Chl-binding+protein+from+Brussels+sprouts+(Brassica+oleracea+var.+gemmifera).">Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera).</a> Plant Cell Physiol. 53, 879-891 (2012).</li><li>Takahashi, S., Ono, M., Uchida, A., Nakayama, K., Satoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+functional+expression+of+a+water-soluble+chlorophyll-binding+protein+from+Japanese+wild+radish.">Molecular cloning and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild radish.</a> J. 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Chem. 267, 22043-22047 (1992).</li><li>Kamimura, Y., Mori, T., Yamasaki, T., Katoh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+properties+and+a+possible+function+of+a+water-soluble+chlorophyll+a/b-protein+from+brussels+sprouts.">Isolation, properties and a possible function of a water-soluble chlorophyll a/b-protein from brussels sprouts.</a> Plant Cell Physiol. 38, 133-138 (1997).</li><li>Murata, T., Itoh, R., Yakushiji, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+water-soluble+chlorophyll-proteins+from+Lepidium+virginicum.">Crystallization of water-soluble chlorophyll-proteins from Lepidium virginicum.</a> Biochim. Biophys. Acta. 593, 167-170 (1980).</li></ol>

我们的目标是开发用于与疏水性颜料的水溶性叶绿素结合蛋白的组装一个新的通用系统。这表明，基于W / O乳液的新重组系统是被证明为从球芽甘蓝，花椰菜，日本辣根和弗吉尼亚pepperweed在大肠杆菌中重组表达WSCP脱辅基蛋白的组装工作的一般方法大肠杆菌 。这里的结果是从1毫克WSCP的重构带有10倍摩尔过量的叶绿素a的。然而也可以使用较低浓度WSCP用于reconstitutions和不同的Chl /...

疏水性颜料如叶绿素（的Chls），菌绿素（BChls）和类胡萝卜素在光合作用反应中心，捕光蛋白是进行电子传输和光能量采集和传输的主要辅助因子。反应中心和大部分叶绿素结合光收获物是跨膜蛋白。非氧光合绿硫细菌1,2的Fenna -马修斯-奥尔森（FMO）的蛋白质，和甲藻3的甲藻素-叶绿素蛋白（PCP）是水溶性的捕光蛋白的特殊例子。水溶性叶绿素结合蛋白十字花科 ， 蓼 ， 藜科 （WSCPs）和苋植物4,5另一个独特的例子，但在对比FMO和PCP，这些既不参与捕光也不在任何主光合反应的和它们的精确的physiological功能还不清楚5-8。它们的高叶绿素结合亲和力，导致一个建议的功能的Chls瞬态载体和叶绿素衍生物9,10。另外，有人推测WSCP起着受损细胞清除的Chls作用和防止叶绿素引起的光氧化损伤7,11-13。最近，有人提出WSCP用作蛋白酶抑制剂和播放食草动物性过程中的作用，以及花的发育过程中14调节细胞死亡。 WSCPs根据其光物理性质分为两大类。第一类（I类， 如 ：从藜 ）可根据照明进行光转化。类从芸苔属植物，即不发生光转化5,10-二WSCPs，被进一步细分为IIa类（ 例如 ， 甘蓝，萝卜 ）和Ⅱb族（ 例如 ，来自北美独行菜 </eM&gt;）。 IIb类WSCP从北美独行菜的结构用X射线晶体学的2.0 A分辨率8解决。它揭示了一个对称的同源四聚体，其中所述蛋白质亚基形成疏水核心。每个亚基结合的单个叶绿素，这导致在四个紧密堆积的Chls的简单所有叶绿素布置使得WSCPs潜在有用的模型系统用于研究叶绿素 - 蛋白复合物的结合和组装core.This内紧密排列，且相邻的Chls的影响和对个人的Chls的光谱和电子特性蛋白的环境中。此外，其可提供用于构建可用于人工光合作用设备捕光模块人工叶绿素结合蛋白的模板。 天然WSCPs的严谨的研究是不可行的，因为从植物中纯化复合物总是含有与叶绿素a的不同组合四聚体的异质混合物和叶绿素b 9。因此，需要一种用于组装具有的Chls 体外重组表达WSCPs的方法。这是通过的Chls的可忽略的水溶解度，这使得它不可能通过简单地与在水溶液中的颜料混合水溶性脱辅基蛋白聚集在体外复通过混合15被证明与类囊体膜的脱辅基蛋白的挑战。 在体外组装，但这种方法仅限于原生的Chls存在于类囊体的。 Schmidt 等人 。报告了在大肠杆菌中重组表达组氨酸标记的蛋白质组装与来自花椰菜（CaWSCP）WSCP几个Chl和Bchl衍生物大肠杆菌它固定到一个镍亲和柱和引进洗涤剂溶解11叶绿素衍生物。成功复溶从A重组WSCPs的拟南芥 6，和球芽甘蓝（BoWSCP），日野萝卜（RshWSCP）的还报道ð弗吉尼亚pepperweed（LvWSCP）类似的方法由被。 在这里，我们提出了一个新颖，装配与WSCP的Chls不需要标记或固定蛋白质一般情况下，直接的方法。它依赖于从在矿物油中的水溶性脱辅基蛋白的它们的水溶液制备乳液。因此蛋白质被封装在水包油（W / O）具有非常高的表面微滴体积比16。然后疏水辅因子被溶解在油中并且容易引入到从油相的液滴。我们使用方法报到WSCP脱辅基蛋白的几个变体在大肠杆菌重组表达的组装大肠杆菌与叶绿素a。我们证明从中可以用作用于开发新颖叶绿素结合蛋白筛选系统WSCP过表达的细菌粗裂解物的装配。

<table border="0" cellpadding="0" cellspacing="0" width="614">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:119px;">M5904</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Span80</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:119px;">85548</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween80</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:119px;">P8074</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bio-Scale Mini Profinity eXact Cartridges</td>
			<td style="width:83px;">Bio Rad</td>
			<td style="width:119px;">10011164</td>
			<td style="width:196px;">Affinity chromatography for WSCP purification with native sequence.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">His Trap HF column</td>
			<td style="width:83px;">GE Healthcare Life Science</td>
			<td style="width:119px;">17-5248-02</td>
			<td style="width:196px;">Affinity chromatography for WSCP purification with His-tag</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">DEAE Sepharose Fast Flow</td>
			<td style="width:83px;">GE Healthcare Life Science</td>
			<td style="width:119px;">17-0709-01</td>
			<td style="width:196px;">Chromatography medium for chlorophyll purification</td>
		</tr>
	</tbody>
</table>

water in oil emulsions: a new system for assembling water-soluble chlorophyll-binding proteins with hydrophobic pigments

叶绿素（的Chls）和菌绿素（BChls）是进行光合作用的光捕获和电子传递的主要辅助因子。其功能主要取决于他们的大而复杂的多亚基跨膜蛋白复合体中的特定组织。为了在分子水平上，这些配合物如何促进太阳能转换了解，重要的是要了解蛋白质颜料，和颜料颜料的相互作用，以及它们对兴奋动力学效果。获得这样的理解的一种方式是通过构建和学习的Chls的配合物与简单的水溶性的重组蛋白。然而，掺入亲脂性的Chls和BChls成水溶性蛋白是困难的。此外，不存在一般的方法，它可以用于与疏水性颜料的水溶性蛋白质的组装。在这里，我们证明根据水包油乳剂的简单和高通量系统，能使屁股疏水的Chls水溶性蛋白质的embly。的新方法，通过组装与叶绿素a 十字花科植物（WSCP）的水溶性叶绿素结合蛋白的重组形式的验证。我们证明叶绿素的成功组装表达E. WSCP 的使用原油裂解大肠杆菌细胞，其可以用于开发遗传筛选系统，用于新的水溶性叶绿素结合蛋白，以及叶绿素-蛋白相互作用和装配过程的研究。

重组WSCP载脂蛋白组装与根据上一节中描述的方案的W / O乳剂叶绿素a。该协议使用含有任一纯WSCPs，或裂解物的大肠杆菌细胞过度WSCP（ 图1）的水相来实现。该协议是简单，快速，不需要任何特殊的设备，除了组织匀浆。 吸光度和叶绿素的CD谱四个重组WSCP变体，即RshWSCP，CaWSCP，BoWSCP和LvWSCP 一个?...

Watch this Scientific Journal Video about Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments at JoVE.com

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments (Video) | JoVE

该原稿描述用于装配与基于水包油乳液的疏水颜料的水溶性蛋白质的简单和高通量方法。我们证明该方法在本地叶绿素大会的有效性与重组水溶性叶绿素的四种变体结合蛋白（WSCPs）的芸苔属植物在大肠杆菌中表达大肠杆菌 。

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Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

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水介质中水溶性钌烯烃置换催化剂的异质化去除与寄主交互作用

A highly efficient transition metal catalyst removal method is developed. The water-soluble catalyst contains a newly-designed NHC ligand for the catalyst ...

Stereolithographic 3D Printing with Renewable Acrylates

Stereolithographic 3D Printing with Renewable Acrylates (Video) | JoVE

Stereolithographic 3D 印刷用可再生丙烯酸酯

The accessibility of cost-competitive renewable materials and their application in additive manufacturing is essential for an efficient biobased economy. ...

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy (Video) | JoVE

一种利用19f 核磁共振光谱测定脂质性 (日志 p) 的新方法

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates (Video) | JoVE

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...