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。丢弃洗脱橙/绿色部分。 注意:如果橙级分没有与100ml丙酮洗脱继续用丙酮洗涤该细胞直至绿色分数开始洗脱。 </li><li>用100％甲醇交换丙酮并收集含有叶绿素a的绿色部分。洗脱FRACT的体积离子可以50-100毫升之间变化。在开始时，将洗脱级分具有深绿颜色，这在洗脱结束时变成浅绿色。当洗脱馏分的颜色变成浅绿色停止洗脱。 </li><li>使用旋转蒸发器，直到萃取液完全干燥蒸发甲醇。不要使用热到解决方案;确保蒸发器的水浴温度不超过30℃。 注:蒸发的时间取决于甲醇馏分的体积被蒸发上，并且可以为10-60分钟之间变化。它完全干燥的提取物是很重要的。 </li><li>溶解从干燥的提取物中的颜料的乙醚（约5-10毫升）的小体积，并通过脱脂棉过滤。确保颜料​​过滤前完全溶于乙醚。 </li><li>蒸发乙醚，直至颜料完全干燥（10-30分钟）。 注意:干颜料可以与吹扫和氮气或氩气下保持在-20℃下，在黑暗中，直到进一步处理。 </li><li>溶于最小音量可能100％的甲醇（约1毫升）的干颜料，即使不是一切都完全暂停。 4毫升丙酮添加到溶液中，并以完全溶解该色素轻轻拂去玻璃。 </li><li>使用巴斯德吸管轻轻装载样品到DEAE柱在100％丙酮琼脂糖平衡。 </li><li>洗脱类胡萝卜素（橙黄色带）用100％丙酮。然后，洗脱叶绿素a（绿色带）以3:1体积/体积的丙酮/甲醇混合物。 注意:丙酮和丙酮/甲醇混合物的体积是大约相当于DEAE的体积琼脂糖装在柱上。 </li><li>验证叶绿素使用68用薄层色谱纯度 :25:5:2二氯甲烷/正-己烷/异丙醇/甲醇（体积/体积）混合物作为洗脱液17。 </li><li>使用旋转蒸发器蒸发溶剂，直至叶绿素a是完全干燥（10-60分钟）。 注意:干叶绿素a可以用氮气或氩气吹扫，并在黑暗中在-20℃氩气氛下贮存。 </li><li>通过再溶解干燥的叶绿素a在2-4毫升100％乙醇的制备叶绿素原液 。 注:叶绿素a的在663纳米的消光系数是74400厘米-1 M -1（83.3 -1（毫克/毫升）-1）的乙醇。一个典型的库存溶液应具有1860对应于25mM的（23毫克/毫升）的浓度的OD。加入20微升这股到含有5ml有机相，并在1ml的水相的结果1毫克WSCP中的混合物与10倍摩尔过量的叶绿素的对比乳液WSCP。 </li></ol> 2.准备乳液的有机相注意:该乳液的有机相由含有4.5％（体积/体积）跨度80的矿物油，和0.4％（体积/体积）吐温80。 <ol><li>称重在50ml管0.2克的T吐温80，1.8克司盘80的，与38克矿物油。拌匀所有组件和降温冰上。 注意:将有机相可被存储在4℃至一周。 </li></ol> 3.准备乳液的水相注意:该乳液的水相可由任何纯化WSCP，或细菌过度WSCP的粗提取物。 <ol><li>制备含有纯化WSCP水相。 <ol><li>生长含WSCP质粒12在1升的LB培养基中，在37℃，直至0.3-0.6 OD 大肠杆菌 BL21细菌。 </li><li>通过添加1mM的IPTG诱导蛋白质的表达。诱导后细菌生长在30℃，12〜16小时。 </li><li>收获细菌离心5000 xg离心在4℃下10分钟。 </li><li>溶解在冰上的结合缓冲液和超声颗粒（30秒时，15秒关，五次）。使用10个毫升缓冲液从250毫升离心获得的沉淀LB培养基与细胞过度的蛋白质。 注意:根据纯化方法，该结合缓冲液可以由的100mM磷酸缓冲液，pH 7.2或的50mM Tris，pH值7.5，500 mM氯化钠，5mM的EDTA中。 </li><li>降速细胞裂解物在12000×g离心30分钟，在4℃下。 </li><li>通过亲和层析纯化WSCP。取决于融合到WSCP标签，使用适当的可商购的​​亲和层析系统的蛋白质纯化按照生产商的说明纯化重组蛋白。 </li><li>于乳液制剂中，使用在50mM纯化WSCP磷酸盐缓冲液，pH 7.8。确保用于重建的最终蛋白量为0.5-1.0毫克每1ml缓冲液中。 </li></ol></li><li>制备含有与WSCP粗细菌裂解液的水相。 <ol><li>生长含于250毫升LB培养基的质粒表达WSCP在37℃，直到OD 0.3-0.6 大肠杆菌 BL21细胞。 </li><li>诱导与蛋白表达1mM的IPTG，在30℃过夜生长的细菌。 </li><li>收获细菌细胞通过离心5000 xg离心在4℃下10分钟。 </li><li>溶解沉淀在1-2毫升的50mM磷酸钠缓冲液pH 7.8，声处理（在30秒钟，15秒关，三次）和离心机以12,000 xg离心在4℃下30分钟。 </li><li>通过混合0.125毫升上清液与0.875毫升50mM磷酸钠缓冲液pH 7.8的制备乳液的水相。 </li></ol></li></ol> 4. WSCP的大会在乳液叶绿素a <ol><li>转印5毫升油 - 表面活性剂混合物到玻璃小瓶中，并冷却在冰上。移液之前，验证该有机相的所有组分充分混合并有表面活性剂和矿物油之间没有相分离。 </li><li>加入1毫升冰冷的水相的如第3制备至5ml有机相。 </li><li>在9500转在冰上混合使用2分钟组织匀浆两个阶段。</li><li>关键的一步:从这个阶段上，执行以尽量减少光损伤下绿光（520纳米）的所有进一步的步骤。加入20μl的25mM叶绿素的储备液（见1.13），以该乳液。通过轻弹和反相的玻璃小瓶分散。确保叶绿素均匀地分布在乳液中。 </li><li>孵育乳剂在黑暗中在冰上1-2小时。 </li><li>为了打破从有机相中的乳液和单独的水滴转移乳剂1.5毫升的塑料管和离心机在14,000rpm xg离心在室温下5分钟。 注意:如果组件是成功的，下层水相应该具有绿色。 </li><li>配置上油相和加入1 ml矿物油。由涡或彻底翻转管沉淀的乳液拌匀矿物油。降速样品以14,000 xg离心在室温下5分钟。重复此步骤，直到一个明确的半月板分离水和矿工人油相，而没有任何中间的乳液得到的。 </li><li>执行在化学罩此步骤。水性和矿物油相清楚地分离后，除去矿物油和加入1ml水饱和的乙醚。涡旋并在14000 xg离心降速样品在室温下5分钟。重复此步骤两次。 </li><li>第二次离心后，除去乙醚，留下打开引擎盖5-20分钟的管子。 </li><li>最后，装载包含WSCP /叶绿素复合水相到脱盐柱上，用适当的进一步的实验缓冲液洗脱。 注:蛋白质是在宽范围的pH（6.0-7.5）的磷酸盐和Tris缓冲液稳定。样品可以储存在避光长达一个月4℃。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

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><tbody><tr><td>Mineral oil</td><td>Sigma</td><td>M5904</td><td></td></tr><tr><td>Span80</td><td>Sigma</td><td>85548</td><td></td></tr><tr><td>Tween80</td><td>Sigma</td><td>P8074</td><td></td></tr><tr><td>Bio-Scale Mini Profinity eXact Cartridges</td><td>Bio Rad</td><td>10011164</td><td>Affinity chromatography for WSCP purification with native sequence.</td></tr><tr><td>His Trap HF column</td><td>GE Healthcare Life Science</td><td>17-5248-02</td><td>Affinity chromatography for WSCP purification with His-tag</td></tr><tr><td>DEAE Sepharose Fast Flow</td><td>GE Healthcare Life Science</td><td>17-0709-01</td><td>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

油包水乳液：用疏水颜料组装水溶性叶绿素结合蛋白的新系统化学|视频

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

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 。

水在油乳剂:与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

water-oil-emulsions-new-system-for-assembling-water-soluble

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14.6K 次观看.  Weizmann Institute of Science. 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 。 

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

A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

A&#39;即插即用&#39;的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

科研

化学

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

生物化学

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

<meta charset="utf8"></head><body>从植物组织中纯化活性光系统 I-Light Harvesting Complex I

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

从植物组织中纯化活性光系统 I-Light Harvesting Complex I

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

神经科学

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds &#8211; PP2, rottlerin, and curcumin &#8211; in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.

This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings.

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic ...

医学

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

生物工程

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.

水在油乳剂:与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

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此视频中的章节

A'即插即用'的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

从植物组织中纯化活性光系统 I-Light Harvesting Complex I

从植物组织中纯化活性光系统 I-Light Harvesting Complex I

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

体外重组巨型单层囊泡内的肌动蛋白细胞骨架

用于生物工程的片上辛醇辅助脂质体组装

水在油乳剂:与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

摘要

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此视频中的章节

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类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

从植物组织中纯化活性光系统 I-Light Harvesting Complex I

从植物组织中纯化活性光系统 I-Light Harvesting Complex I

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

用于生物工程的片上辛醇辅助脂质体组装

体外重组巨型单层囊泡内的肌动蛋白细胞骨架