Name: characterization of membrane transporters by heterologous expression in e. coli and production of membrane vesicles
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1. 生成具有 P1 转导的大肠杆菌双敲出突变剂
<ol><li>从大肠杆菌遗传存量中心<a href="http://cgsc.biology.yale.edu" target="_blank">（http://cgsc.biology.yale.edu）</a>获得大肠杆菌单敲除突变菌株[PotE和+CadB] 注：βPotE菌株耐卡那霉素26，βCadB菌株抗四环素27。</li>
	<li>使用P1转导协议28构建PotE/CadB</e...

<ol>
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在本研究中，我们概述了一种对抗分子进行表征的方法，首先在大肠杆菌中表达蛋白质，然后生成膜囊泡，从而可以在无细胞系统中对异位表达的蛋白质进行测定。除了在大多数分子生物学实验室中发现的设备外，这一策略还要求使用法国压力机、超离心机和进入进行放射性同位素检测的设施。
该技术的一个基本要求是异质蛋白正确定位在大肠杆菌的血浆膜上?...

参与代谢物贩运的蛋白质构成生理调节的基本水平，但绝大多数植物膜转运者尚未具有功能特征。已经实施了几种策略来描述新的运输蛋白。大肠杆菌和真核细胞（如酵母、异体卵母细胞、哺乳动物细胞、昆虫细胞和植物细胞）中的异体表达都被用来确定其迁移活性1。真核细胞是真核蛋白表达的宠儿，因为基本的细胞组成、信号转导途径、转录和翻译机与原生条件兼容。
酵母是植物中新型运输蛋白表征的重要模型。第一个植物运输蛋白，成功地表达在酵母（糖霉菌）是六氯苯（HUP1）从小球藻2。自那时以来，许多植物运输蛋白一直使用酵母表达系统进行功能特征化。其中包括植物糖运输机（SUC1和SUC2 3、VfSUT1和VfSTP14）和辅助糖运输机（AUX1和PIN5）。利用酵母表达植物蛋白的缺点包括，由于酵母缺乏这种细胞器，而蛋白原体局部蛋白的活性受损，靶向错误，以及由于膜蛋白7、8、9的过度表达而在酵母中形成错误折叠的聚集体和激活应激反应。
异体表达在异体细胞中的运输蛋白已被广泛用于运输器10的电生理表征。第一个植物运输蛋白，在Xenopus卵母细胞中使用异体表达为特征的是阿拉伯拟钾通道KAT110和阿拉伯拟南芥六角体运输机STP111。自那时以来，Xenopus卵母细胞已被应用于许多植物运输蛋白的特征，如血浆膜运输器12，真空蔗糖运输器SUT413和真空麻黄素运输机ALMT914。转移性卵母细胞用于运输测定的一个重要限制是细胞内代谢物的浓度不能纵1 。此外，需要专业知识来准备Xenopus卵母细胞，并且卵母细胞批次的变异性难以控制。
模型有机体大肠杆菌中的异构表达是新植物运输蛋白表征的理想系统。大肠杆菌的分子和生理特征是众所周知的。 分子工具和技术已经建立起来。此外，不同的表达载体，非致病菌株和突变体可用17，18，19。此外，大肠杆菌具有高生长率，在实验室条件下很容易生长。许多蛋白质在大肠杆菌9中易于表达和纯化。当蛋白质不能直接在细胞系统中被测定时，将蛋白质重组成脂质体也是一次成功的创新，尽管对于纯化膜蛋白的表征来说也是一项成功的创新。利用该模型系统20、21，完成了植物线粒体运输蛋白的功能表征，包括大豆、玉米、水稻和阿拉伯兰多普西的磷酸盐输送剂等溶质运输剂，在阿拉伯拟南芥中实现了二甲苯甲酸酯-三甲苯酯载体的功能特征。然而，在重组实验中发现番茄蛋白SICAT9的重组蛋白不起作用，而CAT运输体家族的其他成员在Xenopus卵母细胞测定22中被发现无功能。因此，需要额外的分子工具来表征膜输送机。
在大肠杆菌23中发现了5个多胺运输系统。其中包括两名ABC运输商，负责调解精氨酸和普赖辛的摄入，一个放流剂/苯基交换器，一个尸体/赖氨酸交换器，一个精氨酸出口商和一个放流剂进口商。Putrescine交换器PotE最初使用囊泡测定，其中内向外囊泡由裂化细胞与法国印刷机和测量放射性标记putresin到囊泡的吸收，以换取卵泡24。囊泡测定也用于表征钙输送机，该因子在质子梯度25的响应下调节钙的传递。这些实验促使我们制定了一种对其他多胺交换器进行表征的策略。我们首先在PotE和CadB交换器中制造了大肠杆菌缺乏菌株。在这里，我们演示了植物多胺抗剂的功能表征，通过修饰后的大肠杆菌菌株中的异质表达、使用法国印刷机生成膜囊泡以及放射性标记检测。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>3H-putrescine</td>
			<td>PerkinElmer</td>
			<td>NET185001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>3H-spermidine</td>
			<td>PerkinElmer</td>
			<td>NET522001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>4-chloro-1-naphthol</td>
			<td>Sigma-Aldrich</td>
			<td>C8890</td>
			<td></td>
		</tr>
		<tr>
			<td>14C arginine</td>
			<td>Moravek Inc.</td>
			<td>MC137</td>
			<td></td>
		</tr>
		<tr>
			<td>Arginine</td>
			<td>Sigma-Aldrich</td>
			<td>A-5006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-His (C-term)-HRP antibody</td>
			<td>ThermoFisher</td>
			<td>R931-25</td>
			<td>Detects the C-terminal polyhistidine (6xHis) tag, requires the free carboxyl 
			group for detection</td>
		</tr>
		<tr>
			<td>Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>ThermoFisher</td>
			<td>23227</td>
			<td>Pierce BCA protein asay kit.</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Bio-Rad</td>
			<td>161-0404</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxypeptidase B</td>
			<td>Sigma-Aldrich</td>
			<td>C9584-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td></td>
			<td>SS-34 fixed angle rotor and GA-6 fixed angle rotor</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>LabGenome</td>
			<td>7777-7</td>
			<td>Corning 7777-7 pyrex homogenizer with pour spout.</td>
		</tr>
		<tr>
			<td>Ecoscint-H</td>
			<td>National Diagnostics</td>
			<td>LS275</td>
			<td>scintillation cocktail</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration manifold</td>
			<td>Hoefer</td>
			<td>FH225V</td>
			<td></td>
		</tr>
		<tr>
			<td>French Pressure Cell</td>
			<td>Glen Mills</td>
			<td>FA-080A120</td>
			<td></td>
		</tr>
		<tr>
			<td>GABA</td>
			<td>Sigma-Aldrich</td>
			<td>A2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G6904</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software</td>
			<td></td>
			<td></td>
			<td><a href="http://www.graphpad.com/prism/Prism.htm" target="_blank">http://www.graphpad.com/prism/Prism.htm</a></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>KROGER</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>J.T. Baker</td>
			<td>3040-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintillation counter</td>
			<td>Beckman</td>
			<td>LS-6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Maleate</td>
			<td>Sigma-Aldrich</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane filters</td>
			<td>Merck Millipore</td>
			<td>hawp02500</td>
			<td>0.45 &#181;M</td>
		</tr>
		<tr>
			<td>PCR clean up kit</td>
			<td>Genscript</td>
			<td></td>
			<td>QuickClean II</td>
		</tr>
		<tr>
			<td>Potassium Phosphate dibasic</td>
			<td>ThermoFisher</td>
			<td>P290-500</td>
			<td></td>
		</tr>
		<tr>
			<td>putrescine</td>
			<td>fluka</td>
			<td>32810</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate monobasic</td>
			<td>J.T.Baker</td>
			<td>4008</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-aldrich</td>
			<td>S2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Strains :E. coli &#916;potE740(del)::kan, &#916;cadB2231::Tn10</td>
			<td>This manuscript</td>
			<td>Available upon request.</td>
			<td>Strain is deficient in the PotE and CadB polyamine exchangers.</td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Research Products</td>
			<td>T60040-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Sorvall MTX 150</td>
			<td>46960</td>
			<td>Thermo Fisher S150-AT fixed angle rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>ThermoFisher</td>
			<td>45237</td>
			<td>Centrifuge tubes for S150-AT rotor</td>
		</tr>
		<tr>
			<td>Vector: pBAD-DEST49</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>Gateway expression vector for E. coli</td>
		</tr>
	</tbody>
</table>

characterization of membrane transporters by heterologous expression in e. coli and production of membrane vesicles

已经开发出几种方法，以在功能上表征新型膜输送机。多胺在所有生物体中普遍存在，但植物中的多胺交换剂尚未确定。在这里，我们概述了一种使用从大肠杆菌细胞的解毒中产生膜囊泡来描述多胺抗结剂的方法，这种细胞异构体表达植物抗结剂。首先，我们在缺乏多胺和精氨酸交换输送机的大肠杆菌菌株中异构地表示AtBAT1。囊泡是使用法国印刷机生产的，通过超离心净化，并用于标记基材的膜过滤测定，以证明输送机的基板特异性。这些测定表明，AtBAT1是精氨酸、β-氨基丁酸（GABA）、普赖辛和精氨酸的质子介导物。为AtBAT1测定而开发的突变菌株可用于其他植物和动物多胺交换器家族的功能分析。我们还假设，只要这些蛋白质可以在细菌细胞膜中表达，这种方法就可以用来描述许多其他类型的抗剂。大肠杆菌是一种对新型运输者进行表征的良好系统，因为有多种方法可用于诱变性原生运输者。

此协议中的主要步骤在图 1中进行了图中的图中概述。简单地说，所有聚胺交换器和表达AtBAT1中缺乏的大肠杆菌细胞被培养、离心、用缓冲液清洗，并使用法国印刷机进行细胞解压。莱西斯倾向于产生囊泡，主要是从内到外，并捕获细胞外的缓冲液。细胞碎片通过离心去除，第二个超离心步骤用于收集膜颗粒。膜颗粒在Tris-Maleate缓冲液pH...

Watch this Scientific Journal Video about Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles at JoVE.com

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

我们描述了一种在大肠杆菌中的异质表达和利用法国印刷机对细胞进行分理的膜囊泡制剂中质子驱动膜输送剂的表征方法。

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

这种技术对膜运输器的特性的主要优点之一是，您可以确定特定基材的运输器的相对亲和力，并深入了解运输机制。使用蛋白质梯度驱动基质传输的膜蛋白的表征特别适用于我们在此演示的这种从内到外的囊泡测定。虽然这种测定需要一些专门的设备，如法国印刷机和获得超离心剂，但我们认为，从技术上讲，这种检测比需要将膜运输机重组为人造脂质体的检测更容易。该技术的主要优点是，它使我们能够量化不同基材的相对亲和力，并进行竞争分析，以测试竞争基材对运输格栅的影响。在用于说明此测定的 BAT1 运输机的情况下，我们还能够通过精氨酸显示运输器的基板激活。为了开始这个程序，培养准备好的大肠杆菌突变细胞，通过用适当的抗生素在5毫升聚胺3培养基中接种单个菌落，并在37摄氏度下过夜生长，来表达目标蛋白质。第二天，将这种培养物转移到500毫升的新鲜聚胺3培养，补充0.0002%的阿拉伯糖，并生长，直到细胞培养达到OD600之间的0.6和0.8。在此之后，离心机在2000倍G和4摄氏度15分钟收集细胞颗粒。重新悬浮颗粒，用缓冲液洗涤三次，每次在2000次G和4摄氏度下离心15分钟。缓冲区一中第三次旋转后的最终细胞体积应为 10 毫升。设置一个35毫升的法国压力细胞，并倒入细胞的悬浮液到细胞的身体。打开机器，打开设备背面的 RHS 开关，将比率选择器开关设置为高。打开泵开关。活塞将开始从底座上升，以置换腔室中的空气。顺时针转动压力增加阀，直到仪表达到 640 PSI，以增加压力。这将产生10，000 PSI的内部压力。一旦电池达到目标压力，通过逆时针转动流动阀组件，稍微打开该阀总成。调整阀门的开口，使流量每分钟只有 10 滴左右。当活塞体上的止油管到达流水单元顶部时，泵关闭。将比率选择器开关向下放置，然后打开泵开关，降低底板。当底板完全缩回时，通过完全逆时针转动压力增加阀，将系统压力降至零。关闭泵开关并关闭机器。将法国人的除心机在1万倍G和4摄氏度下离心15分钟，以清除不间断的细胞和细胞碎片。丢弃颗粒，将上流液转移到超离心管中。超离心在15万倍G和4摄氏度下产生上流水，一小时来颗粒膜囊。在缓冲液二中，在不重新悬浮的情况下清洗膜囊泡一次。然后，使用 Dounce 组织研磨机在缓冲器二中重新悬浮膜囊泡。在1.5毫升离心管中准备100微升等分膜制剂，并将其储存在零下80摄氏度。首先，在pH 7.8的30毫摩尔中清洗和重新悬浮囊泡，其中含有0.1毫摩尔的钴二氯化物。在每个100微升样品中加入一微升的碳氧培液 A，三氧化二中，在20摄氏度下孵育20分钟。在PH 7.5中加入含有EDTA钠和2-墨二甲醇溶液的5微升，以阻止消化。在室温下孵育溶液一小时，使酶完全灭活。接下来，在样品的pH 7.5至100微升下加入5至10微升含有硫酸锂、甘油、溴酚蓝色和Tris的溶液，然后通过电泳分析样品。为了进行氨基印迹，在聚丙烯酰胺凝胶上铺设一个用25毫摩尔磷酸氢钠润湿的硝基纤维素过滤器。将这些放在两个潮湿的纤维素过滤器之间，最后，放在两个潮湿的塑料冲刷垫之间。在2至4摄氏度的温度下，将这种组合放在含有25毫摩尔磷酸氢钠的腔室的电极之间。电转蛋白质在20伏特和2至3安培三小时。在摄氏2度下，在一夜之间阻断缓冲液中阻断硝基纤维素膜。第二天，孵育20毫升的硝基纤维素，稀释一至五千剂抗血性C-终端HRP抗体，阻断缓冲液两小时，用50毫升缓冲液洗涤两次，共60分钟。为了可视化氨基印迹，将6毫克4-1-纳普托霍尔溶解在20毫升变性酒精中，并加入80毫升15毫摩尔三酯和50微升30%过氧化氢。在此基板溶液中将滤纸洗洗 20 分钟。当颜色足够时，用水冲洗膜，让其干燥。首先，在12摄氏度下孵育100微升膜囊泡的等分，孵育5分钟。在1.5毫升微离质管中，将含有放射性标记多胺的缓冲液3加入，最终浓度为50微摩尔，加入膜囊泡，以启动运输。在 12 摄氏度下进行运输检测一分钟。之后，将反应混合物转移到过滤歧管中，并通过0.45微米硝基纤维素膜过滤器进行过滤。加入三毫升的冰冷测定缓冲液，含有10倍高浓度的未标记聚胺，然后是三毫升的检测缓冲液，不含多胺，以减少非特异性结合。将洗净的过滤器转移到含有 10 毫升闪烁液体的 20 毫升一次性闪烁小瓶中，并使用液体闪烁计数器确定放射性。计算文本协议中概述的聚胺净吸收。通过测量放射性标记基板的吸收到表达目标蛋白质的囊泡中，并使用非线性回归方法计算 Michaelis-Menten 动力学，确定基板的Km。在竞争实验中，在检测缓冲液中添加在检测缓冲液中制成的非标记竞争性基板，在12摄氏度下加入含有100微升囊泡的离心管，同时添加50微摩尔的放射性标记聚胺。测量囊泡内捕获的放射性，如前所述。通过测量放射性标记聚胺的吸收和 100 微摩尔或更高无标记基材的存在，确定竞争基材的父KM，并使用非线性回归方法绘制曲线。在这项研究中，抗波特的特点是首先在大肠杆菌中表达蛋白质，然后产生膜囊泡，使异源表达的蛋白质可以在无细胞系统中检测。西方的印迹用于验证 AtBAT1 是否被转定位到囊泡。用抗他的C-终端抗体探测印迹后，发现一种聚变构造蛋白约为72.3千吨。SDS-PAGE 之前的囊泡消化导致减少，但探针信号没有完全丢失。在12摄氏度下，囊泡的放射性标记精激素在1分钟内最高，在三分钟内保持线性。因此，运输测定的孵化时间固定在一分钟。一分钟后，由于囊泡膜上没有质子梯度，因此在pH 8.0中准备并储存的膜囊泡没有对同位素的净吸收。为了证明人工质子梯度的耗散效果，膜囊泡在添加标记基板之前在pH 8.0缓冲液中孵育10分钟，从而对放射性标记基板的吸收最小。综合起来，这些结果表明，质子驱动的精子酶的吸收是由于BAT1蛋白。为了确定蛋白质的基底特异性，通过测量不同浓度的放射性标记基质的吸收率来计算 Km 值。精氨酸、聚氨基辛和精氨酸的Km值表明这种蛋白质是一种高亲和力的多胺和精氨酸交换器。通过使用竞争分析，也可以间接确定特定基材的运输器的亲和力。这些测定表明，GABA是一种具有竞争力的精子素抑制剂。此外，测量50微摩尔放射性标记精氨酸在不同浓度的不同氨基酸存在的情况下的摄取量表明，AtBAT1也能够以毫摩尔浓度运输谷氨酸和丙氨酸。执行此过程时，膜传输器与细菌膜的集成显然至关重要。验证感兴趣的蛋白质是否被集成到膜中，可以使用针对C-终端标签的抗体进行验证。人类运输者的基因变异可以影响代谢物和药物的运输，而代谢物和药物是特定运输者基质。因此，全药变异可以同时影响药物的吸收和排泄。许多基因变异可以通过表达载体中传输器基因的侧向突变快速生成。然后，可以使用从内到外的囊泡测定，在非常可控的条件下测试这些变异的功能性检测。膜运输机占所有蛋白质的8到10个，而且大多数蛋白质在任何模型生物体中都未完全被描述。局部到细胞膜的运输器在真核细胞中以异质表达来描述特别具有挑战性。如果交换类型或质子介质的运输器可以本地化到此大肠杆菌膜，这些运输器可以使用此体外测定功能特征。囊泡的质量和产量受到法国媒体细胞碎片的洗脱率的影响。在用同位素进行囊泡的运输测定时，重要的是要有一个实验装置，促进实验复制。有许多方法可以做到这一点，但我们认为，这将有助于我们向您展示我们是如何做到这一点。

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Unit 1

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Cell Membrane Structure and Functions

Membrane Structure and Components

SCIENTISTS IN ACTION

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;：设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

C2C12 Myotubes 测量反式等离子体膜电子传输

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

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 ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

热通菌膜-双磷酶抑制剂的筛选

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...