A Plug-Based Microfluidic System for Dispensing Lipidic Cubic Phase (LCP) Material Validated by Crystallizing Membrane Proteins in Lipidic Mesophases.
A Plug-Based Microfluidic System for Dispensing Lipidic Cubic Phase (LCP) Material Validated by Crystallizing Membrane Proteins in Lipidic Mesophases.
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DOI：
10.1007/s10404-009-0512-8
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发表时间：
2010-06
影响因子：
2.8
通讯作者：
Ismagilov, Rustem F.
中科院分区：
文献类型：
--
作者：
Li, Liang;Fu, Qiang;Kors, Christopher A.;Stewart, Lance;Nollert, Peter;Laible, Philip D.;Ismagilov, Rustem F.
本文提出了一种基于塞子的微流控系统，用于分配纳升体积的脂类立方相(LCP)材料的塞子，并随后将LCP塞子与水塞合并。通过结晶包括LCP在内的脂类中间相中的膜蛋白，验证了该体系的有效性。该系统能够准确地分配纳升体积的LCP材料，通过将LCP封装在塞子中防止由于脱水而可能发生的意外相变，并与使用膜蛋白样品形成LCP材料的传统方法兼容，如盐生盐杆菌细菌视紫质的成功结晶所表明的那样。在相图中描述了LCP塞的形成条件。该系统还使用了两种不同的引入膜蛋白的方法：1)使用膜蛋白样品生成LCP材料的传统方法和2)LCP形成后并入(PLI)方法，该方法包括制作不含蛋白质的LCP材料，将膜蛋白样品从外部添加到LCP材料中，并允许蛋白质扩散到LCP材料或可能由相变引起的其他脂类中间相中。用PLI法获得了球形红杆菌和绿色芽孢杆菌的细菌光合作用中心晶体。这种基于插头的、LCP辅助的微流控系统，结合PLI方法将膜蛋白引入LCP，将有助于最大限度地减少样品的消耗，拓宽膜蛋白结晶参数空间的筛选。
This paper presents a plug-based microfluidic system to dispense nanoliter-volume plugs of Lipidic Cubic Phase (LCP) material and subsequently merge the LCP plugs with aqueous plugs. This system was validated by crystallizing membrane proteins in lipidic mesophases, including LCP. This system allows for accurate dispensing of LCP material in nanoliter volumes, prevents inadvertent phase transitions that may occur due to dehydration by enclosing LCP in plugs, and is compatible with the traditional method of forming LCP material using a membrane protein sample, as shown by the successful crystallization of bacteriorhodopsin from Halobacterium salinarum. Conditions for the formation of LCP plugs were characterized and presented in a phase diagram. This system was also implemented using two different methods of introducing the membrane protein: 1) the traditional method of generating the LCP material using a membrane protein sample and 2) Post LCP-formation Incorporation (PLI), which involves making LCP material without protein, adding the membrane protein sample externally to the LCP material, and allowing the protein to diffuse into the LCP material or into other lipidic mesophases that may result from phase transitions. Crystals of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis were obtained using PLI. The plug-based, LCP-assisted microfluidic system, combined with the PLI method for introducing membrane protein into LCP, should be useful for minimizing consumption of samples and broadening the screening of parameter space in membrane protein crystallization.
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DOI：
10.1038/nbt1183-784
发表时间：
1983-01-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
SIMON, R;PRIEFER, U;PUHLER, A
通讯作者：
PUHLER, A
影响因子：
3.4
作者：
Cherezov, V;Fersi, H;Caffrey, M
通讯作者：
Caffrey, M
影响因子：
3.2
作者：
DAVIS, J;DONOHUE, TJ;KAPLAN, S
通讯作者：
KAPLAN, S
影响因子：
2.9
作者：
TAGUCHI, AKW;STOCKER, JW;WOODBURY, NW
通讯作者：
WOODBURY, NW
影响因子：
3.8
作者：
Perry, Sarah L.;Roberts, Griffin W.;Tice, Joshua D.;Gennis, Robert B.;Kenis, Paul J. A.
通讯作者：
Kenis, Paul J. A.