期刊
MICROFLUIDICS AND NANOFLUIDICS
卷 8, 期 6, 页码 789-798出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s10404-009-0512-8
关键词
Droplet; Plugs; Lipidic cubic phase; Membrane protein; Protein crystallization
资金
- National Institute of General Medical Sciences (NIGMS), National Center for Research Resources [U54 GM074961, Y1-GM-1104]
- National Institutes of Health Roadmap for Medical Research [R01 GM075827, P01 GM75913]
- University of Chicago/Argonne National Laboratory (ANL) Collaborative Seed
- U.S. Department of Energy, Basic Energy Sciences, Office of Science [DE-AC02-06CH11357]
- National Cancer Institute [Y1-CO-1020]
This article 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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