Formation of Giant Unilamellar Vesicles from Block Copolymers and Study of AqpZ and KvAP Protein Incorporation with Light Microscopy
J.T. Patti, E.K. Brooks and C.D. Montemagno
University of California, Los Angeles, US
polymer vesicle, protein incorporation, GUV, AqpZ, KvAP
We have formed giant unilamellar vesicles (GUV’s) using two types of block copolymers—PMOXZ-PDMS-PMOXZ triblock and PBD-PEO diblock—and studied incorporation of labeled, transmembrane proteins using aquaporin (AqpZ), a water channel protein, and KvAP, a voltage-gated potassium channel. GUV’s self-assemble from a variety of natural lipids. Their large size enables the use of fluorescence and micromanipulation techniques, such as patch-clamp, to study membrane proteins in a controlled environment. Vesicle-forming polymers which support membrane protein incorporation utilize polymer robustness and other unique features, with several basic science, device, and pharmaceutical applications. To form polymer GUV’s, we adapted several techniques originally used to form giant lipid vesicles, including sonication, detergent-removal, and film hydration. In different incorporation tests, proteins were labeled with fluorescent dyes, beads, and gold nano-particles. Vesicle formation and protein insertion was observed using DIC, fluorescence, and confocal microscopy, and the polymer presented unique challenges for imaging proteins. GUV’s ranging from 1-50um formed from each of the polymers. There was a strong tendency for the PMOXZ-PDMS-PMOXZ triblock to form multilamellar vesicles, while the film hydration technique consistently produced GUV’s using the PBD-PEO diblock. Future experiments will determine the effect of polymer membranes on protein function.
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Nanotech 2007 Conference Program Abstract