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7 March 2019 Cell-based biosynthesis of linear protein nanoarrays
Hiroaki Ishikawa; Jefer E. Yu; Jie Tian; Sindy K. Y. Tang; Hongmin Qin; Wallace F. Marshall
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Proceedings Volume 10893, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI; 108930F (2019) https://doi.org/10.1117/12.2510226
Event: SPIE BiOS, 2019, San Francisco, California, United States
Abstract
We describe an approach for using the flagella axoneme as the basis for biological self-assembling protein nanoarrays. The axoneme is the insoluble protein core of the eukaryotic flagellum or cilium. By attaching a protein of interest to particular axonemal proteins, it is possible to exploit the intraflagellar transport system to incorporate those proteins into the axoneme as it assembles. Using the axoneme as a protein array confers several advantages, such as high protein loading capacity compared to other bioparticle systems; genetically programmed self-assembly without the need for any linking steps; single-step purification of particles without the need for cell lysis, allowing retention and re-use of biomass; and choice of isolating the particle as a membrane enclosed vesicle or as an exposed protein array. Here we test several potential axonemal proteins as adaptor proteins, using green fluorescent protein as a test case. We find that FAP20 is an ideal scaffold protein for this purpose in that it shows high incorporation and uniform localization. We verify that FAP20-GFP constructs are stably associated with the axoneme during purification and storage, that the GFP moiety can be released by protease cleavage, and that the flagellar array can be easily encapsulated in aqueous-oil emulsion droplets for use in microfluidic assays.
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Hiroaki Ishikawa, Jefer E. Yu, Jie Tian, Sindy K. Y. Tang, Hongmin Qin, and Wallace F. Marshall "Cell-based biosynthesis of linear protein nanoarrays ", Proc. SPIE 10893, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI, 108930F (7 March 2019); https://doi.org/10.1117/12.2510226
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