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Cell-based biosynthesis of linear protein nanoarrays
Author(s): Hiroaki Ishikawa; Jefer E. Yu; Jie Tian; Sindy K. Y. Tang; Hongmin Qin; Wallace F. Marshall
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Paper 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.

Paper Details

Date Published: 7 March 2019
PDF: 16 pages
Proc. SPIE 10893, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI, 108930F (7 March 2019); doi: 10.1117/12.2510226
Show Author Affiliations
Hiroaki Ishikawa, Univ. of California, San Francisco (United States)
NSF Ctr. for Cellular Construction (United States)
Jefer E. Yu, Texas A&M Univ. (United States)
Jie Tian, Texas A&M Univ. (United States)
Sindy K. Y. Tang, NSF Ctr. for Cellular Construction (United States)
Stanford Univ. (United States)
Hongmin Qin, Texas A&M Univ. (United States)
Wallace F. Marshall, Univ. of California, San Francisco (United States)
NSF Ctr. for Cellular Construction (United States)


Published in SPIE Proceedings Vol. 10893:
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI
Samuel Achilefu; Ramesh Raghavachari, Editor(s)

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