Download the full-sized PDF
Permanent link (DOI): https://doi.org/10.7939/R32D78
This file is in the following communities:
|Graduate Studies and Research, Faculty of|
This file is in the following collections:
|Theses and Dissertations|
Development of monomeric fluorescent proteins and fluorescent protein-based biosensors Open Access
- Other title
- Type of item
- Degree grantor
University of Alberta
- Author or creator
- Supervisor and department
Campbell, Robert E. (Chemistry)
- Examining committee member and department
Loppnow, Glen R. (Chemistry)
Simmonds, Andrew (Cell Biology)
Li, Liang (Chemistry)
Cairo, Christopher W. (Chemistry)
Prescott, Mark (Department of Biochemistry and Molecular Biology, Monash University)
Department of Chemistry
- Date accepted
- Graduation date
Doctor of Philosophy
- Degree level
Fluorescent protein (FP) technology is now an indispensible tool of biomedical research. Nevertheless, only a few members of the hundreds of existing FPs are generally regarded as the preferred options for most imaging applications. Accordingly, new FPs with ever-improved properties are in demand and worth pursuing, as these efforts may lead to variants that are closer to the “ideal” FP. Also there remain a tremendous number of opportunities for developing FP-based biosensors for probing biological process in vivo. This thesis describes our effort on engineering new FPs with improved properties, and further modifying them to create novel biosensors. Directed evolution and semi-rational protein engineering are the main techniques used to develop these new FPs and FP-based biosensors.
The first class of FPs addressed in this thesis are the green-to-red photoconvertible FPs (pcFPs). In an effort to overcome the limitations imposed by the oligomeric structure of natural pcFPs, we created a new monomeric pcFP based on consensus design. Subsequent optimization yielded mClavGR2 and mMaple, two monomeric pcFPs displaying superior performance in folding and maturation, brightness, photoconversion efficiency and photostability. We demonstrate the application of mClavGR2 for dynamic monitoring of protein trafficking. Furthermore, in collaboration with researchers from several other groups, mMaple was demonstrated to be a multi-model probe that is suitable for use in several conventional and super-resolution fluorescence imaging modalities.
Using mMaple as a template for single-FP biosensor design, we successfully combined the two most important implementations of FPs, the “highlightable” trait and the Ca2+ sensing capability, into one construct. Optimization, characterization and live cell imaging of the resulting green-to-red highlightable Ca2+ indicators are described.
Another class of FPs that are of interest in this thesis are the true yellow emitting FPs that fill the spectral gap between monomeric greenish-yellow FPs and monomeric orange FPs. By disrupting the inter-subunit interfaces of zFP538, a FP with a distinct three-ring chromophore and an emission maximum at 538 nm, we successfully obtained its monomeric version and named it as mPapaya1. Again, characterization and live cell imaging application of mPapaya1 are described.
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
- Citation for previous publication
Hoi, H.; Shaner, N. C.; Davidson, M. W.; Cairo, C. W.; Wang, J.; Campbell, R. E. A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. J. Mol. Biol. 2010, 401, 776-791.McEvoy, A. L.; Hoi, H.; Bates, M.; Platonova, E.; Cranfill, P. J.; Baird, M. A.; Davidson, M. W.; Ewers, H.; Liphardt, J.; Campbell, R. E. mMaple: a photoconvertible fluorescent protein for use in multiple imaging modalities. PLoS one 2012, 7, e51314.Hoi, H.; Matsuda, T.; Nagai, T.; Campbell, R. E. Highlightable Ca2+ indicatros for live cell imaging. J. Am. Chem. Soc. 2012, DOI: 10.1021/ja310184a.
- Date Uploaded
- Date Modified
- Audit Status
- Audits have not yet been run on this file.
File format: pdf (Portable Document Format)
Mime type: application/pdf
File size: 9072641
Last modified: 2015:10:12 11:56:48-06:00
Filename: Hoi_Hiofan_Spring 2013.pdf
Original checksum: 5ed2dbae5aafef4a90ab17a7a765fed4
Well formed: false
Status message: Unexpected error in findFonts java.lang.ClassCastException: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject cannot be cast to edu.harvard.hul.ois.jhove.module.pdf.PdfDictionary offset=2738
Status message: Invalid Annotation list offset=1389881