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Sci Rep. 2018 Jan 12;8(1):667. doi: 10.1038/s41598-017-18045-y.

A Novel Ultra-Stable, Monomeric Green Fluorescent Protein For Direct Volumetric Imaging of Whole Organs Using CLARITY.

Author information

1
The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, 3052, Australia. daniel.scott@florey.edu.au.
2
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3010, Australia. daniel.scott@florey.edu.au.
3
The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, 3052, Australia.
4
IBM Research, Southbank, Victoria, 3006, Australia.
5
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3010, Australia.
6
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia.
7
ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria, 3052, Australia.
8
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3010, Australia. mgriffin@unimelb.edu.au.
9
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, 3010, Australia. mgriffin@unimelb.edu.au.

Abstract

Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.

PMID:
29330459
PMCID:
PMC5766548
DOI:
10.1038/s41598-017-18045-y
[Indexed for MEDLINE]
Free PMC Article

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