scholarly journals Directed molecular evolution to design advanced red fluorescent proteins

2011 ◽  
Vol 8 (12) ◽  
pp. 1019-1026 ◽  
Author(s):  
Fedor V Subach ◽  
Kiryl D Piatkevich ◽  
Vladislav V Verkhusha
Keyword(s):  
Molecular Evolution ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins ◽  
Directed Molecular Evolution

scholarly journals Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

2021 ◽  
Author(s):  
Kiryl D Piatkevich ◽  
Siranush Babakhanova ◽  
Erica Jung ◽  
Kazuhiko Namikawa ◽  
Hanbin ZHANG ◽  
...  
Keyword(s):  
Molecular Evolution ◽  
Mammalian Cells ◽  
High Performance ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Model Organisms ◽  
Multicolor Imaging ◽  
Directed Molecular Evolution

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2x107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

scholarly journals Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

2021 ◽  
Author(s):  
Siranush Babakhanova ◽  
Erica E. Jung ◽  
Kazuhiko Namikawa ◽  
Hanbin Zhang ◽  
Yangdong Wang ◽  
...  
Keyword(s):  
Molecular Evolution ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Directed Molecular Evolution

scholarly journals Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

2021 ◽  
Author(s):  
Siranush Babakhanova ◽  
Erica Jung ◽  
Kazuhiko Namikawa ◽  
Hanbin Zhang ◽  
Yangdong Wang ◽  
...  
Keyword(s):  
Molecular Evolution ◽  
Mammalian Cells ◽  
High Performance ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Practical Method ◽  
Fluorescent Labeling ◽  
Model Organisms ◽  
Directed Molecular Evolution

Abstract In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2·107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~ 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multi-color imaging of small model organisms.

POSSIBLE APPLICATION OF METAL SENSITIVE RED FLUORESCENT PROTEINS IN ENVIRONMENTAL MONITORING

2012 ◽  
Vol 11 (1) ◽  
pp. 193-198 ◽  
Author(s):  
Laszlo Szilagyi ◽  
Maria Szabo (Palfi) ◽  
Judit Petres ◽  
Ildiko Miklossy ◽  
Beata Abraham ◽  
...  
Keyword(s):  
Environmental Monitoring ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

Generation and Characterization of a Rat Monoclonal Antibody Specific for Multiple Red Fluorescent Proteins

Hybridoma ◽  
2008 ◽  
Vol 27 (5) ◽  
pp. 337-343 ◽  
Author(s):  
Andrea Rottach ◽  
Elisabeth Kremmer ◽  
Danny Nowak ◽  
Heinrich Leonhardt ◽  
M. Cristina Cardoso
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

scholarly journals Monomerization of far-red fluorescent proteins

2018 ◽  
Vol 115 (48) ◽  
pp. E11294-E11301 ◽  
Author(s):  
Timothy M. Wannier ◽  
Sarah K. Gillespie ◽  
Nicholas Hutchins ◽  
R. Scott McIsaac ◽  
Sheng-Yi Wu ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Biological Markers ◽  
Fluorescent Proteins ◽  
Biological Tissues ◽  
Biological Applications ◽  
Emission Maximum ◽  
Naturally Occurring ◽  
Red Fluorescent Proteins ◽  
Animal Imaging ◽  
Structural Perturbations

Anthozoa-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λem ∼ 600–700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the monomerization of red-shifted oligomeric RFPs. Among the resultant variants is mKelly1 (emission maximum, λem = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) Chem Commun (Camb) 53:979–982], forms a class of bright, monomeric, far-red FPs.

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