scholarly journals Fluorescent Proteins: Green-Light-Activated Photoreaction via Genetic Hybridization of Far-Red Fluorescent Protein and Silk (Adv. Sci. 6/2018)

2018 ◽  
Vol 5 (6) ◽  
pp. 1870033
Author(s):  
Jung Woo Leem ◽  
Jongwoo Park ◽  
Seong-Wan Kim ◽  
Seong-Ryul Kim ◽  
Seung Ho Choi ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Light ◽  
Red Fluorescent Protein

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

ESTABLISHMENT OF VISIBLE ANIMAL METASTASIS MODELS FOR HUMAN NASOPHARYNGEAL CARCINOMA BASED ON A FAR-RED FLUORESCENT PROTEIN

2012 ◽  
Vol 05 (03) ◽  
pp. 1250019 ◽  
Author(s):  
YING ZHENG ◽  
CHUAN HUANG ◽  
ZHIYONG CHENG ◽  
MIN CHEN
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Animal Models ◽  
Tumor Growth ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Vectors ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Metastasis Model

Background and aims: The spectral properties of enhanced green fluorescent protein (EGFP) used in current visualizable animal models for nasopharyngeal carcinoma (NPC) result in a limited imaging depth. Far-red fluorescent proteins have optimal spectral wavelengths that allow deep tissue penetration, thus are well-suited for the imaging of tumor growth and metastases in live animals. This study aims to establish an imageable animal model of NPC using far-red fluorescent proteins. Methods: Eukaryotic expression vectors of far-red fluorescent proteins, mLumin and Katushka S158A, were separately transfected into 5-8F NPC cells, and cell lines stably expressing the far-red fluorescent proteins were obtained. These cells were intraperitoneally or intravenously injected into mice, and their tumorigenic and metastatic potential were examined through fluorescence imaging. Finally, factors affecting their tumorigenic ability were further assessed through testing side population (SP) cells proportion by flow cytometry. Results: NPC cell line with high tumorigenicity and metastasis (5-8F-mL2) was screened out, which stably expressed far-red fluorescent protein. Intraperitoneal and intravenous injection of 5-8F-mL2 cells resulted in an abdomen metastasis model and a lung metastasis model. In addition, NPC cell line without tumorigenicity (5-8F-Katushka S158A) was screened out. The percentage of SP cells between 5-8F-mL2 and 5-8F-Katushka S158A was found different, suggesting that the SP cell proportion may play a key role in the determination of cell tumorigenic ability. Conclusion: We successfully established animal models for NPC with high tumorigenicity and metastasis using a super-bright far-red fluorescent protein. Owing to the super-brightness and excellent wavelength parameters, these models may be applied as useful tools for intuitive and efficient monitoring of tumor growth and metastasis, as well as assessing the efficacy of nasopharyngeal cancer drugs.

scholarly journals Green-Light-Activated Photoreaction via Genetic Hybridization of Far-Red Fluorescent Protein and Silk

2018 ◽  
Vol 5 (6) ◽  
pp. 1700863 ◽  
Author(s):  
Jung Woo Leem ◽  
Jongwoo Park ◽  
Seong-Wan Kim ◽  
Seong-Ryul Kim ◽  
Seung Ho Choi ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Green Light ◽  
Red Fluorescent Protein

scholarly journals mCherry contains a fluorescent protein isoform that interferes with its reporter function

2021 ◽  
Author(s):  
Maxime Fages-Lartaud ◽  
Lisa Tietze ◽  
Florence Elie ◽  
Rahmi Lale ◽  
Martin Frank Hohmann-Marriott
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Red Fluorescent Protein ◽  
Functional Protein ◽  
Red Fluorescent Proteins ◽  
Expression Studies ◽  
Protein Isoform

AbstractFluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

scholarly journals Sorting out antibiotics' mechanisms of action: a double fluorescent protein reporter for high throughput screening of ribosome and DNA biosynthesis inhibitors

2016 ◽  
pp. AAC.02117-16 ◽  
Author(s):  
Ilya A. Osterman ◽  
Ekaterina S. Komarova ◽  
Dmitry I. Shiryaev ◽  
Ilya A. Korniltsev ◽  
Irina M. Khven ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cost Effective ◽  
High Signal ◽  
Red Fluorescent Protein ◽  
Reporter System ◽  
Ribosome Stalling

In order to accelerate drug discovery, a simple, reliable and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double reporter system for not only antimicrobial activity detection, but also for simultaneous sorting of potential antimicrobials into those that cause ribosome stalling, and others that induce SOS response due to DNA damage. In this reporter system the red fluorescent protein generfpwas placed under the control of the SOS-induciblesulApromoter. The far-red fluorescent protein genekatushka2Swas inserted downstream the tryptophan attenuator where two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator, to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to any ribosome stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need in enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.

scholarly journals Red Fluorescent Protein (DsRed) as a Reporter inSaccharomyces cerevisiae

2001 ◽  
Vol 183 (12) ◽  
pp. 3791-3794 ◽  
Author(s):  
Fernando Rodrigues ◽  
Martijn van Hemert ◽  
H. Yde Steensma ◽  
Manuela Côrte-Real ◽  
Cecı́la Leão
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Dsred Protein ◽  
Green Fluorescent

ABSTRACT We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

scholarly journals Selective Immobilization of Fluorescent Proteins for the Fabrication of Photoactive Materials

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2775 ◽  
Author(s):  
Benítez-Mateos ◽  
Mehravar ◽  
Velasco-Lozano ◽  
RadmilaTomovska ◽  
Salassa ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Fluorescent Proteins ◽  
Green Light ◽  
Metal Chelates ◽  
Ph Indicator ◽  
Carboxylic Groups ◽  
Photoactive Materials ◽  
Technological Developments

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

scholarly journals mBeRFP, a versatile fluorescent tool to enhance multichannel live imaging and its applications

2022 ◽  
Author(s):  
Emmanuel Martin ◽  
Magali Suzanne
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Live Imaging ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Living Organisms ◽  
Illumination Source

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and the development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins, such as mBeRFP, offers interesting new perspectives to bypass this limitation. Here, we constructed a set of mBeRFP-expressing vectors and provided a detailed characterization of this fluorescent protein for in vivo live imaging and its applications in Drosophila. Briefly, we showed that a single illumination source is sufficient to simultaneously stimulate mBeRFP and GFP. We demonstrated that mBeRFP can be easily combined with classical green and red fluorescent protein without any crosstalk. We also showed that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano biologists in their current research.

scholarly journals A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

2020 ◽  
Author(s):  
Benjamin B. Kim ◽  
Haodi Wu ◽  
Yukun A. Hao ◽  
Michael Pan ◽  
Mariya Chavarha ◽  
...  
Keyword(s):  
High Performance ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
High Gain ◽  
Motion Artifacts ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Transmembrane Voltage ◽  
Site Mutagenesis ◽  
Green Fluorescent

AbstractA ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in cells that are undergoing motion. To create a high-performance ratiometric GEVI, we explored the possibility of adding a voltage-independent red fluorophore to ASAP3, a high-gain green fluorescent GEVI. We performed combinatorial multi-site mutagenesis on the cyan-excitable red fluorescent protein mCyRFP1 to enhance brightness and monomericity, creating mCyRFP3. Among red fluorescent proteins tested, mCyRFP3 proved to be the least perturbing when fused to ASAP3. We demonstrate that the red fluorescence of ASAP3-mCyRFP3 (ASAP3-R3) provides an effective reference channel to remove motion artifacts from voltage-induced changes in green fluorescence. Finally we use ASAP3-R3 to visualize membrane voltage changes throughout the cell cycle of motile cells.

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