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Animal Biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Green fluorescent protein (GFP) is a protein isolated from coelenterates such as the Pacific jellyfish, Aequoria victoria, or from the sea pansy, Renilla reniformis. Although many other marine organisms have similar GFPs, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. In modified forms, it has been used to make biosensors, and many animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism. The GFP gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector, or cell transformation. To date, the GFP gene has been introduced and expressed in many bacteria, yeast and other fungi, fish (such as zebrafish), plants, flies, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on October 10, 2008, for their discovery and development of the GFP.
Animal biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
GFP is a protein isolated from coelenterates such as the Pacific jellyfish, Aequorea victoria, or from the sea pansy, Renilla reniformis. Although many other marine organisms have similar GFPs, GFP traditionally refers to the protein first isolated from the jellyfish A. victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The GFP from the sea pansy (R. reniformis) has a single major excitation peak at 498 nm. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. In modified forms, it has been used to make biosensors, and many animals have been created that express GFP as a proof of concept that a gene can be expressed throughout a given organism. The GFP gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector, or cell transformation. To date, the GFP gene has been introduced and expressed in many bacteria, yeast and other fungi, fish (such as zebrafish), plant, fly, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in chemistry on October 10, 2008, for their discovery and development of the GFP.
Fluorescent Proteins
Published in Guy Cox, Fundamentals of Fluorescence Imaging, 2019
The increasing need to understand the function of genes and proteins in cells and organisms and the advances in imaging platforms to image these events in radically new ways have contributed to the explosive development of fluorescent protein technologies in the last quarter of the century. Numerous proteins exhibit fluorescence, but the term fluorescent proteins (FPs) generally defines a diverse group of proteins found in marine organisms that are homologues of the prototypical jellyfish (Aequorea victoria) green fluorescent protein (GFP, avGFP, or wild-type wtGFP). This 28-kDa protein has bright green fluorescence at UV or blue light excitation. The main advantage of GFP-like proteins over other fluorescent proteins, stains or tags is their ability to form an internal chromophore without requiring any accessory cofactors, enzymes or substrates other than molecular oxygen [1–4]. This unique property makes possible the formation of the chromophore in live organisms, tissues and cells. Consequently, one of the most common uses for GFPs is as gene tags fused to proteins or polypeptides of interest so that the expression of the target protein becomes accompanied by the fluorescent glow of GFP. What was previously invisible becomes visible, and standard tools of fluorescence and confocal microscopy can be used to observe the target protein in a living cell as it performs its function. GFP-like FPs have fueled a new era in cell biology and biomedicine and are widely used as genetically encoded fluorescent labels to visualize structures and dynamic processes in living cells and organisms [2, 5–8].
Exploring the potential of microalgae cell factories for generation of biofuels
Published in Biofuels, 2023
Dixita Chettri, Ashwani Kumar Verma, Anil Kumar Verma
In another approach, the intracellular spectral recomposition (ISR) technique was developed in Phaeodactylum tricornutum to mediate the absorption of excess blue light by intracellular emission to improve the efficiency of light utilization. The technique was used to facilitate the expression of green fluorescent protein (GFP). The resulting genetically modified algae showed a 28% improved photosynthetic rate with reduced non-photochemical quenching (NPQ) and increased quantum yield. In addition, the transformed P. tricornutum showed a significant improvement in biomass yield when cultured in open ponds [93]. Recently, a 3-fold increase in in photosynthetic efficiency was achieved by reducing the optical cross-section of LHA by selectively decreasing the content of chlorophyll b and subunits of the peripheral complex. In this study, we described a translational control system to adjust the size of LHA by expressing the CAO gene encoding chlorophyllide-a oxygenase with an extension in the 5′ mRNA encoding the binding site for the translational repressor Nab1 in the CAO knockout line. The resulting strain showed a 2-fold increase in biomass productivity and the ability to facilitate NPQ and out-state transitions [94].
A photoelectron imaging and quantum chemistry study of the deprotonated cyan fluorescent protein chromophore anion
Published in Molecular Physics, 2019
Michael A. Parkes, Anabel Bennett, Helen H. Fielding
Green fluorescent protein (GFP), and its family of variants, have revolutionised many areas of the life sciences by enabling in vivo monitoring of biological and biochemical processes [1–5]. The chromophore that lies at the heart of GFP is formed by intramolecular cyclisation of a serine-tyrosine-glycine sequence, at positions 65–67 of the protein, and it is localised in the centre of a β-barrel structure. The chromophore exists in neutral and anionic (deprotonated) forms which have different hydrogen-bond networks around the phenolic oxygen on the tyrosine residue and different spectral properties. The anionic form absorbs light around 480 nm and fluoresces around 508 nm with a high quantum yield () [6]. The neutral form absorbs light around 395 nm, after which it can undergo ultrafast excited state proton transfer to form the deprotonated anionic form of the chromophore which then fluoresces around 508 nm.
Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells
Published in Preparative Biochemistry and Biotechnology, 2019
Omid Mohammadian, Masoumeh Rajabibazl, Es’hagh Pourmaleki, Hadi Bayat, Roshanak Ahani, Azam Rahimpour
Second generation HIV-1 derived vector particles pseudotyped with the envelope of vesicular stomatitis virus (VSV-G) were produced by co-transfection of HEK293T cells with lentiviral transfer vector (pCDH-mAb or pCDH-mAb-SAR), together with psPAX2 (packaging plasmid expressing the viral gag and pol genes, Addgene plasmid #12260) and pMD2G (envelope plasmid expressing of VSV-G glycoprotein Addgene plasmid #12259) in 6-well plates using TurboFect transfection reagent (Thermo Fisher Scientific, USA). Plasmids were combined at the ratio of 3:2:1, for the transfer vector:psPAX2:pMD2G, respectively. 48 and 72 h post-transfection, cell culture supernatants were harvested, pooled, passed through a 0.45 µm pore size filter to remove cell debris, and stored at −80 °C. Transfection was verified by observing the expression of GFP protein in transfected cells using fluorescence microscopy.