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Fluorescent Proteins
Published in Guy Cox, Fundamentals of Fluorescence Imaging, 2019
The first step in what has often been termed as the “GFP revolution” in cell biology was made by Osamu Shimomura in 1961, when he discovered a green fluorescent protein accidentally in a bioluminescent jellyfish Aequorea victoria (avGFP) while studying aequorin, a chemoluminescent calcium-activated photoprotein [13]. Aequorin and GFP were found localized in the jellyfish light organs and blue fluorescent emissions by aequorin were shown to excite green GFP fluorescence by Förster resonance energy transfer between the two molecules [14]. Another GFP-type protein was found in a bioluminescent relative of corals, the sea pansy Renilla reniformis [15], and it likewise transforms blue light emitted by luciferase into green light. Since both proteins form components of a bioluminescent system, it was assumed that GFPs were restricted to bioluminescent organisms.
An Overview of Immunoassays
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Caroline Murphy, Sarah Gilgunn, Richard O’Kennedy
Many bright photoproteins have been discovered in the darkest places. Aqueorin was originally found in the jellyfish Aequoria victoria [43] and is a stable complex composed of an apoprotein (apoaequorin), a chromophore (coelenterazine) and molecular oxygen [60]. Recombinant aequorin has successfully been used in immunoassays to detect the hormone cortisol [61]. Recombinant aequorin is chemically conjugated to cortisol at different molar ratios, and the resulting conjugates are assessed for the highest level of bioluminescence [61]. The development of a chimeric antibody incorporating aequorin has also been shown to be useful. The apoaequorin gene was sub-cloned into a mammalian expression vector and the constant domain of the antibody replaced by the photoprotein. This method is more reliable than chemically linking the photoprotein to the antibody as chemical modifications can often lead to ‘batch-to-batch’ variations or loss of antibody binding activity [45]. When aequorin was first discovered in 1962, Shimomura et al. simultaneously discovered a second protein that has also become a fundamental bioimaging tool [43]. Green fluorescence protein (GFP) has been frequently used as a genetically encoded fluorescence marker. The fluorescence protein (FP) family has expanded to over 1000 variant forms, each exhibiting different excitation and emission wavelengths, thus, all displaying different colours (e.g. monomeric Orange (mOrange) (excitation 548 nm/emission 562 nm) to mRuby (ex. 558 nm/em. 605 nm)). A review by Nienhaus and colleagues contains a selection of some of the most widely used FPs [62].
Introduction
Published in Shoogo Ueno, Bioimaging, 2020
Aequorin and green fluorescent protein (GFP) were discovered by Osamu Shimomura (1928–2018) in Japan in 1962 (Shimomura et al., 1962). Using the jelly fish Aequorea victoria, Shimomura’s target was a luminescent substance, aequorin. GFP was isolated as a by-product of aequorin owing to its bright conspicuous fluorescence. Both are unusual proteins but they had no particular importance when Shimomura and his co-authors first reported them; 40 years after their discovery, they are well known and widely used, aequorin as a calcium probe and GFP as a marker protein (Shimomura, 2005).
Improving the soluble expression of aequorin in Escherichia coli using the chaperone-based approach by co-expression with artemin
Published in Preparative Biochemistry and Biotechnology, 2018
Elaheh Khosrowabadi, Zeinab Takalloo, Reza H. Sajedi, Khosro Khajeh
Aequorin is a photoprotein originally isolated from the bioluminescent jellyfish Aequorea victoria.[20] This calcium-activated photoprotein is widely used for its unique advantages, such as detection within few seconds at the attomole level, high sensitivity and low background noise, high quantum yield, being active at physiological pH, and harmless application.[13–15] Additionally, aequorin has numerous applications in cell trafficking, biosensing, and immunoassays.[20] Despite the vast application of aequorin, this protein is susceptible to aggregation and proteolysis degradation[15] and a high level of the protein mostly accumulates in an insoluble form when it is over-expressed in bacterial cells. Therefore, in some previous studies, several approaches were used to improve its soluble expression.[21–23] In the present work, we have attempted to increase the yield of soluble, correctly folded aequorin produced in E. coli cells following its simultaneous expression with artemin using the single and multiple plasmid-based strategies. Therefore, artemin along with aequorin was co-expressed under the optimal expressing conditions to decrease the inclusion body formation of aequorin and increase its soluble content.