Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy

Santos, Nathalia Vieira dos; Saponi, Carolina Falaschi; Greaves, Tamar L.; Pereira, Jorge Fernando Brandão

doi:10.1039/c9ra02567g

Cited by 16 publications

(6 citation statements)

References 26 publications

(40 reference statements)

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“…Unexpectedly, most of the stress response genes were not significantly induced in our study even though these were significantly upregulated in the previous study, despite the similarity in blue light treatment, age, and eye color of the flies used ( Figure 3 A). Because the EGFP used in the GFP KASH nuclear membrane tag absorbs blue light in the 440–500 nm range with a λ max of 488 nm [ 50 ], we initially wondered if the high levels of GFP KASH protein present in the photoreceptors might diminish the negative impact of blue light on photoreceptor survival in the aging white-eyed flies, as well as our blue light exposure, which has a λ max of 465 nm [ 20 ]. We note that the white LEDs present in the incubator used to raise these flies have a strong peak in the blue wavelengths ( Figure S1 ), which is a common feature of commercially available white LEDs [ 51 , 52 , 53 ].…”

Section: Resultsmentioning

confidence: 99%

Aging and Light Stress Result in Overlapping and Unique Gene Expression Changes in Photoreceptors

Escobedo

Stanhope

Dong

et al. 2022

Genes

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Advanced age is one of the leading risk factors for vision loss and eye disease. Photoreceptors are the primary sensory neurons of the eye. The extended photoreceptor cell lifespan, in addition to its high metabolic needs due to phototransduction, makes it critical for these neurons to continually respond to the stresses associated with aging by mounting an appropriate gene expression response. Here, we sought to untangle the more general neuronal age-dependent transcriptional signature of photoreceptors with that induced by light stress. To do this, we aged flies or exposed them to various durations of blue light, followed by photoreceptor nuclei-specific transcriptome profiling. Using this approach, we identified genes that are both common and uniquely regulated by aging and light induced stress. Whereas both age and blue light induce expression of DNA repair genes and a neuronal-specific signature of death, both conditions result in downregulation of phototransduction. Interestingly, blue light uniquely induced genes that directly counteract the overactivation of the phototransduction signaling cascade. Lastly, unique gene expression changes in aging photoreceptors included the downregulation of genes involved in membrane potential homeostasis and mitochondrial function, as well as the upregulation of immune response genes. We propose that light stress contributes to the aging transcriptome of photoreceptors, but that there are also other environmental or intrinsic factors involved in age-associated photoreceptor gene expression signatures.

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Section: Resultsmentioning

confidence: 99%

Aging and Light Stress Result in Overlapping and Unique Gene Expression Changes in Photoreceptors

Escobedo

Stanhope

Dong

et al. 2022

Genes

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“…From these studies, histidine‐, arginine‐ and lysine‐rich peptides were identified as potentially suitable affinity purification/immobilization tags for both iron oxide and silica. [ 19,22,24,26,27 ] However, gaps still exist regarding the study of these interactions in multi‐compound solutions such as cellular lysates and the comparative performance of different tags in similar conditions. In such complex mixtures, the well‐known His6 tag and the recently developed (HR)4/(RH)4 tag have been effectively used as affinity purification/immobilization tags for both BIONs and BSiNs.…”

Section: Discussionmentioning

confidence: 99%

Recombinant protein purification and immobilization strategies based on peptides with dual affinity to iron oxide and silica

Aguiar

Domingues

2023

Biotechnology Journal

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Iron oxide and silica‐based materials have emerged as attractive protein purification and immobilization matrices. His6 has been reported as an effective affinity tag for both iron oxide and silica. Here, the silica‐binding tags CotB1p and Car9 were shown to work as effectively as iron oxide‐binding tags. Using EGFP as a model protein, commercially available bare iron oxide (BIONs) or silicon dioxide (BSiNs) nanoparticles as low‐cost purification/immobilization matrices, and non‐hazardous and mild binding and elution conditions, adsorption and desorption studies were performed with lysates from Escherichia coli‐producing cells to compare the performance of these dual‐affinity tags. Under the conditions tested, the His6 tag stood out as the best‐performing tag, followed by CotB1p. Our findings concluded the promising combination of these tags, BIONs and BSiNs for one‐step purification of recombinant proteins, and two‐step purification and immobilization of recombinant proteins without intermediate buffer exchange. This proof of concept work set the ground for future evaluation of these purification and immobilization strategies using other proteins with different properties, which will be of interest to expand their utility and applicability.

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“…To define the best wavelengths for levansucrase quantification by fluorescence with minimal interference of the ABS phase-forming agents, the enzyme and the different ABS phases were evaluated by 3D fluorescence spectroscopy. The 3D spectra were acquired at 25 °C with a variable wavelength range of excitation from 220 to 550 nm and emission from 280 to 560 nm (interval of 2.0 nm for the excitation and emission range), excitation bandwidth of 10 nm, and emission bandwidth of 1 nm, using the spectrofluorophotometer RF-6000 (Shimadzu, Kyoto, Japan). , The 3D spectrum of levansucrase is presented in Figure S1, while the spectra of the different ABS phases and comparison with levansucrase are shown in Figure S2 (Supporting Information). The calibration curves of the levansucrase quantification methods are illustrated in Figure S3.…”

Section: Methodsmentioning

confidence: 99%

Use of Tunable Copolymers in Aqueous Biphasic Systems for Extractive Bioconversion Aimed at Continuous Fructooligosaccharide Production

Mendonça

Veríssimo

Pereira

et al. 2023

ACS Sustainable Chem. Eng.

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Aqueous biphasic systems (ABSs) based on sodium polyacrylate (NaPA), ethylene oxide/propylene oxide (EO/PO) polymers, and (EO) x -(PO) y -(EO) x triblock copolymers were prepared and applied aiming at continuous fructooligosaccharide (FOS) production and separation. EO/PO hydrophilicity/hydrophobicity balance had a significant effect on ABS formation. To develop an integrated process including the continuous enzymatic (levansucrase) production of FOSs and their purification while improving the production yield by further glucose separation, the potential of these novel polymer-based ABSs as alternative platforms was investigated. They were used for the partitioning of different carbohydrates (FOS, sucrose, d-fructose, and d-glucose) and levansucrase. Results revealed a highly polymer-dependent partition of carbohydrates and a poorly dependent one of the enzymes. Changing EO/PO and copolymers, FOS was purified with high yields (72.94–100.0%). Using polypropylene glycol 400 + NaPA 8000-based ABS, the FOS was precipitated in the interphase and separated from the other components. Pluronic PE-6800 + NaPA 8000 was identified as the best ABS for FOS continuous production and in situ purification, while minimizing levansucrase inhibition by d-glucose. This system allowed selective partition of FOSs and d-glucose toward the top phase and that of levansucrase and its substrates toward the bottom one. COnductor-like Screening MOdel for Real Solvent (COSMO-RS) suggested that ABS formation may have been due to NaPA and polymer/copolymer competition to form hydrogen bonds with water molecules. Moreover, the partition of FOSs and sugar may have been the result of a subtle balance between hydrogen bonding of sugar and polymer/copolymer and electrostatic misfit of solute with NaPA. Finally, two integrated processes were proposed to be applied with real FOS extracts obtained by chemical or enzymatic hydrolysis of inulin or by transfructosylation of concentrated sucrose solutions using bacterial levansucrases.

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Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy

Abstract: Three-dimensional fluorescence spectroscopy as a powerful tool to identify a new fluorescence peak of Enhanced Green Fluorescent Protein (EGFP).

Cited by 16 publications

References 26 publications

Aging and Light Stress Result in Overlapping and Unique Gene Expression Changes in Photoreceptors

Aging and Light Stress Result in Overlapping and Unique Gene Expression Changes in Photoreceptors

Recombinant protein purification and immobilization strategies based on peptides with dual affinity to iron oxide and silica

Use of Tunable Copolymers in Aqueous Biphasic Systems for Extractive Bioconversion Aimed at Continuous Fructooligosaccharide Production

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