Denaturation studies reveal significant differences between GFP and blue fluorescent protein

Saeed, Ibtesam; Ashraf, S. Salman

doi:10.1016/j.ijbiomac.2009.05.010

Cited by 36 publications

(30 citation statements)

References 20 publications

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“…To do so, blue fluorescent protein (BFP)-expressing plasmids, containing AOX1 promoter, zeocin resistance cassette and each of the three different ARS sequences, were constructed starting from the commercial vector pSEC-SUMO (see ‘‘ Methods ’’ section). BFP was selected as reporter gene, due to its fast maturation, high photostability and pH-stability [ 24 – 26 ].…”

Section: Resultsmentioning

confidence: 99%

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications

et al. 2016

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BackgroundRecombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence (“panARS”) in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells.ResultsA panARS-based episomal vector was evaluated using blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence from colonies carrying panARS-BFP outperformed the level of signal obtained from integrative controls by several-fold, whereas endogenous sequences, identified from the P. pastoris genome, were not as efficient in terms of protein production. At the single cell level, panARS-BFP clones showed lower interclonal variability but higher intraclonal variation compared to their integrative counterparts, supporting the idea that heterologous protein production could benefit from episomal plasmids. Finally, efficiency of 2-fragment and 3-fragment in vivo recombination was tested using varying lengths of overlapping regions and molar ratios between fragments. Upon optimization, minimal background was obtained for in vivo assembled vectors, suggesting this could be a quick and efficient method to generate of episomal plasmids of interest.ConclusionsAn expression vector based on the panARS sequence was shown to outperform its integrative counterparts in terms of protein productivity and interclonal variability, facilitating recombinant protein expression and screening. Using optimized fragment lengths and ratios, it was possible to perform reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the applicability of panARS episomal vectors for synthetic biology approaches.

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Section: Resultsmentioning

confidence: 99%

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications

et al. 2016

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“…The median fluorescence intensity of the T cells treated with RNAlater declined by 80% as measured by flow cytometry. Cellular proteins differ widely in their conformational stabilities, and studies have shown that the fluorescence of GFP and its variants is pH dependent [13]. It has been reported that 80% of GFP fluorescence is lost at pH 6.5 and lower [13].…”

Section: Resultsmentioning

confidence: 99%

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

et al. 2010

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BackgroundFlow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail.FindingsTo address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment.ConclusionsWhen considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.

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“…In principle, there are two sources of tissue protein loss: protein denaturation due to the clearing solution and protein release in the clearing solution. Since the clearing solution (pH 8.5 containing SDS) is not likely to denature biological fluorophores (Saeed and Ashraf, 2009 ), the loss of fluorescent protein from the slice can be assumed equal to that released in the clearing solution. In this study, to quantify GFP loss, at each time point, 200 μL samples of the clearing solution were analyzed in triplicate with a plate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany, Ex: 485 nm and Em: 544 nm).…”

Section: Methodsmentioning

confidence: 99%

Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue

et al. 2016

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Tissue clarification has been recently proposed to allow deep tissue imaging without light scattering. The clarification parameters are somewhat arbitrary and dependent on tissue type, source and dimension: every laboratory has its own protocol, but a quantitative approach to determine the optimum clearing time is still lacking. Since the use of transgenic mouse lines that express fluorescent proteins to visualize specific cell populations is widespread, a quantitative approach to determine the optimum clearing time for genetically labeled neurons from thick murine brain slices using CLARITY2 is described. In particular, as the main objective of the delipidation treatment is to clarify tissues, while limiting loss of fluorescent signal, the "goodness" of clarification was evaluated by considering the bulk tissue clarification index (BTCi) and the fraction of the fluorescent marker retained in the slice as easily quantifiable macroscale parameters. Here we describe the approach, illustrating an example of how it can be used to determine the optimum clearing time for 1 mm-thick cerebellar slice from transgenic L7GFP mice, in which Purkinje neurons express the GFP (green fluorescent protein) tag. To validate the method, we evaluated confocal stacks of our samples using standard image processing indices (i.e., the mean pixel intensity of neurons and the contrast-to -noise ratio) as figures of merit for image quality. The results show that detergent-based delipidation for more than 5 days does not increase tissue clarity but the fraction of GFP in the tissue continues to diminish. The optimum clearing time for 1 mm-thick slices was thus identified as 5 days, which is the best compromise between the increase in light penetration depth due to removal of lipids and a decrease in fluorescent signal as a consequence of protein loss: further clearing does not improve tissue transparency, but only leads to more protein removal or degradation. The rigorous quantitative approach described can be generalized to any clarification method to identify the moment when the clearing process should be terminated to avoid useless protein loss.

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Denaturation studies reveal significant differences between GFP and blue fluorescent protein

Cited by 36 publications

References 20 publications

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue

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