Stability Of Green Fluorescent Protein Research Articles

Histidine-Specific Bioconjugation for Single-Molecule Force Spectroscopy.

Atomic force microscopy (AFM) based single-molecule force spectroscopy (SMFS) is a powerful tool to study the mechanical properties of proteins. In these experiments, site-specific immobilization of proteins is critical, as the tether determines the direction and amplitude of forces applied to the protein of interest. However, existing methods are mainly based on thiol chemistry or specific protein tags, which cannot meet the need of many challenging experiments. Here, we developed a histidine-specific phosphorylation strategy to covalently anchor proteins to an AFM cantilever tip or the substrate via their histidine tag or surface-exposed histidine residues. The formed covalent linkage was mechanically stable with rupture forces of over 1.3 nN. This protein immobilization method considerably improved the pickup rate and data quality of SMFS experiments. We further demonstrated the use of this method to explore the pulling-direction-dependent mechanical stability of green fluorescent protein and the unfolding of the membrane protein archaerhodopsin-3.

Experimental mapping of short-wavelength phonons in proteins

Phonons are quasi-particles, observed as lattice vibrations in periodic materials, that often dampen in the presence of structural perturbations. Nevertheless, phonon-like collective excitations exist in highly complex systems, such as proteins, although the origin of such collective motions has remained elusive. Here we present a picture of temperature and hydration dependence of collective excitations in green fluorescent protein (GFP) obtained by inelastic neutron scattering. Our results provide evidence that such excitations can be used as a measure of flexibility/softness and are possibly associated with the protein’s activity. Moreover, we show that the hydration water in GFP interferes with the phonon propagation pathway, enhancing the structural rigidity and stability of GFP.

Purification of the Recombinant Green Fluorescent Protein Using Aqueous Two-Phase System Composed of Recyclable CO2-Based Alkyl Carbamate Ionic Liquid.

The formation of aqueous two-phase system (ATPS) with the environmentally friendly and recyclable ionic liquid has been gaining popularity in the field of protein separation. In this study, the ATPSs comprising N,N-dimethylammonium N′,N′-dimethylcarbamate (DIMCARB) and thermo-responsive poly(propylene) glycol (PPG) were applied for the recovery of recombinant green fluorescent protein (GFP) derived from Escherichia coli. The partition behavior of GFP in the PPG + DIMCARB + water system was investigated systematically by varying the molecular weight of PPG and the total composition of ATPS. Overall, GFP was found to be preferentially partitioned to the hydrophilic DIMCARB-rich phase. An ATPS composed of 42% (w/w) PPG 1000 and 4.4% (w/w) DIMCARB gave the optimum performance in terms of GFP selectivity (1,237) and yield (98.8%). The optimal system was also successfully scaled up by 50 times without compromising the purification performance. The bottom phase containing GFP was subjected to rotary evaporation of DIMCARB. The stability of GFP was not affected by the distillation of DIMCARB, and the DIMCARB was successfully recycled in three successive rounds of GFP purification. The potential of PPG + DIMCARB + water system as a sustainable protein purification tool is promising.

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems

Abstract The production of recombinant biopharmaceuticals is highly dependent of a proper choice of the downstream processing stages. Particularly, the purification that must ensure that all the endotoxins (lipopolysaccharides - LPS) are efficiently removed from the final product. The efficient removal of LPS has a direct impact on the manufacturing of therapeutic biopharmaceuticals, since LPS is naturally presented in Gram-negative bacterial expression systems. In order to provide a more simple and faster technique for the purification of green fluorescent protein (GFP) and LPS removal, aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and poly(acrylic acid) (NaPA) and electrolytes were studied. Firstly, the binodal curves of PEG/NaPA + salt systems were established using NaCl, Li2SO4, KI, and KNO3 as additives. It was demonstrated that the formation of ATPS is enhanced following the anion tendency: SO42− > Cl− ≫ NO3− > I−. The stability of GFP in the presence of all different phase forming agents (polymers and salts) was evaluated, and the high biocompatibility of these ATPS demonstrated by the maintenance of GFP fluorescence in all conditions under study. Then, the GFP extraction and LPS removal aptitude of each ATPS was investigated. GFP and LPS were preferentially partitioned into the top (PEG-rich) phase (KGFP > 20), but with a removal of 35% of LPS was attained. Hydrophobic and electrostatic interactions were found to be the major driving forces for GFP partitioning and LPS removal. Moreover, a new effect was found, the presence of high loads of LPS can affect (decrease) the KGFP values (KGFP without LPS > KGFP with 104 EU/mL > KGFP with 106 EU/mL). The ATPS with best GFP extraction performance was selected for the recovery directly from the cell lysates of Escherichia coli. In this experiment, the system composed of 12 wt% PEG 1000 g/mol, 12 wt% NaPA 8000 g/mol, and 0.25 M Li2SO4 lead to a LPS removal (REMLPS) of 13%, KGFP of 20.2, and high selectivity relatively to the total proteins (S = 20), since the majority of contaminants proteins were preferentially partitioned into the bottom (NaPA-rich) phase (purification factor of 4-fold). It is here demonstrated that the ATPS composed of PEG/NaPA + salts as additives can be used as a first step for the recovery of GFP and removal of contaminants (LPS in part, and most contaminant proteins) from cell lysates by applying a system with low polymer content and using mild conditions.

3P065 変異体解析を用いた緑色蛍光蛋白質の安定化機構におけるヒスチジン残基の役割に関する研究(01C. 蛋白質:物性,ポスター,第52回日本生物物理学会年会(2014年度))

3P065 変異体解析を用いた緑色蛍光蛋白質の安定化機構におけるヒスチジン残基の役割に関する研究(01C. 蛋白質:物性,ポスター,第52回日本生物物理学会年会(2014年度))

Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase

The Fc-III tag is a newly developed fusion tag that can be applied to protein purification and detection. In the present work, we use the Fc-III-tagged green fluorescent protein (GFP) and human muscle creatine kinase (CK) as model systems to investigate effects of the Fc-III tag on activities and stabilities of the expressed multicysteine-containing proteins. Our results show the Fc-III tag has no adverse effects on the fluorescence of GFP and reduces the occurrence of GFP misfolding due to incorrect Cys oxidation compared with the His-tagged protein. The activity and stability of the Fc-III-tagged CK is slightly lower than that of the tag-free CK, but is higher than that of the His-tagged CK as determined by the ratio of the oxidized versus reduced CK. A major portion of His-tagged CK is in its oxidized form, while that of the Fc-III-tagged CK is in its reduced form. A folding model of CK with different tags was proposed, which may provide insights into the effect of the Fc-III tag on the conformations of disulfide-bridged proteins.

Characterization of the Influence of the Ionic Liquid 1-Butyl-3-methylimidazolium Chloride on the Structure and Thermal Stability of Green Fluorescent Protein

Ionic liquids (ILs) are finding a vast array of applications as novel solvents for a wide variety of processes that include enzymatic chemistry, particularly as more biocompatible ILs are designed and discovered. While it is assumed that a native or near-native structure is required for enzymatic activity, there is some evidence that ILs alter protein structure and oligomerization states in a manner than can negatively impact function. The IL 1-butyl-3-methylimidazolium chloride, [bmim]Cl, is a well-studied, water-miscible member of the popular 1-alkyl-3-methylimidazolium IL family. To improve our understanding of the impact of water-miscible ILs on proteins, we have characterized the structure and oligomerization state of green fluorescent protein (GFP) in aqueous solutions containing 25 and 50 vol % [bmim]Cl using a combination of optical spectroscopy and small-angle neutron scattering (SANS). Measurements were also performed as a function of temperature to provide insight into the effect of the IL on the thermal stability of GFP. While GFP exists as a dimer in water, the presence of 25 vol % [bmim]Cl causes GFP to transition to a monomeric state. The SANS data indicate that GFP is a great deal less compact in 50 vol % [bmim]Cl than in neat water, indicative of unfolding from the native structure. The oligomerization state of the protein in IL-containing aqueous solution changes from a dimer to a monomer in response to the IL, but does not change as a function of temperature in the IL-containing solution. The SANS and spectroscopic results also demonstrate that the addition of [bmim]Cl to the solution decreases the thermal stability of GFP, allowing the protein to unfold at lower temperatures than in aqueous solution.

Effect of polyethylene glycol on the thermal stability of green fluorescent protein

Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70-95 degrees C. Thermal stability was expressed in decimal reduction time. It was observed that the D-values obtained were almost constant for temperatures of 85, 90, and 95 degrees C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75 degrees C, PEG 600 and 4,000 g/mol stabilized GFP.

Thermus thermophilus-derived protein tags that aid in preparation of insoluble viral proteins

The expression and solubilization of insoluble proteins have been facilitated by the introduction of protein tags. In our analyses of viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1), however, several conventional tag proteins enhanced its expression but failed to solubilize it. Therefore, we decided to explore whether proteins derived from Thermus thermophilus HB8 ( T. th.), a highly heat-stable bacterium, could be used as tag proteins to enhance the solubilization of Vpr. Based on the data accumulated during the recent structural genomics project of T. th., we selected 15 T. th. proteins with high expression levels and solubilities. From this group, we identified a T. th. tag protein that expressed Vpr in a soluble form. Furthermore, two T. th. tag proteins, including the identified one, were found to solubilize the extremely insoluble membrane-spanning domain of the envelope protein of HIV-1. When green fluorescent protein (GFP) was used as a passenger protein of T. th. tags, the brightness and stability of GFP were similar to those of untagged GFP, suggesting that the T. th. tags do not negatively affect the function of the passenger protein. Thus, data of structural genomics can be applied to generate a customized versatile protein tag for protein analyses.

Understanding the role of Arg96 in structure and stability of green fluorescent protein

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide-induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5- and 50-times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl-induced unfolding both in kinetics and in the quasi-equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP.

Comparison of endoplasmic reticulum targeted and non-targeted cytoplasmic GFP as a selectable marker in citrus protoplast transformation

In order to improve transformation efficiency in citrus two green fluorescent protein ( GFP) constructions, an endoplasmic reticulum targeted ( GFP-ER) and cytoplasmic targeted (Cy- GFP), were used to study expression and stability of GFP in transgenic plants. Results from citrus protoplast-GFP transformation showed that GFP-ER had brighter fluorescence than the Cy- GFP. Although both of these constructs gave transient expression at the protoplast level, the non-targeted GFP gave less stable expression than targeted GFP at the micro-calli or plant level. Stable expression of GFP after 4 weeks of culture was observed in about 1.0% and 0.1% of the initial GFP positive cells in GFP-ER and Cy- GFP experiments, respectively. At the mature plant level, there were distinct fluorescent differences between shoots transformed with the two GFP constructs. Moreover, the fluorescence intensity varied among different parts of the plant, being higher in new expanding leaf flush than in old leaves, even when using the same construct. These results demonstrate that GFP-ER-targeted gene expression in citrus tissues is more stable than the Cy- GFP, thereby improving transformation efficiency and the subsequent recovery of transgenic plants. The protoplast-GFP transformation method in citrus could have an advantage over the routinely used Agrobacterium-mediated citrus transformation system that always requires the use of an antibiotic resistance gene.

Study on the thermal stability of green fluorescent protein (GFP) in glucose parenteral formulations

Large volume parenteral solutions (LVPS) that are widely used in the healthcare system must be processed by moist-heat treatment to an assured sterility level in which the efficacy is measured by a bioindicator (BI) that provides fast, accurate and reliable results. This study evaluated the thermal stability of green fluorescent protein (GFP) into glucose-based LVPS (1.5–50%) solutions to determine its utility as a BI for thermal processes. GFP, expressed by Escherichia coli, isolated/purified by TPP/HIC, was diluted in buffered (each 10 mM: Tris–EDTA, pH 8; phosphate, pH 6 and 7; acetate, pH 5) and in water for injection (WFI; pH 6.70 ± 0.40) glucose solutions (1.5–50%) and exposed to constant temperatures from 80 °C to 95 °C. The thermal stability was expressed in decimal reduction time ( D-value, time required to reduce 90% of the GFP fluorescence intensity). At 95 °C, the D-values for GFP in 1.5–50% glucose were: (i) 1.63 ± 0.23 min (pH 5); (ii) 2.64 ± 0.26 min (WFI); (iii) 2.50 ± 0.18 min (pH 6); (iv) 3.24 ± 0.28 min (pH 7); (v) 2.89 ± 0.44 min (pH 8). By the convenient measure of fluorescence intensity and its thermal stability, GFP has the potential as a BI to assay the efficacy of moist-heat processing of LVPS at temperatures ≤100 °C.

Stability of Green Fluorescent Protein (GFP) in Chlorine Solutions of Varying pH

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.

Stability of Green Fluorescent Protein (GFP) in Chlorine Solutions of Varying pH

Stability of Green Fluorescent Protein (GFP) in Chlorine Solutions of Varying pH

Temperature-pressure stability of green fluorescent protein: a Fourier transform infrared spectroscopy study.

Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high-pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild-type GFP (wtGFP) and three of its red shift mutants (AFP, GFP(mut1), and GFP(mut2)) using Fourier transform infrared spectroscopy. For the wt GFP, GFP(mut1), and GFP(mut2) we found that up to 13-14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3-D structure is held responsible for this high-pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure-induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants.

Stability of green fluorescent protein using luminescence spectroscopy: is GFP applicable to field analysis of contaminants?

Green fluorescent protein (GFP) was first isolated in the early 1970s for experimental use from coelenterates or the Pacific jellyfish, Aequorea victoria (Morin and Hastings, 1971). GFP has since become a favored biomarker in the photophysical analysis of molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (Creemers et al., 2000). Here we report using Bacillus subtilis expressing GFP to evaluate the influence of different environmental pH conditions on GFP fluorescence. Emission acquisitions were configured to excite at 471 nm and detect at an emission from 490 to 650 nm at 1-nm increments. Fluorescence intensity was significantly better at pH 7 (4.2×105 cps; P-value<0.01) than at acid or alkaline conditions. GFP is a good biomarker for environments near netural conditions: however, GFP may be unsuitable where soils or waters are below or above pH 7 because of loss in fluorescence intensity. Alternative fluorescent markers and delivery systems must be examined in different environments to optimize responses from bioreporter molecules.

Green Fluorescent Protein as a Reporter for Macromolecular Localization in Bacterial Cells

Green fluorescent protein (GFP) is a highly useful fluorescent tag for studying the localization, structure, and dynamics of macromolecules in living cells, and has quickly become a primary tool for analysis of DNA and protein localization in prokaryotes. Several properties of GFP make it an attractive and versatile reporter. It is fluorescent and soluble in a wide variety of species, can be monitored noninvasively by external illumination, and needs no external substrates. Localization of GFP fusion proteins can be analyzed in live bacteria, therefore eliminating potential fixation artifacts and enabling real-time monitoring of dynamics in situ. Such real-time studies have been facilitated by brighter, more soluble GFP variants. In addition, red-shifted GFPs that can be excited by blue light have lessened the problem of UV-induced toxicity and photobleaching. The self-contained domain structure of GFP reduces the chance of major perturbations to GFP fluorescence by fused proteins and, conversely, to the activities of the proteins to which it is fused. As a result, many proteins fused to GFP retain their activities. The stability of GFP also allows detection of its fluorescence in vitro during protein purification and in cells fixed for indirect immunofluorescence and other staining protocols. Finally, the different properties of GFP variants have given rise to several technological innovations in the study of cellular physiology that should prove useful for studies in live bacteria. These include fluorescence resonance energy transfer (FRET) for studying protein–protein interactions and specially engineered GFP constructs for direct determination of cellular ion fluxes.

32] Spectral variants of green fluorescent protein

Publisher Summary The use of green fluorescent protein (GFP) for many applications in biological sciences strongly depends on its spectral properties. GFP originates from the jellyfish Aequorea victoria, in which its fluorescence serves to convert the blue light produced by aequorin to green light. Purified protein that retains fluorescence has been the object of intense investigations for many years. Denaturation by harsh treatment, however, can abolish fluorescence and change the absorption spectrum of GFP. The spectrum of the native protein is also affected by many other parameters, such as mutations, protein concentration, ionic strength, temperature, and pH. However, the stability of GFP in a wide pH range (pH 5-12), at high temperature (T m = 78°), in chaotropic reagents (8 M urea), and against proteolysis makes it attractive for practical use. Expression has been optimized by mutations that change the promoter, ribosome binding site, or codon usage; eliminate splicing sites; or enhance folding. Even though such procedures can drastically increase the overall fluorescence by GFP per cell, as the spectra of purified GFP remain the same in all these cases. Numerous mutations in and around the chromophore, however, do change the spectra. Taken together, these advances enable the use of GFP for detection of promoter activity and for localization of fusion proteins. New applications such as simultaneous localization of two fusion proteins and fluorescence resonance energy transfer (FRET) for detection of Ca 2+ concentration or proteolytic activity have become possible through the use of GFP mutants of different colors.