RESUMO
This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb(3+) and from sensitized Tb(3+) to acceptor--the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.
Assuntos
Técnicas Biossensoriais/métodos , Caspase 3/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/química , Térbio/farmacologia , Sequência de Aminoácidos , Caspase 3/genética , Proteínas Luminescentes/genética , Metaloproteínas/química , Metaloproteínas/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Térbio/química , Proteína Vermelha FluorescenteRESUMO
Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator's dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.
Assuntos
Proteínas de Escherichia coli/genética , Corantes Fluorescentes , Peróxido de Hidrogênio/análise , Proteínas Repressoras/genética , Células 3T3 , Animais , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , Microscopia Confocal , Mutação , Oxirredução , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
GFP-like fluorescent proteins (FPs) are crucial in biological and biomedical studies. The majority of FP purification techniques either include multiple time-consuming chromatography steps with a low yield of the desired product or require prior protein modification (addition of special tags). In the present work, we propose an alternative ethanol extraction-based technique previously used for GFP purification and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purification of any amount of protein and requires no expensive reagents and equipment.
Assuntos
Etanol/química , Proteínas de Fluorescência Verde/isolamento & purificação , Animais , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein's hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.
Assuntos
Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Aminoácidos/genética , Proteínas Luminescentes/química , Espectrometria de Massas , Proteínas Recombinantes/químicaRESUMO
The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.
Assuntos
Herpesvirus Suídeo 1/isolamento & purificação , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Cricetinae , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Imunoensaio/métodos , Luminescência , Proteínas Luminescentes/genética , Fases de Leitura Aberta , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Viral/genética , RNA Viral/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.
Assuntos
Células Eucarióticas/patologia , Transferência Ressonante de Energia de Fluorescência/métodos , Substâncias Luminescentes , Proteínas Luminescentes , Imagem Corporal Total/métodos , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Caspase 3/metabolismo , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Camundongos , Camundongos Nus , Proteína Vermelha FluorescenteRESUMO
The yellow fluorescent protein from coral (zFP538) forms aggregates in water solutions. According to dynamic light scattering and gel filtration data, the aggregation number is approximately 1000-10000 at pH 8-9 and protein concentration 1 mg/mL. Gel filtration demonstrated that dissociation of the aggregates takes place upon dilution, and the molecular weight of the aggregates decreases with pH. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were used to obtain images of zFP538 in the solid state. It was shown that protein films are comprised of fluorescent ellipsoidal granules with a 50-300 nm major axis and a 30-130 nm minor axis. The dependence of zFP538 fluorescence on protein concentration between 1.2 x 10(-)(9) and 5.5 x 10(-)(7) M can be divided in two linear regions with different slopes indicating the existence of at least two different forms of zFP538. The fluorescence of zFP538 decreases with time upon acidification, and the decrease depends on pH and protein concentration. Between pH 3.5 and pH 5.5, relative residual fluorescence is higher for concentrated zFP538 solutions (about 10(-)(6) M) as compared with diluted ones (10(-)(7) M and below). Aggregation makes zFP538 more stable against fluorescence quenching upon acidification: the decrease in zFP538 fluorescence at protein concentration 1 mg/mL is completely reversible, unlike that observed for less concentrated solutions. This phenomenon may be due to the decrease in the freedom of chromophore mobility in zFP538 aggregates.
Assuntos
Antozoários/química , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Animais , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Força Atômica/métodos , Modelos Químicos , Peso Molecular , Dinâmica não Linear , Desnaturação Proteica , Soluções , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Análise Espectral/métodos , TitulometriaRESUMO
Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.
Assuntos
Escherichia coli/genética , Proteínas de Insetos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/citologia , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Proteínas Luminescentes/genética , Proteínas Luminescentes/isolamento & purificação , Proteínas Luminescentes/metabolismo , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Fatores de TempoRESUMO
Green fluorescent protein (GFP) is widely used as an excellent reporter module of the fusion proteins. The unique structure of GFP allows isolation of the active fluorescent protein directly from the crude cellular sources by extraction with organic solvents. We demonstrated the stable expression of four short polypeptides fused to GFP in Escherichia coli cells, including antimicrobial cationic peptides, which normally kill bacteria. EGFP module protected fusion partners from the intracellular degradation and allowed the purification of the chimerical proteins by organic extraction. The nature of the polypeptide fused to GFP, as opposed to the order of GFP and the polypeptide modules in the fusion protein, influenced the efficiency of the described purification technique.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Expressão Gênica , Proteínas Luminescentes/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/isolamento & purificação , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/isolamento & purificação , Acetil-CoA Carboxilase/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Ácido Graxo Sintase Tipo II , Proteínas de Fluorescência Verde , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.
Assuntos
Bioensaio , Biotina/análise , Medições Luminescentes , Cifozoários/química , Equorina/química , Equorina/genética , Equorina/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.