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1.
Nat Commun ; 11(1): 5199, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060572

RESUMO

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.


Assuntos
ADP-Ribosilação/fisiologia , Adenosina Difosfato Ribose/metabolismo , Engenharia de Proteínas/métodos , Proteínas/metabolismo , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/genética , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutagênese , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/isolamento & purificação , Proteômica/métodos
2.
J Chromatogr A ; 1627: 461415, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823113

RESUMO

A molecularly imprinted polymeric monolith was synthesized in an aqueous environment in 15 min via UV-irradiation. The imprinted monolith was composed of hydroxyethyl methacrylate as monomer, dimethyl amino ethyl methacrylate as functional monomer, methylene bisacrylamide and piperazine diacrylamide as crosslinkers and human serum albumin as template molecule. The synthesis took place in a PDMS-based device (2.5 cm long) yielding a micro-solid phase extraction column (3 × 5 mm) with two built-in fingertight connectors for an infusion pump and fraction collector. The imprinted monolith displayed the characteristic features of a porous polymeric monolith, had dimethyl amino ethyl methacrylate and human serum albumin as functional groups within the monolith and showed high permeability (0.51 × 10-13 m2). 85% of the imprinted cavities were readily available for rebinding of human serum albumin with an imprinting factor of 1.3. In comparison to a non-imprinted monolith, molecular imprinting increased human serum albumin adsorption by > 30%. Imprinted monolith displayed selectivity for human serum albumin over other competing proteins (human transferrin, ovalbumin and carbonic anhydrase) with similar or different isoelectric points and size. Human serum albumin was adsorbed (in dynamic mode) with > 98% selectivity from diluted human plasma using the imprinted monolith device. Device to device reproducibility and reusability of the device for 5 cycles showcase the imprinted monolith micro-device efficiency.


Assuntos
Impressão Molecular , Proteínas/isolamento & purificação , Microextração em Fase Sólida/instrumentação , Adsorção , Etilaminas/química , Humanos , Metacrilatos/química , Permeabilidade , Polímeros/química , Porosidade , Reprodutibilidade dos Testes , Albumina Sérica Humana/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier
3.
J Chromatogr A ; 1627: 461420, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823115

RESUMO

Monitoring preparative protein chromatographic steps by in-line spectroscopic tools or fraction analytics results in medium or large sized data matrices. Multivariate Curve Resolution (MCR) serve to compute or to estimate the concentration values of the pure components only from these data matrices. However, MCR methods often suffer from an inherent solution ambiguity which underlies the factorization problem. The typical unimodality of the chromatographic profiles of pure components can support the chemometric analysis. Here we present the pure components estimation process within the framework of the area of feasible solutions, which is a systematic approach to represent the range of all possible solutions. The unimodality constraint in combination with Pareto optimization is shown to be an effective method for the pure component calculation. Applications are presented for chromatograms on a model protein mixture containing ribonuclease A, cytochrome c and lysozyme and on a two-dimensional chromatographic separation of a monoclonal antibody from its aggregate species. The root mean squared errors of the first case study are 0.0373, 0.0529 and 0.0380 g/L compared to traditional off-line analytics. The second case study illustrates the potential of recovering hidden components with MCR from off-line reference analytics.


Assuntos
Produtos Biológicos/análise , Cromatografia/métodos , Preparações Farmacêuticas/análise , Anticorpos Monoclonais/isolamento & purificação , Estudos de Viabilidade , Análise dos Mínimos Quadrados , Análise Multivariada , Proteínas/isolamento & purificação , Reprodutibilidade dos Testes
4.
Nat Protoc ; 15(8): 2341-2386, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690956

RESUMO

Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules are therefore of major significance in basic and translational biology. However, detection and quantification of protein complexes on a proteome-wide scale is technically challenging. We have recently extended the targeted proteomics rationale to the level of native protein complex analysis (complex-centric proteome profiling). The complex-centric workflow described herein consists of size exclusion chromatography (SEC) to fractionate native protein complexes, data-independent acquisition mass spectrometry to precisely quantify the proteins in each SEC fraction based on a set of proteotypic peptides and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler (https://github.com/CCprofiler/CCprofiler). Complex-centric proteome profiling captures most proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to cultured cells and can potentially also be adapted to primary tissue and does not require any genetic engineering of the respective sample sources. At present, it requires ~8 d of wet-laboratory work, 15 d of mass spectrometry measurement time and 7 d of computational analysis.


Assuntos
Cromatografia em Gel , Espectrometria de Massas , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteômica/métodos , Células HEK293 , Humanos
5.
J Chromatogr A ; 1625: 461309, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709351

RESUMO

The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion. Cooperative adsorption resulted in negative or positive deviations from the Langmuir model for both single and multicomponent isotherms. The model was used to assess possible contributions of different adsorption mechanisms of proteins and their structurally different forms to the overall adsorption pattern, as well as to simulate chromatographic band profiles under different loading conditions. For proteins with unstable structure, the overall adsorption isotherm was dominated by binding of unfolded species at low surface coverage and by positive cooperative adsorption at high surface coverage. Furthermore, regardless of structural stability, exclusion effects influenced strongly adsorption equilibrium, particularly at low surface coverages. In case of chromatographic elution, i.e. under dynamic conditions, unfolding, negative cooperative adsorption, and kinetic effects governed the retention behavior and determined peak shapes, whereas the effect of positive cooperative adsorption was negligible.


Assuntos
Cromatografia/métodos , Interações Hidrofóbicas e Hidrofílicas , Proteínas/isolamento & purificação , Adsorção , Animais , Calibragem , Galinhas , Cinética , Lactalbumina/isolamento & purificação , Muramidase/isolamento & purificação , Ligação Proteica , Soroalbumina Bovina/isolamento & purificação , Temperatura
6.
J Vis Exp ; (160)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32568242

RESUMO

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.


Assuntos
Análise de Dados , Proteômica , Manejo de Espécimes , Espectrometria de Massas em Tandem , Animais , Clorofórmio/química , Indicadores e Reagentes , Metanol/química , Camundongos , Peptídeos/metabolismo , Proteínas/isolamento & purificação , Proteoma/análise
7.
Science ; 368(6494): 980-987, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32467387

RESUMO

Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.


Assuntos
Peptídeos/síntese química , Proteínas/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Peptídeos/química , Peptídeos/isolamento & purificação , Domínios Proteicos , Dobramento de Proteína , Proteínas/química , Proteínas/isolamento & purificação
8.
J Chromatogr A ; 1621: 461064, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32336499

RESUMO

The performance of columns packed with 1.7 µm particles for aqueous size-exclusion chromatography was assessed at high-pressure conditions and linked to particle- and column-bed integrity. Decreasing the particle size from 3.5 µm to 1.7 µm increases the resolution due to the improved mass-transfer characteristics, allowing to significantly speed-up analysis without compromising the selectivity. A sub-minute separation of intact proteins was realized on a 4.6 mm i.d × 75 mm long column packed with 1.7 µm SEC particles applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. Ultra-high pressure operation (exceeding manufacturer's recommendations) resulted in peak deformation, a shift towards earlier retention times, and an alteration in selectivity. To gain insights in the mechanisms of column deterioration, short 30 mm long columns were operated at UHPLC conditions, maximizing the pressure drop over individual particles. This resulted in the presence of fractured particles situated at the column outlet, as verified by scanning electron micrographs. Mercury-intrusion porosimetry and argon-adsorption measurements did not reveal significant differences in intraparticle volume between particle batches sampled before and after pressure stress testing. As particles at the column outlet fracture (but not collapse) at high pressure operation, a void was formed at the column inlet. The degradation of the separation performance appeared to be the result of a decrease in interparticle pore volume.


Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Tamanho da Partícula , Proteínas/análise , Proteínas/isolamento & purificação , Água/química
9.
J Chromatogr A ; 1621: 461051, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32268955

RESUMO

The strength of the biotin/avidin interaction makes it an ideal tool for the purification of biotin-labeled proteins via avidin-coupled resin with high specificity and selectivity. Nevertheless, this tight binding comes at an extra cost of performing the elution step under denaturing conditions. Weakening the biotin/avidin interaction improves the elution conditions, but only to mild or harsh denaturing buffers with the drawback of reducing the specificity and selectivity of this interaction. Here, we present two chromatographic protein purification schemes that are well-suited for application under native conditions thus preserving the strength of the biotin/avidin interaction. In the first scheme, we introduce a biotin-labeled SUMO-tag to each of human flap endonuclease 1 and Escherichia coli replication termination protein Tus, and elute both proteins by performing on-resin cleavage using SUMO protease. In the second scheme, we immobilize biotin-labeled human proliferating cell nuclear antigen (PCNA) on the avidin-coupled resin and use the resulting resin as a tag-free affinity method to purify the PCNA-binding protein human DNA Ligase 1. Furthermore, we streamlined the protein biotinylation protocol by constructing a single plasmid expression system that ensures high level of expression and solubility for each of the target protein bearing the biotin-tag and the enzyme responsible for the in vivo biotinylation reaction. Both chromatographic schemes resulted in a high yield of pure proteins in their native form.


Assuntos
Avidina , Biotina , Cromatografia de Afinidade/métodos , Cromatografia/métodos , Proteínas/isolamento & purificação , Biotinilação , DNA Ligase Dependente de ATP/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Endonucleases Flap/isolamento & purificação , Humanos , Plasmídeos , Antígeno Nuclear de Célula em Proliferação , Proteínas/genética , Proteína SUMO-1
10.
PLoS One ; 15(3): e0230334, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32168352

RESUMO

AIM: The aim of the present study was to assess the feasibility and diagnostic contribution of protein profiling using MALDI-TOF mass spectrometry applied to saliva, gingival crevicular fluid (GCF) and dental plaque from periodontitis and healthy subjects. We hypothesized that rapid routine and blinded MALDI-TOF analysis could accurately classify these three types of samples according to periodontal state. MATERIALS AND METHODS: Unstimulated saliva, GCF and dental plaque, collected from periodontitis subjects and healthy controls, were analyzed by MALDI-TOF MS. Based on the differentially expressed peaks between the two groups, diagnostic decision trees were built for each sample. RESULTS: Among 141 patients (67 periodontitis and 74 healthy controls), the decision trees diagnosed periodontitis with a sensitivity = 70.3% (± 0.211) and a specificity = 77.8% (± 0.165) for saliva, a sensitivity = 79.6% (± 0.188) and a specificity = 75.7% (± 0.195) for GCF, and a sensitivity = 72.1% (± 0.202) and a specificity = 72.2% (± 0.195) for dental plaque. The sensitivity and specificity of the tests were improved to 100% (CI 95% = [0.91;1]) and 100% (CI 95% = [0.92;1]), respectively, when two samples were tested. CONCLUSION: We developed, for the first time, diagnostic tests based on protein profiles of saliva, GCF and dental plaque between periodontitis patients and healthy subjects. When at least 2 of these samples were tested, the best results were obtained.


Assuntos
Periodontite/diagnóstico , Proteínas/genética , Saliva/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Periodontite/genética , Periodontite/patologia , Proteínas/isolamento & purificação , Dente/metabolismo , Dente/patologia
12.
J Chromatogr A ; 1619: 460871, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32044126

RESUMO

Affinity adsorbents have been the cornerstone in protein purification. The selective nature of the molecular recognition interactions established between an affinity ligands and its target provide the basis for efficient capture and isolation of proteins. The plethora of affinity adsorbents available in the market reflects the importance of affinity chromatography in the bioseparation industry. Ligand discovery relies on the implementation of rational design techniques, which provides the foundation for the engineering of novel affinity ligands. The main goal for the design of affinity ligands is to discover or improve functionality, such as increased stability or selectivity. However, the methodologies must adapt to the current needs, namely to the number and diversity of biologicals being developed, and the availability of new tools for big data analysis and artificial intelligence. In this review, we offer an overview on the development of affinity ligands for bioseparation, including the evolution of rational design techniques, dating back to the years of early discovery up to the current and future trends in the field.


Assuntos
Cromatografia de Afinidade , Proteínas/isolamento & purificação , Inteligência Artificial , Ligantes
13.
Braz J Med Biol Res ; 53(1): e9001, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939598

RESUMO

Due to its various structures in bio-compounds, snake venom is the indisputable result of evolutionary stages of molecules with an increasingly complex structure, high specificity, and of great importance for medicine because of their potential. The present study proposed an underpinning examination of venom composition from nine species of venomous snakes using a useful and replicable methodology. The objective was the extension of the evaluation of protein fractions in the field up to 230 kDa to permit possible identification of some fractions that are insufficiently studied. The gel capillary electrophoresis method on the chip was performed using an Agilent 2100 bioassay with the 80 and 230-LabChip Protein kits. Interpretation of electrophoresis was performed using the Protein 2100 expert (Agilent) test software as follows: a) Protein 80 (peak size scale): 1.60, 3.5, 6.50, 15.00, 28.00, 46.00, 63.00, 95.00 kDa; b) Protein 230 (peak size scale): 4.50, 7.00, 15.00, 28.00, 46.00, 63.00, 95.00, 150.00, 240.00 kDa. The screening revealed the presence of compounds with a molecular weight greater than 80 kDa, in the case of Vipera aspis and Vipera xantina palestinae. For V. aspis, a 125 kDa molecular weight pro-coagulant protein was identified, known as being involved in the reduction of plasma clotting time without any direct activity in the fibrinogen coagulation process. The samples examined on the Protein 230-LabChip electrophoresis chip can be considered as a novelty with possible uses in medicine, requiring further approaches by advanced proteomics techniques to confirm the intimate structural features and biological properties of snake venoms.


Assuntos
Proteínas/química , Venenos de Víboras/química , Viperidae/classificação , Animais , Eletroforese Capilar , Proteínas/análise , Proteínas/isolamento & purificação , Proteoma/química , Proteoma/classificação , Proteômica/métodos , Venenos de Víboras/análise
14.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 1): 20-24, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929182

RESUMO

Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects ß-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48 Šusing wavelengths of 1.0 and 2.1 Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.


Assuntos
Aplysia/química , Cristalização/métodos , Proteínas/química , Animais , Aplysia/enzimologia , Aplysia/genética , Aplysia/metabolismo , Cristalografia por Raios X , Hidrólise , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Domínios Proteicos/genética , Proteínas/isolamento & purificação
15.
Biosens Bioelectron ; 151: 111978, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999585

RESUMO

Rapid and accurate detection of proteins in biological fluids is increasingly required in the biomedical environment. Actually, it is performed with conventional techniques, which are generally run by robotized platforms at centralized laboratories. In this work, molecular dynamics calculations and an experimental procedure were conducted to set up electrochemical sensors based on polypyrrol (PPy) molecular imprinted polymers (MIP) for proteins detection. Here, prostate-specific antigen (PSA) was selected as a template model. Computational calculations indicate that for any PPy conformation and any amino-acid location in the protein, PSA molecules remain strongly inserted in the PPy polymer without biological alterations. One from possible orientations, appeared to be most probable as it presents the lowest absorption energy (-363 kcal mol-1) and largest contact area (4034.1 Å2). The device was then elaborated by in situ electropolymerization of PPy films. MIP's thickness and extraction duration were optimized by chronoamperometry. Square wave voltammetry technique was investigated for PSA detection in standard solution in the concentration range of 3x10 -8 ng.ml-1- 300 ng ml-1. According to the Hill equation, the equilibrium dissociation constant Kdbetween PSA and its imprint was estimated at Kd = (1.02 ±â€¯0.54) × 10-14 M, confirming the strong binding between the designed MIP and the protein as predicted by the computational study. PSA concentration values directly measured in 35 human serum samples were found closely correlated to those measured by the ELISA technique. The promising fast and low-cost sensor might be used successfully for proteins detection at low concentrations with high selectivity and reproducibility.


Assuntos
Técnicas Biossensoriais , Impressão Molecular , Antígeno Prostático Específico/isolamento & purificação , Proteínas/isolamento & purificação , Humanos , Limite de Detecção , Conformação Molecular , Polímeros/química , Antígeno Prostático Específico/genética , Proteínas/genética
16.
Biochem J ; 477(3): 727-745, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31957806

RESUMO

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.


Assuntos
Aminopeptidases , Dipeptidil Peptidases e Tripeptidil Peptidases , Lipofuscinoses Ceroides Neuronais , Proteínas , Serina Proteases , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/isolamento & purificação , Aminopeptidases/metabolismo , Animais , Células CHO , Clonagem Molecular , Cricetulus , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Terapia de Reposição de Enzimas , Estabilidade Enzimática , Humanos , Linfócitos , Mutação , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Cultura Primária de Células , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo
17.
Methods Mol Biol ; 2102: 163-176, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989554

RESUMO

Two-dimensional difference gel electrophoresis (2D-DIGE) remains to be one of the most popular and versatile methods of protein separation among many proteomics technologies. Similar to traditional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the proteins are separated based on their charges and molecular weight by 2D-DIGE. Different from 2D-PAGE, proteins are pre-labeled with different fluorescent dyes, and different protein samples are run in one gel by this method. Therefore, 2D-DIGE not only carries the advantages of 2D-PAGE but also eliminates gel-to-gel variation and achieves high resolution, sensitivity, and reproducibility.


Assuntos
Proteoma/análise , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Líquidos Corporais/química , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Humanos , Proteínas/análise , Proteínas/isolamento & purificação , Reprodutibilidade dos Testes , Fluxo de Trabalho
18.
Methods Mol Biol ; 2102: 509-528, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989574

RESUMO

Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can "reactivate" or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair (TCR), a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences. This method features two ways to perform the assay using expression vectors with luciferase and beta galactosidase, as well as with firefly luciferase and Renilla luciferase using the same luminometer.


Assuntos
Reparo do DNA/genética , Luciferases/metabolismo , Transfecção/métodos , Linhagem Celular , Genes Reporter , Técnicas Genéticas , Vetores Genéticos , Humanos , Luciferases/genética , Fosfatidiletanolaminas , Plasmídeos/efeitos da radiação , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas/metabolismo , Transcrição Genética , Raios Ultravioleta , Fluxo de Trabalho , beta-Galactosidase/metabolismo
19.
Ultrason Sonochem ; 62: 104852, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31806557

RESUMO

This study evaluates the effect of ultrasound and ozone pretreatments for the subsequent recovery of Desmodesmus sp. biocomponents-lipids, proteins, and carbohydrates-using a response surface methodology. Both pretreatments impact on the recovered lipids quality, solvent waste production and extraction time is analysed for process intensification purposes. For ultrasound pretreatment, independent parameters were energy applied (50-200 kWh/kg dry biomass), biomass concentration (25-75 g/L), and ultrasonic intensity (0.32 and 0.53 W/mL). While for ozone pretreatment, independent parameters were ozone concentration (3-9 mg O3/L), biomass concentration (25-75 g/L), and contact time (5-15 min). In the case of ultrasound pretreatment, recovery yield reached 97 ±â€¯0.4%, 89 ±â€¯3%, and 73 ±â€¯0.6% for proteins, carbohydrates and lipids respectively. Given process required: energy applied of 50 kWh/kg dry biomass, 75 g/L of biomass concentration, 0.32 W/mL of ultrasonic intensity, and 56 min of time process. Ultrasound caused high cell disruption releasing all proteins, thereby obviating downstream processing for its recovery. Ozone pretreatment recovery yield was 85 ±â€¯2%, 48 ±â€¯1.4%, and 25 ±â€¯1.3%, for carbohydrates, lipids and proteins respectively, under the following conditions: 9 mg O3/L of ozone concentration, 25 g/L of biomass concentration, and 5 min of contact time that depicts an energy consumption of 30.64 kWh/kg dry biomass. It was found that ultrasound and ozone pretreatments intensified the lysis and biocomponents recovery process by reducing solvent consumption by at least 92% and extraction time between 80% and 90% compared with extraction of untreated biomass biocomponents. Both pretreatments improve the composition of the recovered lipids. It was noted that the yield of neutral lipids increased from 28% to 67% for ultrasound pretreatment while for ozone pretreatment from 49% to 63%. The method used for lipid extraction may also have an effect but here it was kept constant.


Assuntos
Carboidratos/isolamento & purificação , Lipídeos/isolamento & purificação , Microalgas/metabolismo , Ozônio/química , Proteínas/isolamento & purificação , Sonicação , Águas Residuárias/química , Biomassa , Cromatografia Gasosa-Espectrometria de Massas
20.
J Chromatogr A ; 1609: 460491, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31481295

RESUMO

The development of multifarious stationary phases is still a growing demand so as to solve the tasks of ever evolving actual applications. Herein, with D-2-allylglycine hydrochloride (AG·HCl) as the hydrophilic monomer, diene ionic liquid 1-allyl-3-vinylimidazolium bromide (AVI·Br) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) as the dual crosslinkers, the highly cross-linked imidazolium-bridged POSS-AVI-AG hybrid monolithic column was fabricated via the "one-pot" free radical copolymerization. The AG·HCl embedded POSS-AVI-AG column displays typical reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention mechanisms. Both hydrophobic phenols, alkylbenzenes, aromatic amines and hydrophilic nucleosides/nucleic acid bases, amides and thioureas were successfully separated with high column efficiencies (up to 571,000 plates/m for amides), outperforming our previously reported AVI·Br modified POSS-AVI column. Moreover, the column was also explored for the separation of cytochrome c tryptic digests and egg white protein extraction. All these results demonstrate that the POSS-AVI-AG column has a good potential in separation of both small molecules and complex biological samples with multiple mechanisms.


Assuntos
Alilglicina/química , Imidazóis/química , Substâncias Macromoleculares/isolamento & purificação , Compostos de Organossilício/química , Citocromos c/isolamento & purificação , Nucleosídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Polimerização , Proteínas/isolamento & purificação
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