Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.177
Filtrar
1.
J Chromatogr A ; 1625: 461304, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709347

RESUMO

A twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been developed for the purification of a therapeutic peptide, glucagon, from a crude synthetic mixture. This semi-continuous process uses two identical columns operating either in interconnected or in batch mode, thus enabling the internal recycle of the portions of the eluting stream which do not comply with purity specifications. Because of this feature, which actually results in the simulated countercurrent movement of the stationary phase with respect to the mobile one, the yield-purity trade-off typical of traditional batch preparative chromatography can be alleviated. Moreover, the purification process can be completely automatized. Aim of this work is to present a simple procedure for the development of the MCSGP process based on a single batch experiment, in the case of a therapeutic peptide of industrial relevance. This allowed to recover roughly 90% of the injected glucagon in a purified pool with a purity of about 90%. A comparison between the performance of the MCSGP process and the classical single column batch process indicates that percentage increase in the recovery of target product is +23% when transferring the method from batch conditions to MCSGP, with an unchanged purity of around 89%. This improvement comes at the expenses of a reduction of about 38% in productivity.


Assuntos
Distribuição Contracorrente/métodos , Peptídeos/isolamento & purificação , Solventes/química , Cromatografia Líquida de Alta Pressão , Glucagon/isolamento & purificação , Fatores de Tempo
2.
Proc Natl Acad Sci U S A ; 117(32): 19168-19177, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719135

RESUMO

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Clostridiales/química , Peptídeos/química , Peptídeos/farmacologia , Antibacterianos/isolamento & purificação , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Peptídeos/isolamento & purificação
3.
J Chromatogr A ; 1624: 461227, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32540069

RESUMO

Affinity chromatography is generally regarded as a powerful tool allowing the single step purification of recombinant proteins with high purity and yields. However, for most protein products, affinity purification methods for industrial applications are not readily available, mainly due to the lack of specific and robust natural counterparts that could function as affinity ligands. In this study, we explored the applicability of nanobody-based peptide-tag immunorecognition systems as a platform for affinity chromatography. Two typical nanobodies (BC2-nb and Syn2-nb) that are capable of recognizing specifically a particular peptide-tag, were prepared through prokaryotic expression and proved to be able to bind with nanomolar affinity to their cognate tag fused to enhanced green fluorescent protein (eGFP). Through an epoxy-based immobilization reaction, the two nanobodies were coupled on a Sepharose CL-6B matrix under the same conditions. The remaining antigen binding activity of the immobilized BC2-nb and Syn2-nb was determined to be 83.1% and 42.9%, yielding the resins with the dynamic binding capacity (DBC) of 21.4 mg/mL and 5.9 mg/mL, respectively. The immobilized affinity ligands exhibited high binding specificity towards their respective target peptides, yielding a product purity above 90% directly from crude bacterial lysates in one single chromatographic step. However, for the both affinity complexes, desorption has been found difficult, and effective recovery of the bound products could be only achieved with competitive elution or after employing harsh conditions such as 10 mM NaOH solution, which will compromise the reuse cycles of the affinity resins. This study shows the potential of nanobody-based affinity chromatography for efficient purification of recombinant proteins especially from complex feedstocks and reveals the primary issues to be addressed to develop a successful application.


Assuntos
Cromatografia de Afinidade/métodos , Peptídeos/imunologia , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Cromatografia Líquida de Alta Pressão , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Peptídeos/química , Peptídeos/isolamento & purificação , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo
4.
Nature ; 582(7813): 592-596, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555458

RESUMO

Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported1, advances in mass-spectrometry-based proteomics2 have enabled increasingly comprehensive identification and quantification of the human proteome3-6. However, there have been few comparisons across species7,8, in stark contrast with genomics initiatives9. Here we use an advanced proteomics workflow-in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system-for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides from Bacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at www.proteomesoflife.org.


Assuntos
Classificação , Aprendizado Profundo , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma/química , Proteoma/isolamento & purificação , Proteômica/métodos , Animais , Bacteroides/química , Bacteroides/classificação , Metabolismo dos Carboidratos , Cromatografia , Glicólise , Homeostase , Transporte de Íons , Proteínas com Ferro-Enxofre/metabolismo , Oxirredução , Fotossíntese , Biossíntese de Proteínas , Dobramento de Proteína , Proteólise , Especificidade da Espécie
5.
J Chromatogr A ; 1622: 461152, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32376024

RESUMO

The chiral separations of small peptides is an important challenge in the biological and medical sciences, because different stereoisomers of chiral drugs can often possess different pharmacological, pharmacokinetic, and/or toxicological activities. Commercially available crown ether chiral stationary phases based on S-(3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 (CROWNPAK CR-I (+)) and (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (ChiroSil RCA (+)) have been successfully used for separating enantiomers of various racemic compounds containing primary amino groups. In this investigation, enantioresolution of more complex model analyte - tetrapeptide Tyr-Arg-Phe-Lys-NH2, has been reported on crown ether chiral stationary phases. Organic and acidic modifier content in aqueous mobile phase was tested. All Tyr-Arg-Phe-Lys-NH2 stereoisomers showed U-shaped retention plots, based on ACN content in mobile phase. Increased retention of tetrapeptide stereoisomers was observed at low (<35%) and at high (>70%) acetonitrile content in the mobile phase, indicating that different separation mechanisms are most likely involved. As a result, baseline separation of all eight tetrapeptide enantiomer pairs was achieved under isocratic elution mode on both chiral columns.


Assuntos
Éteres de Coroa/química , Peptídeos/química , Peptídeos/isolamento & purificação , Acetonitrilos/química , Padrões de Referência , Estereoisomerismo , Fatores de Tempo
6.
Food Chem ; 327: 127059, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32447138

RESUMO

The aim of this study was to purify and identify antioxidant peptides from watermelon seed protein hydrolysates (WSPHs-I: Mw < 1 kDa) and further evaluate their cytoprotective effects against H2O2-induced oxidative stress in HepG2 cells. After purification by Sephadex G-15 and semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC), five peptides, RDPEER (P1), KELEEK (P2), DAAGRLQE (P3), LDDDGRL (P4), and GFAGDDAPRA (P5) were sequenced by LC-MS/MS and synthesized with solid-phase synthesis method. These peptides showed desirable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity (IC50: 0.216 ± 0.01-0.435 ± 0.03), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity (IC50: 0.54 ± 0.02-1.23 ± 0.03), and oxygen radical absorbance capacity (ORAC) (82.36 ± 1.2-130.67 ± 2.2 µM TE/mg). Among them, peptide P1 exhibited the strongest antioxidant capacity. Moreover, the results suggested that peptide P1 may protect HepG2 cells from H2O2-induced oxidative damage by significantly inhibiting reactive oxygen species (ROS), [Ca2+]i, malondialdehyde (MDA) levels and increasing antioxidative enzyme activities.


Assuntos
Citrullus/química , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/química , Hidrolisados de Proteína/química , Sequência de Aminoácidos , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão , Citrullus/metabolismo , Células Hep G2 , Humanos , Oxirredução , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteínas de Plantas/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sementes/química , Sementes/metabolismo , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem
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 ; 1622: 461093, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32340726

RESUMO

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia de Fase Reversa , Bases de Dados Factuais , Peptídeos/isolamento & purificação , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Insulina/química , Peptídeos/química , Análise de Componente Principal , Dióxido de Silício/química
9.
Chemosphere ; 253: 126728, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32298913

RESUMO

Nile perch wastewater was biodegraded using two Bacillus species to recover bioactive substances to enhance its reutilization value. The two Bacillus species successfully produced low-molecular-weight substances with a 47.8% degree of hydrolysis. The antioxidant activities of the Nile perch wastewater increased as the biodegradation proceeded, and the culture supernatant exhibited the highest DPPH (80.1%), ABTS (93.1%) and Fe2+ chelating (88.5%) antioxidant activities at 60 h. The antioxidant potential of the biodegraded Nile perch wastewater was found to be higher than those of other fish hydrolysates. Moreover, the biodegraded Nile perch wastewater exhibited effective antimicrobial activity against Vibrio vulnificus, exhibiting a minimal inhibitory concentration of 585 µg mL-1. Two-dimensional thin layer chromatography analysis revealed the specific amino acids responsible for the antioxidant activity, and molecular-weight cut-off ultrafiltration revealed that the <2-kDa fraction exhibited the highest antioxidant activity with the lowest IC50 values (0.43 and 0.22 mg mL-1 for DPPH and ABTS antioxidant activities, respectively). This is the first report of the reutilization of Nile perch wastewater as a natural antioxidant and antimicrobial ingredient for nutraceuticals.


Assuntos
Antibacterianos/isolamento & purificação , Antioxidantes/isolamento & purificação , Bacillus/metabolismo , Peptídeos/isolamento & purificação , Percas , Águas Residuárias/química , Aminoácidos/isolamento & purificação , Aminoácidos/farmacologia , Animais , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Biodegradação Ambiental , Compostos de Bifenilo/química , Quelantes/isolamento & purificação , Quelantes/farmacologia , Pesqueiros , Hidrólise , Peso Molecular , Peptídeos/farmacologia , Percas/crescimento & desenvolvimento , Picratos/química , Ultrafiltração , Vibrio vulnificus/efeitos dos fármacos
10.
J Dairy Sci ; 103(6): 4919-4928, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32229119

RESUMO

An angiotensin-converting enzyme inhibitory (ACEI) peptide with a median inhibitory concentration (IC50) of 1.26 mg/mL was purified from whey proteins resulting from a fermentation using Lactobacillus plantarum QS670. The peptide was subsequently derived from an αS1-casein, κ-casein, ß-lactoglobulin, or serum albumin fraction. Analysis via liquid chromatography tandem mass spectrometry indicated that it had an amino acid sequence of Gly-Ala (GA). The GA dipeptide was also synthesized using an Fmoc solid-phase method. The GA dipeptide exhibited an IC50 of 1.22 mg/mL and was shown to be stable across both temperature (20 to 60°C) and pH (2 to 12). Digestive enzymes including pepsin, trypsin, and chymotrypsin had negligible effects on activity. The whey exerted hypotensive effects when fed to spontaneously hypertensive rats (SHR), which exhibited a blood pressure drop of 2.33 kPa. A 4-wk gavage treatment resulted in greater decreases of 7.46 kPa. Results of this study indicate that milk fermented using Lb. plantarum QS306 has potential to be used as a functional food to help prevent or reduce hypertension-associated diseases.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Lactobacillus plantarum/metabolismo , Leite/química , Peptídeos/isolamento & purificação , Proteínas do Soro do Leite/química , Animais , Anti-Hipertensivos/isolamento & purificação , Anti-Hipertensivos/farmacologia , Caseínas/análise , Digestão , Fermentação , Humanos , Lactoglobulinas/química , Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Ratos
11.
Artigo em Inglês | MEDLINE | ID: mdl-32172172

RESUMO

Purification of small bioactive peptides from complex biological samples is a difficult task due to the interference of concentrated large biomolecules. In this study, a magnetic immobilized metal affinity chromatography matrix modified by poly (ethylene glycol) methyl ether (IMACM@mPEG) was prepared and applied for the rapid purification of angiotensin I-converting enzyme (ACE) inhibitory peptides from casein hydrolysate. The proposed IMACM@mPEG considerably reduced the non-specific adsorption of large proteins and exhibited improved purification efficiency towards ACE inhibitory peptides. A novel peptide with moderate ACE inhibitory activity (IC50 value of 274 ± 5 µM) was identified as LLYQEPVLGPVR. Lineweaver-Burk plot confirmed the non-competitive inhibition pattern of LLYQEPVLGPVR. The purified peptide was digested after simulated gastrointestinal digestion and produced shorter peptides which contributed to enhanced ACE inhibitory activity. These results indicated that the IMACM@mPEG is an effective method for the prepurification of ACE inhibitory peptide and the purified peptide LLYQEPVLGPVR may have potential as nutraceutical ingredient in functional foods for hypertension treatments.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Caseínas/química , Cromatografia de Afinidade/métodos , Éteres/química , Peptídeos/isolamento & purificação , Polietilenoglicóis/química , Adsorção , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/análise , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Cobre/química , Óxido Ferroso-Férrico/química , Microesferas , Peptídeos/análise , Peptídeos/metabolismo , Hidrolisados de Proteína , Dióxido de Silício/metabolismo , Propriedades de Superfície
12.
Food Chem ; 319: 126534, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32193058

RESUMO

The antioxidant peptides extracted from duck plasma hydrolysate (DPH) was investigated. The antioxidant activity of DPH, which was isolated and purified via ultrafiltration, size exclusion chromatography, and reversed-phase high-performance liquid chromatography, was evaluated using its free radical scavenging ability. Nano-liquid chromatography-tandem mass spectrometry was conducted to identify the DPH fractions with the highest antioxidant ability. Seven novel peptides: LDGP, TGVGTK, EVGK, RCLQ, LHDVK, KLGA, and AGGVPAG (400.43, 561.63, 431.48, 260.14, 610.71, 387.47, and 527.57 Da, respectively) were identified and synthesized using a solid-phase peptide produce to evaluate their antioxidant activities. Of these, EVGK exhibited the highest Fe2+ chelating ability (16.35%), and RCLQ presented the highest reducing power, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt scavenging activity, and 1,1-diphenyl-2-picrylhydrazyl scavenging rate (0.62, 274.83 mM TE/mg, and 95.12%, respectively). Our results indicated that DPH possessed antioxidant capabilities and could be used to obtain antioxidant peptides, thus adding economic value to duck blood.


Assuntos
Antioxidantes/química , Proteínas Sanguíneas/química , Patos , Peptídeos/química , Animais , Antioxidantes/isolamento & purificação , Compostos de Bifenilo , Proteínas Sanguíneas/isolamento & purificação , Cromatografia em Gel , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/química
13.
J Food Sci ; 85(3): 657-665, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32052448

RESUMO

Red oncom, a fermented product from solid waste of soybean curd process, and black oncom, a similar fermented product but made from defatted peanut cake, have been known to have umami taste. Umami fractions of red oncom and black oncom that are responsible for umami taste have not been investigated yet. The objective of this research was to characterize umami fractions obtained by ultrafiltration and chromatography of both oncoms. The first step, water-soluble extracts of oncoms were ultrafiltered using a membrane with cutoff 3,000 Da and followed by gel filtration chromatography (Sephadex G-25) to obtain umami fractions. Ultrafiltration fractions of red oncom (molecular weight [MW] less than 3,000 Da) and black oncom (MW more than 3,000 Da) had an intense umami taste. The further fractionation by gel filtration chromatography linked to taste dilution analysis yielded umami fractions. Chemical characterization revealed that free glutamic acid, free phenylalanine, and peptides containing their residual amino acids were present in the fractions. PRACTICAL APPLICATION: Umami fractions of red and black oncoms can be used as a source of umami compounds for food industries and food services. The information from this paper can be used by other researchers who will explore umami peptides.


Assuntos
Arachis/química , Alimentos e Bebidas Fermentados/análise , Aromatizantes/química , Soja/química , Aminoácidos/análise , Fracionamento Químico , Cromatografia em Gel , Aromatizantes/isolamento & purificação , Humanos , Indonésia , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Paladar
14.
J Food Sci ; 85(3): 611-617, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32078748

RESUMO

Tartary buckwheat (Fagopyrum tataricum Gaertn.) albumin was hydrolyzed by alkaline protease, and three new antioxidant peptides (P1, P2, and P3) were successfully separated from the hydrolysate (TBAH). The sequences of the three antioxidant peptides were Gly-Glu-Val-Pro-Trp (GEVPW), Tyr-Met-Glu-Asn-Phe (YMENF), and Ala-Phe-Tyr-Arg-Trp (AFYRW), and their molecular weights were 586.65, 702.79, and 741.85 Da, respectively. All three peptides have a good antioxidant capacity, and P3 (AFYRW) demonstrates the best antioxidant activity of the three. The IC50 values of AFYRW for scavenging hydroxyl radicals (OH·) and (1,1-diphenyl-2-picrylhydrazyl) DPPH· free radicals were 0.65 and 0.64 mM, respectively. In addition, AFYRW exhibits the strongest lipid peroxidation inhibition ability and the highest reducing power. The results of this research indicate that the three isolated peptides can be used in the development of various antioxidant additives in the food and pharmaceutical industries.


Assuntos
Albuminas/química , Antioxidantes/química , Fagopyrum/química , Peptídeos/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Antioxidantes/isolamento & purificação , Peptídeos/isolamento & purificação
15.
Exp Parasitol ; 210: 107830, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31917970

RESUMO

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi, which is transmitted by insects of the family Reduviidae. Since conventional treatments with nitroheterocyclic drugs show serious adverse reactions and have questionable efficiency, different research groups have investigated polypeptide-based approaches to interfere with the parasite cell cycle in other Trypanosomatids. These strategies are supported by the fact that surface players are candidates to develop surface ligands that impair function since they may act as virulence factors. In this study, we used a phage display approach to identify peptides from one library-LX8CX8 (17 aa) (where X corresponds to any amino acid). After testing different biopanning conditions using live or fixed epimastigotes, 10 clones were sequenced that encoded the same peptide, named here as EPI18. The bacteriophage expressing EPI18 binds to epimastigotes from distinct strains of T. cruzi. To confirm these results, this peptide was synthetized, biotinylated, and assayed using flow cytometry and confocal microscopy analyses. These assays confirmed the specificity of the binding capacity of EPI18 toward epimastigote surfaces. Our findings suggest that EPI18 may have potential biotechnological applications that include peptide-based strategies to control parasite transmission.


Assuntos
Doença de Chagas/tratamento farmacológico , Peptídeos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Bacteriófagos/isolamento & purificação , Bioprospecção , Biotinilação , Doença de Chagas/parasitologia , Doença de Chagas/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Temperatura , Trypanosoma cruzi/genética
16.
J Chromatogr A ; 1618: 460873, 2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31987525

RESUMO

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH4Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE-MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest.


Assuntos
Eletroforese Capilar , Peptídeos , Proteômica/métodos , Espectrometria de Massas em Tandem , Cromatografia Líquida , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma
17.
Mar Drugs ; 18(1)2020 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-31940927

RESUMO

This study reports the isolation of two novel cysteine-rich antibacterial peptides, turgencin A and turgencin B, along with their oxidized derivatives, from the Arctic marine colonial ascidian Synoicum turgens. The peptides are post-translationally modified, containing six cysteines with an unusual disulfide connectivity of Cys1-Cys6, Cys2-Cys5, and Cys3-Cys4 and an amidated C-terminus. Furthermore, the peptides contain methionine residues resulting in the isolation of peptides with different degrees of oxidation. The most potent peptide, turgencin AMox1 with one oxidized methionine, displayed antimicrobial activity against both Gram-negative and Gram-positive bacteria with a minimum inhibitory concentration (MIC) as low as 0.4 µM against selected bacterial strains. In addition, the peptide inhibited the growth of the melanoma cancer cell line A2058 (IC50 = 1.4 µM) and the human fibroblast cell line MRC-5 (IC50 = 4.8 µM). The results from this study show that natural peptides isolated from marine tunicates have the potential to be promising drug leads.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos/farmacologia , Urocordados/química , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dissulfetos/química , Descoberta de Drogas , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/isolamento & purificação
18.
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
19.
Biosci Biotechnol Biochem ; 84(3): 563-574, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31790634

RESUMO

Two kinds of Kunitz-type protease inhibitors, AKPI1 and AKPI2, were purified from Apios americana tubers by four steps of column chromatographies and their cDNA cloning was performed. AKPI1 cDNA consist of 809 nucleotides, and the matured protein had 190 amino acids with 20,594 Da. AKPI2 cDNA consist of 794 nucleotides, and the matured protein had 177 amino acids with 19,336 Da. P1 site of AKPI2 was Leu88, suggested the target enzyme was chymotrypsin. On the other hand, Gly85-Ile86-Ser87 was positioned around P1 site of AKTI1. Sequence analysis suggested that two forms (single-chain and two-chain form) of AKPI2 protein were present in the tubers. Recombinant AKPI2 expressed by E.coli system showed inhibitory activity toward serine proteases and heat stability. The Ki values toward chymotrypsin and trypsin were 4 × 10-7 M and 6 × 10-6 M, respectively.Abbreviations: AAL: Apios americana lectin; AATI: Apios americana Bowman-Birk type trypsin inhibitor; ACE: angiotensin-converting enzyme; IPTG: isopropyl-ß-D-thio-galactopyranoside; Ki: inhibition constant; KPIs: Kunitz-type protease inhibitors; L-BAPA: Benzoyl-L-arginine p-nitroanilide monohydrochloride; L-BTPA: Benzoyl-L-tyrosine p-nitroanilide; PFLNA: Pyr-Phe-Leu-p-nitroanilide; RP-HPLC: reverse-phase high-performance liquid chromatography; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLIC: sequence and ligation independent cloning; STANA: N-Succinyl-Ala-Ala-Ala-p-nitroanilide; SHR: spontaneously hypertensive rats; TFA: trifluoroacetic acid; UTR: untranslated region.


Assuntos
Clonagem Molecular , DNA Complementar/genética , Fabaceae/metabolismo , Peptídeos/genética , Proteínas de Plantas/genética , Tubérculos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida/métodos , Escherichia coli/genética , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
20.
Nat Prod Res ; 34(6): 754-758, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30470149

RESUMO

Amphibian skin is known to secrete gene-encoded antioxidant peptides of small molecular weight, which play important roles in host defense. However, recognition of such peptides is still in its infancy. Here, we discovered a novel gene-encoded antioxidant peptide (named OM-GF17) from skin secretions of amphibian species, Odorrana margaretae. Produced by the post-translational processing of a 61-residue prepropeptide, the amino acid sequence of OM-GF17 was 'GFFKWHPRCGEEHSMWT', with a molecular mass of 2135.7 Da. Functional analysis revealed that OM-GF17 scavenged ABTS+, DPPH, NO and decreased iron oxidation. Our results also implied that five amino acid residues, including Cys, Pro, Met, Trp, and Phe, be related to the antioxidant activity of OM-GF17. Furthermore, OM-GF17 did not exhibit direct microbe-killing activity. This novel gene-encoded antioxidant peptide could help in the development of new antioxidant agents and increase our understanding of the biological functions of amphibian skin. [Formula: see text].


Assuntos
Antioxidantes/isolamento & purificação , Peptídeos/isolamento & purificação , Ranidae/genética , Pele/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Antioxidantes/química , Oxirredução , Peptídeos/análise , Peptídeos/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA