Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 35.525
Filtrar
1.
Int J Nanomedicine ; 16: 2059-2070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33727813

RESUMO

Purpose: The purpose of this study was to establish a lateral flow immunoassay using selenium nanoparticles (Se-NPs) as a probe to detect ractopamine (RAC) and salbutamol (SAL) in swine urine. Methods: SDS and PEG were used as templates to prepare Se-NPs; anti-RAC monoclonal antibodies or anti-SAL monoclonal antibodies were labelled with Se-NPs; and rapid detection kits were prepared. The sensitivity, specificity, and stability were measured, and actual samples were analysed. Results: The Se-NPs were spherical with a diameter of 40.63 ± 5.91 nm, and were conjugated successfully with an anti-RAC antibody to give a total diameter of 82.33 ± 17.91 nm. The detection limit of a RAC kit in swine urine was 1 ng/mL, and that of a SAL kit was 3 ng/mL. Both procedures could be completed within 5 minutes. No cross-reaction occurred with clenbuterol, bambuterol and phenylethanolamine A. Samples were tested consistently across different batches of kits for swine urine. The results of the kits were identical to those of actual clinical samples analysed by ELISA, and the coincidence rate was 100%. Conclusion: The assay kit does not require any special device for reading the results, and the readout is a simple colour change that can be evaluated with the naked eye. It is easy to operate, sensitive, specific, and stable This kit is suitable for the rapid and real-time detection of RAC and SAL residues in swine urine samples. Clinical Trial Registration: Swine urines samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University.


Assuntos
Albuterol/urina , Cromatografia de Afinidade/métodos , Nanopartículas Metálicas/química , Fenetilaminas/urina , Selênio/química , Animais , Anticorpos/metabolismo , Concentração de Íons de Hidrogênio , Suínos
2.
Sensors (Basel) ; 21(5)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33670868

RESUMO

This paper proposes a combined strategy of using paper-based competitive immunochromatography and a near field communication (NFC) tag for wireless cotinine determination. The glucose oxidase labeled cotinine antibody specifically binds free cotinine in a sample, whereas the unoccupied antibody attached to BSA-cotinine at the test line on a lateral flow strip. The glucose oxidase on the strip and an assistant pad in the presence of glucose generated H2O2 and imposed the Ag oxidation on the modified electrode. This enabled monitoring of immunoreaction by either electrochemical measurement or wireless detection. Wireless sensing was realized for cotinine in the range of 100-1000 ng/mL (R2 = 0.96) in PBS medium. Undiluted urine samples from non-smokers exhibited an Ag-oxidation rate three times higher than the smoker's urine samples. For 1:8 diluted urine samples (smokers), the proposed paper-based competitive immunochromatography coupled with an enzyme-modified electrode differentiated positive and negative samples and exhibited cotinine discrimination at levels higher than 12 ng/mL. This novel sensing platform can potentially be combined with a smartphone as a reader unit.


Assuntos
Técnicas Biossensoriais , Cromatografia de Afinidade , Cotinina , Cotinina/urina , Eletrodos , Peróxido de Hidrogênio
3.
J Vis Exp ; (168)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33720114

RESUMO

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5' extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Cromatografia de Afinidade , Bactérias Gram-Positivas/genética , Análise de Sequência de RNA , Sequência de Bases , Tampões (Química) , Fracionamento Celular , Análise de Dados , Regulação Bacteriana da Expressão Gênica , Humanos , Plasmídeos/genética , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Reprodutibilidade dos Testes , Staphylococcus aureus/genética
4.
J Chromatogr A ; 1640: 461946, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33556682

RESUMO

In previous work, we have established a one-step method to immobilize halo-tagged proteins onto microspheres through the covalent bond formed between the halo-tag and the halide linkers on the support surface. We observe extremely tailed peaks of most of drugs on the immobilized proteins, which is reasoned by the nonspecific interaction between the linkers and the drugs. To prove this, the current work designed five different halide linkers for the immobilization of beta2-adrenoceptor (ß2-AR). We applied the immobilized receptor to systematically realize the effects of these halide linkers on drug-receptor interaction by analyzing peak profiles of five drugs. The retention times and the half-widths of the drugs appeared to be negatively correlated to the atom numbers of the linkers in the range of 6-13 atoms. Subsequent increase of linker atoms resulted in reduced retention times and wider peaks of the drugs. Applying identical linker length, we observed clear reduced retention times and half-widths of the five drugs than the linker in the absence of oxygen atom. Such improvement was dominated by the number of oxygen atoms. These indicated that linker S-4 (2-(2-(2-(2-chloroethoxy) ethoxy) ethoxy) acetic acid) was optimal to eliminate the unwanted non-specific interactions. In comparison with the columns prepared by linker S-1 (6-chlorocaproic acid) and histidine tagged ß2-AR, the drugs on the linker S-4 column gave greater dissociation rate constants (e.g. 60.3±0.3 s-1 for salbutamol), which is closer to the data in literatures. Taking together, we concluded that optimization of the linker structure plays particular role in reducing the non-specific interaction between the immobilized protein and the drugs, thereby making the determination of drug-protein interaction more reliable.


Assuntos
Halogênios/química , Proteínas Imobilizadas/química , Preparações Farmacêuticas/química , Cromatografia de Afinidade , Histidina/química , Cinética , Dinâmica não Linear , Receptores Adrenérgicos beta 2/química , Fatores de Tempo
5.
Methods Mol Biol ; 2261: 55-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33420984

RESUMO

Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteômica , Acetilação , Cromatografia de Afinidade , Cromatografia de Fase Reversa , Glicosilação , Imunoprecipitação , Fosforilação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Titânio/química , Fluxo de Trabalho
6.
Methods Mol Biol ; 2261: 277-289, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33420996

RESUMO

Targeted protein quantification can be challenging in body fluids such as plasma with regard to sensitivity and selectivity. In this chapter, we present a protocol for the quantification of high mobility group box 1 protein (HMGB1) in plasma using an immunoaffinity liquid chromatography mass spectrometric assay (IA-LC-MSMS). The protocol provides detailed assay instructions involving sample proteolysis, peptide-targeted immunoprecipitation, and LC-MSMS-based read out.


Assuntos
Métodos Analíticos de Preparação de Amostras , Coleta de Amostras Sanguíneas , Cromatografia de Afinidade , Ácido Edético/farmacologia , Proteína HMGB1/sangue , Proteômica , Espectrometria de Massas em Tandem , Anticoagulantes/farmacologia , Humanos , Imunoprecipitação , Proteólise
7.
Methods Mol Biol ; 2261: 535-547, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33421013

RESUMO

Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.


Assuntos
Fracionamento Celular , Cloroplastos/metabolismo , Cromatografia de Afinidade , Proteínas de Plantas/análise , Proteoma , Proteômica , Arabidopsis/metabolismo , Proteínas de Arabidopsis/análise , Espectrometria de Massas , Ervilhas/metabolismo , Folhas de Planta/metabolismo
8.
J Chromatogr A ; 1637: 461848, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33421679

RESUMO

Plasmid DNA chromatography is a powerful field in constant development and evolution. The use of this technique is considered mandatory in the production of an efficient and safe formulation to be applied for plasmid-mediated gene therapy. Concerning this, the search for an ideal chromatographic support/ligand combination motivated scientist to pursue a continuous improvement on the plasmid chromatography performance, looking for a progression on the ligands and supports used. The present review explores the different approaches used over time to purify plasmid DNA, ambitioning both high recovery and high purity levels. Overall, it is presented a critical discussion relying on the relevance of the binding capacity versus selectivity of the supports.


Assuntos
DNA/isolamento & purificação , Plasmídeos/isolamento & purificação , Cromatografia de Afinidade , Escherichia coli/genética , Ligantes
9.
Methods Mol Biol ; 2247: 3-16, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301109

RESUMO

Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of membrane proteins and their complexes is of key importance, but the progress in this area is slow because of the difficulties to produce them in sufficient quality and quantity. Overexpression of membrane proteins is often restricted by the limited capability of translocation systems to integrate proteins into the membrane and to fold them properly. Purification of membrane proteins requires their isolation from the membrane, which is a further challenge. The choice of expression system, detergents, and purification tags is therefore an important decision. Here, we present a protocol for expression in bacteria and isolation of a seven-subunit membrane protein complex, the bacterial holo-translocon, which can serve as a starting point for the production of other membrane protein complexes for structural and functional studies.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/isolamento & purificação , Subunidades Proteicas/biossíntese , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes , Cromatografia de Afinidade , Cromatografia em Gel , Escherichia coli/genética , Expressão Gênica , Proteínas de Membrana/química , Plasmídeos , Regiões Promotoras Genéticas , Multimerização Proteica , Subunidades Proteicas/química
10.
Methods Mol Biol ; 2247: 59-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301112

RESUMO

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.


Assuntos
Anticorpos/genética , Formação de Anticorpos/genética , Expressão Gênica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Anticorpos/isolamento & purificação , Baculoviridae/genética , Cromatografia de Afinidade , Ordem dos Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Calicreínas/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Transfecção
11.
Methods Mol Biol ; 2247: 77-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301113

RESUMO

Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise "uncrystallizable" proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.


Assuntos
Cromatografia de Afinidade , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/isolamento & purificação , Proteínas Recombinantes de Fusão , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Baculoviridae/genética , Sequência de Bases , Linhagem Celular , Cromatografia de Afinidade/métodos , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Ordem dos Genes , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Modelos Moleculares , Plasmídeos/genética , Conformação Proteica , Proteólise , Células Sf9 , Relação Estrutura-Atividade
12.
Methods Mol Biol ; 2200: 425-440, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33175391

RESUMO

Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to the environment. However, a main limitation of traditional approaches to study protein interactions, such as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of interactions include many of the protein complexes that mediate the perception and response to cellular stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells, including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins (within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the study of proteomes in numerous organisms, its application to plant systems has only recently been realized. In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Biotina/química , Complexos Multiproteicos , Mapeamento de Interação de Proteínas , Proteômica , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Biotinilação , Cromatografia de Afinidade , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica
13.
Food Chem ; 345: 128607, 2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-33310562

RESUMO

Dexamethasone (DEX) is a synthetic long-acting glucocorticoid, which can increase the risk of hypertension and diabetes if it is abused or used improperly. In this study, lateral flow immunoassays (LFIA) based on black and blue latex microspheres (LMs), integrated with a strip reader, was developed for quantitative detection of DEX in milk and pork. The results could be achieved within 15 min. The visible limits of detection (vLODs) were 0.3 ng/mL and 0.7 µg/kg for milk and pork, respectively. The quantitative limits of detection (qLODs) were 0.047 ng/mL and 0.087 µg/kg, respectively. The recoveries ranged from 80.0% to 122.8%. 20 milk and 10 pork samples were analyzed to confirm the performance of the on-site application. The detection results were consistent with the data from LC-MS/MS, indicating the practical reliability of our established assay. The developed LMs-LFIA provides a promising technical support for rapid, sensitive, and on-site detection of DEX.


Assuntos
Cromatografia de Afinidade/métodos , Dexametasona/análise , Contaminação de Alimentos/análise , Látex/química , Microesferas , Leite/química , Carne de Porco/análise , Animais , Cromatografia Líquida , Limite de Detecção , Reprodutibilidade dos Testes , Suínos
14.
J Chromatogr A ; 1637: 461831, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33373794

RESUMO

In this work, the porous cellulose-agarose microspheres with high specific surface area and enhanced mechanical strength are prepared by a novel chemical crosslinking method. The crosslinking reaction homogeneously proceeds between polysaccharides, and the covalent bonding network is generated to replace the inherent hydrogen bonding network of cellulose. The prepared microspheres exhibit low crystallinity of 12.45%, which means high content of amorphous regions. The micro-meso-macroporous structure of microspheres in morphology is conducive to high permeability and adsorption capacity, and the microspheres possess high specific surface area of 183.81 m2/g. The affinity chromatographic microspheres are prepared by immobilizing Cu2+, which exhibits high adsorption capacity of 197.65 mg/g for bovine hemoglobin (BHb), fast adsorption rate wihin 40 minutes, well-selectivity, and excellent recyclability in ten cycles. We expect that this work to provide an outstanding candidate for the high performance of biomacromolecular purification.


Assuntos
Microesferas , Proteínas/isolamento & purificação , Adsorção , Animais , Bovinos , Celulose/química , Cromatografia de Afinidade/métodos , Hemoglobinas/isolamento & purificação , Ligação de Hidrogênio , Permeabilidade , Porosidade , Sefarose/química
15.
J Chromatogr A ; 1636: 461773, 2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33316564

RESUMO

Extracellular vesicles (EVs) are heterogenous membrane-bound vesicles released from various origins. EVs play a crucial role in cellular communication and mediate several physiological and pathological processes, highlighting their potential therapeutic and diagnostic applications. Due to the rapid increase in interests and needs to elucidate EV properties and functions, numerous isolation and separation approaches for EVs have been developed to overcome limitations of conventional techniques, such as ultracentrifugation. This review focuses on recently emerging and modern EV isolation and separation techniques, including size-, charge-, and affinity-based techniques while excluding ultracentrifugation and precipitation-based techniques due to their multiple limitations. The advantages and drawbacks of each technique are discussed together with insights into their applications. Emerging approaches all share similar features in terms of being time-effective, easy-to-operate, and capable of providing EVs with suitable and desirable purity and integrity for applications of interest. Combination and hyphenation of techniques have been used for EV isolation and separation to yield EVs with the best quality. The most recent development using an automated on-line system including selective affinity-based trapping unit and asymmetrical flow field-flow fractionation allows reliable isolation and fractionation of EV subpopulations from human plasma.


Assuntos
Vesículas Extracelulares/química , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese , Fracionamento por Campo e Fluxo , Humanos , Ultracentrifugação
16.
J Chromatogr A ; 1635: 461632, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33333349

RESUMO

Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as ß-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.


Assuntos
Produtos Biológicos/isolamento & purificação , Química Farmacêutica/métodos , Cromatografia de Afinidade , Ligantes , Anticorpos/isolamento & purificação , Características da Família , Humanos , Peptídeos/isolamento & purificação , Peptoides/química , Proteínas/isolamento & purificação
17.
Food Chem ; 337: 127802, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32795851

RESUMO

Deoxynivalenol (DON) and T-2 toxin are major trichothecenes contaminated in cereals, which might bring harmful effects to humans. In this research, mixed anti-DON and anti-T-2 mAb were used for multiple immunoaffinity columns (mIACs) preparation. Under the optimal conditions, column capacities were tested at 1280 ng/mL for DON and 1160 ng/mL for T-2 toxin. Regeneration investigation showed mIACs capacities were over 510 ng/mL for DON and 440 ng/mL for T-2 toxin in 10 recycle usages. Good performances were obtained when applying mIACs purification coupled UHPLC-MS/MS for spiked samples with limit of detection at 3-13 µg/kg and mean recoveries at 79.0-97.6%. Applying to estimate the exposure of DON and T-2 toxin in commercial samples, maize samples were 100% DON positive and rice samples were 40% DON positive while T-2 toxin was negative in all tested samples. The proposed method is reliable and suitable for monitoring DON and T-2 toxin in cereal samples.


Assuntos
Cromatografia de Afinidade/métodos , Grão Comestível/química , Contaminação de Alimentos/análise , Toxina T-2/análise , Espectrometria de Massas em Tandem/métodos , Tricotecenos/análise , Limite de Detecção , Zea mays/química
18.
Biochim Biophys Acta Gen Subj ; 1865(1): 129761, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33086119

RESUMO

BACKGROUND: Not only strong biomolecular interactions but also weak interactions play important roles in ensuring appropriate operations of many biological systems. Although a thorough investigation of the latter is essential in understanding life science, few suitable research tools are available because of inherent difficulties. SCOPE OF REVIEW: Frontal affinity chromatography (FAC) is a versatile method that overcomes the inherent difficulties to provide accurate information on weak interactions. Since its concept and merit are not widely recognized, a comprehensive interpretation of FAC is provided in this review to encourage its application among researchers. MAJOR CONCLUSION: FAC is based on a unique principle of measuring the binding strength by the delayed migration of an analyte through an affinity column. Its utility was elucidated via the lectin-glycan interactions. GENERAL SIGNIFICANCE: FAC has a great potential as a research tool to solve many difficult problems in general bioscience that are relevant to almost all researchers.


Assuntos
Cromatografia de Afinidade/métodos , Animais , Sítios de Ligação , Cromatografia de Afinidade/instrumentação , Desenho de Equipamento , Humanos , Lectinas/análise , Polissacarídeos/análise , Proteínas/análise
19.
Methods Mol Biol ; 2210: 143-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32815135

RESUMO

OmpA-like proteins located in the outer bacterial membrane are potential virulence factors from the major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia. Our previous studies have shown that OmpA-like proteins are glycosylated by O-linked N-acetylglucosamine (O-GlcNAc) and are strongly reactive to wheat germ agglutinin (WGA) lectin, which shows sugar specificity to GlcNAc. Utilizing this property, we have developed a separation method for OmpA-like proteins by affinity chromatography using WGA lectin-agarose. The purity of enriched native OmpA-like proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. More importantly, the purified OmpA-like proteins formed a unique trimeric structure keeping their bioactivity intact. In this chapter, we describe a detailed procedure to separate OmpA-like proteins, which may be used to further progress the biological studies of OmpA-like proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Cromatografia de Afinidade/métodos , Porphyromonas gingivalis/química , Tannerella forsythia/química , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/análise , Infecções por Bacteroidaceae/microbiologia , Eletroforese em Gel de Poliacrilamida/métodos , Glicosilação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Multimerização Proteica , Aglutininas do Germe de Trigo/química
20.
Methods Mol Biol ; 2199: 45-66, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125644

RESUMO

In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.


Assuntos
Cromatografia de Afinidade , Cromatografia em Gel , Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...