RESUMEN
Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage-derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.
Asunto(s)
Anticuerpos Monoclonales , Técnicas de Visualización de Superficie Celular , Desarrollo de Medicamentos/métodos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos/genética , Especificidad de Anticuerpos/inmunología , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Ingeniería Genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Terapia Molecular Dirigida , Investigación Biomédica Traslacional , Resultado del TratamientoRESUMEN
Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.
Asunto(s)
Anticuerpos Antivirales/aislamiento & purificación , Antígenos Virales/inmunología , Bioprospección/métodos , Técnicas de Visualización de Superficie Celular/métodos , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Proteínas de la Nucleocápside/inmunología , Anticuerpos Antivirales/inmunología , Estudios de Factibilidad , Humanos , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/aislamiento & purificación , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/aislamiento & purificaciónRESUMEN
The cell surface ecto-enzyme CD38 is a promising target antigen for the treatment of hematological malignancies, as illustrated by the recent approval of daratumumab for the treatment of multiple myeloma. Our aim was to evaluate the potential of CD38-specific nanobodies as novel diagnostics for hematological malignancies. We successfully identified 22 CD38-specific nanobody families using phage display technology from immunized llamas. Crossblockade analyses and in-tandem epitope binning revealed that the nanobodies recognize three different non-overlapping epitopes, with four nanobody families binding complementary to daratumumab. Three nanobody families inhibit the enzymatic activity of CD38 in vitro, while two others were found to act as enhancers. In vivo, fluorochrome-conjugated CD38 nanobodies efficiently reach CD38 expressing tumors in a rodent model within 2 hours after intravenous injection, thereby allowing for convenient same day in vivo tumor imaging. These nanobodies represent highly specific tools for modulating the enzymatic activity of CD38 and for diagnostic monitoring CD38-expressing tumors.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/tratamiento farmacológico , Anticuerpos de Dominio Único/inmunología , ADP-Ribosil Ciclasa 1/inmunología , Animales , Camélidos del Nuevo Mundo , Línea Celular Tumoral , Técnicas de Visualización de Superficie Celular , Modelos Animales de Enfermedad , Epítopos/inmunología , Colorantes Fluorescentes , Humanos , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Desnudos , Mieloma Múltiple/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bacteriophage therapy dates back almost a century, but the discovery of antibiotics led to a rapid decline in the interests and investments within this field of research. Recently, the novel threat of multidrug-resistant bacteria highlighted the alarming drop in research and development of new antibiotics: 16 molecules were discovered during 1983-87, 10 new therapeutics during the nineties, and only 5 between 2003 and 2007. Phages are therefore being reconsidered as alternative therapeutics. Phage display technique has proved to be extremely promising for the identification of effective antibodies directed against pathogens, as well as for vaccine development. At the same time, conventional phage therapy uses lytic bacteriophages for treatment of infections and recent clinical trials have shown great potential. Moreover, several other approaches have been developed in vitro and in vivo using phage-derived proteins as antibacterial agents. Finally, their use has also been widely considered for public health surveillance, as biosensor phages can be used to detect food and water contaminations and prevent bacterial epidemics. These novel approaches strongly promote the idea that phages and their proteins can be exploited as an effective weapon in the near future, especially in a world which is on the brink of a "postantibiotic era."
Asunto(s)
Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Infecciones Bacterianas/terapia , Bacteriófagos , Terapia de Fagos , Animales , Antibacterianos/administración & dosificación , Antibacterianos/inmunología , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Bacteriófagos/inmunología , Bacteriófagos/fisiología , Terapia Biológica/métodos , Técnicas de Visualización de Superficie Celular , Ensayos Clínicos como Asunto , Humanos , Ratones , Proteínas Virales/uso terapéuticoRESUMEN
Tyrosine phosphorylation is an essential posttranslational modification in intracellular signaling molecules. Since tyrosine phosphorylation occurs in less than 0.1 % of all phosphorylated amino acids in mammalian cells, it is difficult to detect the nascent phosphotyrosine at a high signal-to-noise ratio due to high intracellular backgrounds (i.e., unexpected crosstalks among endogenous signaling molecules). In order to address this issue, we reconstituted the mammalian signaling pathway involving an extracellular ligand and a receptor tyrosine kinase (RTK) in Saccharomyces cerevisiae, a lower eukaryote that lacks endogenous tyrosine kinases. In this chapter, we describe a method for high-throughput analysis of ligand-receptor interaction by combining the yeast cell-surface display technique with an automated single-cell analysis and isolation system. Yeast cells coexpressing the cell-wall-anchored form of the human epidermal growth factor (EGF) and the human EGF receptor (EGFR) fused with a signal peptide at the N terminus facilitated the interaction of EGF with EGFR in an autocrine manner, followed by EGFR oligomerization and subsequent autophosphorylation. Furthermore, yeast cells expressing cell-wall-anchored forms of a conformationally constrained random peptide library instead of EGF are treated with a fluorophore-labeled anti-phosphorylated EGFR antibody and then subjected to the automated single-cell analysis and isolation system. The yeast cells with the highest level of fluorescence were shown to display novel and efficient EGFR agonistic peptides. Thus, our yeast display technique serves as a quantitative measurement for RTK activation, which is applicable to high-throughput de novo screening of RTK agonistic peptides.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Proteínas Tirosina Quinasas Receptoras/metabolismo , Levaduras/metabolismo , Animales , Línea Celular Tumoral , Técnicas de Visualización de Superficie Celular , ADN Complementario/genética , Activación Enzimática , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Expresión Génica , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Ligandos , Biblioteca de Péptidos , Péptidos/genética , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Proteínas Tirosina Quinasas Receptoras/agonistas , Proteínas Tirosina Quinasas Receptoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análisis de la Célula Individual , Levaduras/genéticaRESUMEN
In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.
Asunto(s)
Anticuerpos Monoclonales/análisis , Técnicas de Visualización de Superficie Celular/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Inmunoglobulina G/análisis , Animales , Elementos Transponibles de ADN , Evaluación Preclínica de Medicamentos/métodos , Vectores Genéticos , HumanosRESUMEN
The studies of microbes have been instrumental in combatting infectious diseases, but they have also led to great insights into basic biological mechanism like DNA replication, transcription, and translation of mRNA. In particular, the studies of bacterial viruses, also called bacteriophage, have been quite useful to study specific cellular processes because of the ease to isolate their DNA, mRNA, and proteins. Here, I review the recent discovery of how properties of the filamentous phage M13 emerge as a novel approach to combat neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/antagonistas & inhibidores , Bacteriófago M13/fisiología , Enfermedad de Parkinson/terapia , Placa Amiloide/terapia , Agregación Patológica de Proteínas/terapia , Sinucleínas/antagonistas & inhibidores , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Terapia Biológica/métodos , Técnicas de Visualización de Superficie Celular , Escherichia coli/virología , Expresión Génica , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Placa Amiloide/genética , Placa Amiloide/metabolismo , Placa Amiloide/patología , Prealbúmina/antagonistas & inhibidores , Prealbúmina/genética , Prealbúmina/metabolismo , Priones/antagonistas & inhibidores , Priones/genética , Priones/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Unión Proteica , Sinucleínas/genética , Sinucleínas/metabolismo , Proteínas Virales/biosíntesis , Proteínas Virales/química , Proteínas Virales/genética , Proteínas tau/antagonistas & inhibidores , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques.
Asunto(s)
Antivenenos/uso terapéutico , Venenos de Abeja/antagonistas & inhibidores , Diseño de Fármacos , Mordeduras y Picaduras de Insectos/tratamiento farmacológico , Proteínas de Insectos/antagonistas & inhibidores , Meliteno/antagonistas & inhibidores , Anticuerpos de Cadena Única/uso terapéutico , Animales , Antivenenos/genética , Antivenenos/metabolismo , Antivenenos/farmacología , Venenos de Abeja/química , Venenos de Abeja/enzimología , Venenos de Abeja/toxicidad , Técnicas de Visualización de Superficie Celular , Células Clonales , Quimioterapia Combinada , Edema/etiología , Edema/prevención & control , Hemólisis/efectos de los fármacos , Humanos , Mordeduras y Picaduras de Insectos/fisiopatología , Proteínas de Insectos/análisis , Proteínas de Insectos/toxicidad , Masculino , Meliteno/análisis , Meliteno/toxicidad , Ratones , Inhibidores de Fosfolipasa A2/farmacología , Inhibidores de Fosfolipasa A2/uso terapéutico , Fosfolipasas A2 Secretoras/antagonistas & inhibidores , Fosfolipasas A2 Secretoras/toxicidad , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Anticuerpos de Cadena Única/farmacología , Tejido Subcutáneo/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
Currently, development of biofuels as an alternative fuel has gained much attention due to resource and environmental challenges. Bioethanol is one of most important and dominant biofuels, and production using corn or cassava as raw materials has become a prominent technology. However, phytate contained in the raw material not only decreases the efficiency of ethanol production, but also leads to an increase in the discharge of phosphorus, thus impacting on the environment. In this study, to decrease phytate and its phosphorus content in an ethanol fermentation process, Saccharomyces cerevisiae was engineered through a surface-displaying system utilizing the C-terminal half of the yeast α-agglutinin protein. The recombinant yeast strain, PHY, was constructed by successfully displaying phytase on the surface of cells, and enzyme activity reached 6.4 U/g wet biomass weight. Ethanol productions using various strains were compared, and the results demonstrated that the specific growth rate and average fermentation rate of the PHY strain were higher 20 and 18 %, respectively, compared to the control strain S. cerevisiae CICIMY0086, in a 5-L bioreactor process by simultaneous saccharification and fermentation. More importantly, the phytate phosphorus concentration decreased by 89.8 % and free phosphorus concentration increased by 142.9 % in dry vinasse compared to the control in a 5-L bioreactor. In summary, we constructed a recombinant S. cerevisiae strain displaying phytase on the cell surface, which could improve ethanol production performance and effectively reduce the discharge of phosphorus. The strain reported here represents a useful novel engineering platform for developing an environment-friendly system for bioethanol production from a corn substrate.
Asunto(s)
6-Fitasa/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Enzimas Inmovilizadas/metabolismo , Etanol/metabolismo , Ingeniería Metabólica/métodos , Ácido Fítico/metabolismo , Saccharomyces cerevisiae/metabolismo , 6-Fitasa/genética , Reactores Biológicos , Enzimas Inmovilizadas/genética , Fermentación , Manihot/metabolismo , Fósforo/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
Anti-idiotype monoclonal antibodies represent a class of reagents that are potentially optimal for analyzing the pharmacokinetics of fully human, anti-infective antibodies that have been developed as therapeutic candidates. This is particularly important where direct pathogen binding assays are complicated by requirements for biosafety level III or IV for pathogen handling. In this study, we describe the development of a recombinant, anti-idiotype monoclonal antibody termed E1 for the detection of a fully human, serotype-specific, therapeutic antibody candidate for the BSLIII pathogen Dengue virus termed 14c10 hG1. E1 was generated by naïve human Fab phage library panning technology and subsequently engineered as a monoclonal antibody. We show that E1 is highly specific for the fully-folded form of 14c10 hG1 and can be employed for the detection of this antibody in healthy human subjects' serum by enzyme linked immunosorbent assay. In addition, we show that E1 is capable of blocking the binding of 14c10 hG1 to dengue virus serotype 1. Finally, we show that E1 can detect 14c10 hG1 in mouse serum after the administration of the therapeutic antibody in vivo. E1 represents an important new form of ancillary reagent that can be utilized in the clinical development of a therapeutic human antibody candidate.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Antivirales/farmacología , Virus del Dengue/inmunología , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/química , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Técnicas de Visualización de Superficie Celular , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos/métodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Células VeroRESUMEN
Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas de la Cápside/inmunología , Diarrea/prevención & control , Gastroenteritis/prevención & control , Inmunización Pasiva/métodos , Anticuerpos de Cadena Única/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Camélidos del Nuevo Mundo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Técnicas de Visualización de Superficie Celular , Diarrea/inmunología , Diarrea/virología , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Gastroenteritis/inmunología , Gastroenteritis/virología , Biblioteca de Genes , Pruebas de Inhibición de Hemaglutinación , Humanos , Sueros Inmunes/química , Inmunización , Masculino , Norovirus/efectos de los fármacos , Norovirus/inmunología , Norovirus/patogenicidad , Unión Proteica , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificación , PorcinosRESUMEN
Phage display involves the expression of selected proteins on the surface of filamentous phage through fusion with phage coat protein, with the genetic sequence packaged within, linking phenotype to genotype selection. When combined with antibody libraries, phage display allows for rapid in vitro selection of antigen-specific antibodies and recovery of their corresponding coding sequence. Large non-immune and synthetic human libraries have been constructed as well as smaller immune libraries based on capturing a single individual's immune repertoire. This completely in vitro process allows for isolation of antibodies against poorly immunogenic targets as well as those that cannot be obtained by animal immunization, thus further expanding the utility of the approach. Phage antibody display represents the first developed methodology for high throughput screening for human therapeutic antibody candidates. Recently, other methods have been developed for generation of fully human therapeutic antibodies, such as single B-cell screening, next-generation genome sequencing and transgenic mice with human germline B-cell genes. While each of these have their particular advantages, phage display has remained a key methodology for human antibody discovery due its in vitro process. Here, we review the continuing role of this technique alongside other developing technologies for therapeutic antibody discovery.
Asunto(s)
Anticuerpos Monoclonales , Técnicas de Visualización de Superficie Celular , Descubrimiento de Drogas , Biblioteca de Péptidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Autoantígenos/inmunología , Biotecnología , Carbohidratos/inmunología , Evaluación Preclínica de Medicamentos , Humanos , Lípidos/inmunología , Proteínas/inmunología , Proteínas/metabolismoRESUMEN
Phage display technology refers to a high-throughput in vitro screening technology for extracting required peptides/ proteins from colonies with mass mutants. Due to its high efficiency, practicability and convenience, it has been widely applied in pharmaceutical research and development, as well as target protein screening for active ingredients of traditional Chinese medicines. Target protein is the binding site of drug molecules in vivo, and good targets are the basis of excellent pharmaceuticals. This article summarizes the advance in studies on the phage display technology and its application in targeted protein screening for active ingredients of Chinese materia medica.
Asunto(s)
Técnicas de Visualización de Superficie Celular/métodos , Medicamentos Herbarios Chinos/química , Sitios de Unión , Tamizaje Masivo/métodos , Extractos Vegetales/química , Proteínas/químicaRESUMEN
Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 108 α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control.
Asunto(s)
Evolución Molecular Dirigida , Inhibidores Enzimáticos/metabolismo , Gossypium , Gorgojos/enzimología , alfa-Amilasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Arabidopsis/genética , Técnicas de Visualización de Superficie Celular , Barajamiento de ADN , Inhibidores Enzimáticos/farmacología , Variación Genética , Control de Insectos , Datos de Secuencia Molecular , Extractos Vegetales/genética , Extractos Vegetales/metabolismo , Plantas Modificadas Genéticamente , Análisis de Secuencia de Proteína , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that generally contain four RNA recognition motifs (RRMs). In potato, six cDNAs encoding full-length PTB proteins have been identified. In the present study Nova1-like protein, designated StNova1, was identified as a potential interacting partner of the StPTB proteins via yeast two-hybrid screening. Nova protein is a RNA-binding protein that contains three K-homology (KH) domains. In humans, these proteins are involved in regulation of neuronal RNA metabolism but the role of Nova-like proteins in plants is poorly understood. We have validated this interaction and mapped the protein binding region on StNova1 and StPTB1 and -6 using a novel domain interaction phage display (DIPP) technique. The interaction between the two RNA-binding proteins StPTB1/6 and StNova1 is mediated through linker regions that are distinctly separated from the RRMs. Furthermore, using a random 21-mer phage-peptide library, we have identified a number of peptides with the consensus sequence motif [S/G][V/I][L/V]G that recognize the StPTB proteins. One over-represented peptide that recognizes StPTB6 contains the GVLGPWP sequence that is similar to the GIGGRYP sequence in the glycine-rich linker region between the KH2 and KH3 domains of StNova1. We show, through site-specific mutations, the importance of glycine and proline residues in StNova1-StPTB interactions.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Técnicas de Visualización de Superficie Celular , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/química , Antígeno Ventral Neuro-Oncológico , Biblioteca de Péptidos , Proteína de Unión al Tracto de Polipirimidina/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas de Unión al ARN/química , Alineación de SecuenciaRESUMEN
Phage display technology refers to a high-throughput in vitro screening technology for extracting required peptides/ proteins from colonies with mass mutants. Due to its high efficiency, practicability and convenience, it has been widely applied in pharmaceutical research and development, as well as target protein screening for active ingredients of traditional Chinese medicines. Target protein is the binding site of drug molecules in vivo, and good targets are the basis of excellent pharmaceuticals. This article summarizes the advance in studies on the phage display technology and its application in targeted protein screening for active ingredients of Chinese materia medica.
Asunto(s)
Sitios de Unión , Técnicas de Visualización de Superficie Celular , Métodos , Medicamentos Herbarios Chinos , Química , Tamizaje Masivo , Métodos , Extractos Vegetales , Química , Proteínas , QuímicaRESUMEN
The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.