Nou marcador per detectar el VIH

Investigadors de l'Institut de Biotecnologia i de Biomedicina de la UAB han aconseguit desenvolupar una nova proteïna, denominada NF795gpC, que actua com un sensor quan entra en contacte amb el sèrum d'un pacient infectat pel VIH (Virus d'Immunodeficiència Humana) i permet detectar-lo fàcilment.Investigadores del Instituto de Biotecnología y Biomedicina de la UABhan conseguido desarrollar una nueva proteína, denominadaNF795gpC, que actúa como un sensor cuando entra en contacto con elsuero de un paciente infectado por el VIH (Virus de InmunodeficienciaHumana) y permite detectarlo fácilmente.Researchers at the Institute of Biotechnology and Biomedicine at theUAB have managed to develop a new protein, called NF795gpC, whichacts as a sensor when it comes into contact with the serum of a patientinfected by HIV (Human Immunodeficiency Virus) and allows it to beeasily detected

Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins

BACKGROUND: Many enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein. RESULTS: By using two structurally and functionally different model enzymes and two fluorescent proteins we show that physiological aggregation in bacteria might only result in a moderate loss of biological activity and that inclusion bodies can be used in reaction mixtures for efficient catalysis. CONCLUSION: This observation offers promising possibilities for the exploration of inclusion bodies as catalysts for industrial purposes, without any previous protein-refolding step

Biosensor para la detección de anticuerpos anti-VIH

Biosensor para la detección de anticuerpos anti-VIH. En la presente invención se provee un biosensor capaz de detectar anticuerpos anti-VIH en una muestra biológica, basado en la utilización combinada de una enzima alostérica, modificada genéticamente, y una matriz con redes de microelectrodos.Consejo Superior de Investigaciones Científicas (España), Universidad Autónoma de BarcelonaA1 Solicitud de patente con informe sobre el estado de la técnic

Development of allosteric biosensors for the diagnosis of infectious diseases

Consultable des del TDXTítol obtingut de la portada digitalitzadaLos biosensores representan actualmente herramientas importantes para la detección de moléculas de interés. Los biosensores proteicos destacan por su fácil producción y uso, permitiendo un desarrollo económico de biosensores que pueden ser utilizados en todo el mundo y sobretodo, útiles en países con falta de recursos y tecnología. Un diagnóstico rápido y eficaz de enfermedades infecciosas permitiría un tratamiento más rápido y un mejor aprovechamiento de los recursos disponibles. En este estudio, el biosensor proteico NF795gpC, útil para la detección de anticuerpos anti-virus de la inmunodeficiencia humana (VIH), ha sido caracterizado. La proteína NF795gpC es una beta-galactosidasa modificada previamente sintetizada en nuestro laboratorio, que contiene el péptido antigénico P1 de la proteína gp41 de VIH insertado entre los residuos 795 y 796. La inserción de péptidos en lugares permisivos expuestos del enzima, permite la síntesis de proteínas que responden a la presencia de anticuerpos específicos. El hecho de que contengan inserciones, hace que estas proteínas tengan una actividad enzimática reducida. Sin embargo, en presencia de anticuerpos específicos para el péptido insertado, estos enzimas híbridos traducen la interacción antígeno-anticuerpo en un incremento de actividad enzimática fácil de medir. Finalmente, y teniendo un ejemplo previo con el virus de la fiebre aftosa, este principio puede extenderse a la detección de otras enfermedades infecciosas que producen una respuesta immune mediada por anticuerpos. Primero se optimizó el ensayo buscando las concentraciones óptimas de proteína y sustrato y también analizando el efecto sobre sensibilidad, ratio señal-fondo y rango de respuesta, de seis sustratos diferentes. La proteína NF795gpC fue también inmovilizada en un soporte para analizar su capacidad de respuesta en estas condiciones, siendo el primer paso para un futuro desarrollo de un sistema de detección útil para hacer medidas de campo. El análisis de los diferentes tipos de anticuerpos específicos presentes en muestras de suero con el sensor mostró una correlación entre la cantidad de inmunoglobulinas IgG4 y la respuesta, considerándose esta inmunoglobulina como la mayormente responsable de la activación del sensor. Finalmente, nuevos enzimas alostéricos fueron sintetizados para mejorar la sensibilidad del sistema previamente descrito. El hecho de que la proteína NF795gpC contiene solamente un único inserto representa una limitación a la hora de diagnosticar la enfermedad, llegando solo a un 94% de coincidencia entre este y el método estándar normalmente utilizado para diagnosticar VIH. Por tanto, la inserción de otros fragmentos antigénicos del virus en la proteína permitiría aumentar el número de anticuerpos VIH específicos y, por tanto, también aumentar la sensibilidad del sistema. El desarrollo de un sistema mucho más completo representaría un gran avance en la salud pública y también sería de gran importancia para entender mejor el mecanismo de regulación enzimática en biosensores alostéricos.Biosensors have become important tools in the detection of different types of molecules of analytical interest. Among them, protein-only biosensors stand out due to their ease production and use, what allow a cheaper development and applicability with low costs, especially important in low resource areas. A better and quicker diagnosis of illnesses would allow a faster treatment and an effective use of the available resources. In this study, the protein-only biosensor NF795gpC, suitable for the detection of anti-human immunodeficiency virus (HIV) antibodies, has been characterized. NF795gpC protein is an engineered beta-galactosidase previously developed in our laboratory, containing the antigenic P1 peptide of gp41 protein of HIV in between the residues 795 and 796. The insertion of small peptides in permissive solvent exposed sites of the enzyme produces hybrid beta-galactosidases with a reduced enzymatic activity but allows the protein to respond enzymatically to peptide-specific antibodies. In presence of anti peptide monoclonal antibodies or polyclonal sera, these hybrid enzymes translate the antigen-antibody interaction into an easily measurable increase of the enzymatic activity. Furthermore, and having a previous example with FMDV, this principle can be extended to other infectious diseases producing an antibody response. First, the sensing assay was optimized looking for the optimal concentrations of NF795gpC protein and substrate generating a higher sensing response, and also analyzing the sensor response regarding sensitivity, signal-background ratio and range of response with six different substrates. Protein NF795gpC was also immobilized into a support to analyze the sensor response in these conditions, being the first step to the further development of a sensor device useful for field measurements. The analysis of the different types of antibodies present in sera samples through the sensor showed a correlation between the amount of IgG4 subpopulation of antibodies of a group of sera samples and the sensing signal, considering this immunoglobulin as the most important for sensor activation. Finally, new allosteric enzymes were constructed in order to improve the sensitivity of the test. The fact that the NF795gpC contains only one type of HIV peptide represents a limitation to the proper diagnosis of HIV infection, arriving only to a 94% of agreement between this test and the standard method normally used for HIV detection. Hence, the insertion of other antigenic fragments of HIV into the beta-galactosidase could allow the detection of other specific antibodies. The development of a more complete device could represent big advances in public health and also be helpful in a better understanding of the mechanism of enzymatic regulation in allosteric biosensors

Insertional protein engineering for analytical molecular sensing

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors

Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins

Background: Many enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein. Results: By using two structurally and functionally different model enzymes and two fluorescent proteins we show that physiological aggregation in bacteria might only result in a moderate loss of biological activity and that inclusion bodies can be used in reaction mixtures for efficient catalysis. Conclusion: This observation offers promising possibilities for the exploration of inclusion bodies as catalysts for industrial purposes, without any previous protein-refolding step

Biosensor para la detección de anticuerpos anti-VIH

[EN] In the present invention, provision is made for a biosensor capable of detecting anti-HIV antibodies in a biological sample, based on the combined use of a genetically modified allosteric enzyme and a matrix with microelectrode networks. One of the advantages of this biosensor is the high sensitivity thereof, the simplicity thereof in terms of detection and the portability thereof.[ES] En Ia presente invención se provee un biosensor capaz de detectar anticuerpos anti-VIH en una muestra biológica, basado en la utilización combinada de una enzima alostérica, modificada genéticamente, y una matriz con redes de microelectrodos. Una de las ventajas de este biosensor es su alta sensibilidad, su sencillez en la detección y su portabilidad.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Autónoma de Barcelona (UAB)A1 Solicitud de patentes con informe sobre el estado de la técnic