Evaluación de estrategias para la producción de proteínas recombinantes solubles de Plasmodium falciparum, en un sistema procariote

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dc.contributor.advisorChaparro Olaya, Jacqueline
dc.contributor.advisorHernández Atehortúa, Paula Constanza
dc.contributor.authorMorales de la Pava, Liliana
dc.date.accessioned2023-03-02T15:23:51Z
dc.date.available2023-03-02T15:23:51Z
dc.date.issued2013
dc.description.abstractLa malaria es la enfermedad parasitaria con el mayor número de casos clínicos y muertes reportadas al año, causados principalmente por Plasmodium falciparum, una de las cinco especies que infecta a humanos. Diversas estrategias de control se han implementado para combatir la enfermedad, desde el desarrollo de campañas de educación para evitar la transmisión, hasta el uso de insecticidas y medicamentos contra el vector y el parásito. En miras de desarrollar nuevas estrategias para bloquear la enfermedad, la invasión del parásito al glóbulo rojo ha despertado gran interés en los últimos años, proceso que es llevado a cabo por un complejo de proteínas denominado Glideosoma, que incluye, entre otras proteínas, un motor actina-miosina. Hasta la fecha se han identificado seis miosinas en P. falciparum y solo una de ellas (PfMyoA) ha sido caracterizada como participante activa en el Glideosoma; sin embargo, resultados previos de nuestro laboratorio acompañan la idea de que otra miosina del parásito posiblemente participa en el proceso de invasión. Para retar dicha hipótesis es necesaria la evaluación de interacciones entre todas las proteínas del Glideosoma y la miosina candidata, para lo cual obtener el repertorio completo de las proteínas es imperativo; de esta manera, la producción de proteínas recombinantes se convierte en la estrategia molecular de elección. Sin embargo, la obtención de proteínas recombinantes solubles de P. falciparum es un desafío, debido a características muy particulares en el genoma del parásito. El objetivo de este trabajo fue evaluar estrategias que permitieran la obtención de proteínas recombinantes solubles, bien sea a partir de la expresión soluble o de la recuperación a partir de cuerpos de inclusión, sin la adición de agentes caotrópicos para la solubilización. Adicionalmente, con el fin de conocer si el genoma de P. falciparum tenía más miosinas (diferentes a las ya descritas) se hizo un análisis bioinformático para la búsqueda de nuevas secuencias. Por otro lado, se realizó una comparación de estructuras terciarias entre PfMyoA y nuestro candidato a homólogo funcional (PfMyoB), para lo cual la identificación de similitudes entre las estructuras, soportaría dicha hipótesis. Los resultados obtenidos en la inducción de la expresión soluble, indicaron que ninguno de los parámetros evaluados en la inducción, como temperatura, concentración de IPTG y densidad óptica (DO), tuvo efecto en la solubilidad de las proteínas recombinantes rPfMyoB y rPfGAP50. Sorprendentemente, el uso de vectores con secuencias fusión y bacterias modificadas para la optimización de la traducción y plegamiento, tampoco ofrecieron ventajas significativas sobre la solubilidad, pues solo un poco de expresión soluble se obtuvo con la combinación del vector pMAL y las bacterias chaperonas; sin embargo, la mayoría de la proteína recombinante se concentró junto con los cuerpos de inclusión; por consiguiente nuestros resultados sugieren que la marcada insolubilidad de las recombinantes producidas, está influenciada por características propias de la secuencia proteica (aún no establecidas) y no a condiciones en la inducción de la expresión. Teniendo en cuenta que las recombinantes siempre se expresaron como proteínas insolubles, se desarrolló una estrategia de electro elusión para la recuperación de recombinantes solubles de P. falciparum desde cuerpos de inclusión. Adicionalmente, con base en la comparación de los resultados en la predicción de solubilidad de programas bioinformáticos y los obtenidos experimentalmente, se concluye que la única manera de conocer el comportamiento soluble de las proteínas recombinantes de P. falciparum producidas en Escherichia coli, es mediante el desarrollo experimental.spa
dc.description.abstractenglishMalaria is a parasitic disease, with millions of clinical cases and deaths reported annually. This mortality is primarily caused by Plasmodium falciparum, one of the five species that cause malaria in humans. Several strategies have been implemented to block the disease but no one of them has been completely successful; as a result, there is a growing interest in studying the biology of the parasite in order to develop new treatment approaches. One of the processes that have gained big interest is the red blood cell invasion, event executed by a protein complex called glideosoma which includes an actin-myosin motor. The myosin A of P. falciparum (PfMyoA) has been identified as the myosin of the glideosome but results from our laboratory suggest that another myosin could be involved in the invasion process. Then, the production of recombinant proteins becomes the molecular strategy of choice to evaluate interactions between glideosome proteins and the candidate myosin. However, the production of soluble recombinant proteins from P. falciparum has become a challenge, because in despite of all the available strategies in genetic engineering, there is not protocol to ensure the production of soluble recombinants. The objective of this study was to evaluate strategies that would permit production of soluble recombinant proteins, either from the expression itself or from solubilized inclusion bodies (without the addition of chaotropic agents for the solubilization). Additionally, in order to determine whether the genome of P. falciparum had more myosins (other than those already described) bioinformatic analysis was performed to look for new sequences. Furthermore, a comparison was made between PfMyoA tertiary structures and our candidate PfMyoB (possible functional homologue), because the identification of similarities in the structures, supports this hypothesis. The results in induction of soluble expression indicated that none of the parameters evaluated in the induction as temperature, concentration of IPTG and optical density (OD) showed effect on the solubility of recombinant proteins rPfGAP50 and rPfMyoB. Surprisingly, neither the use vectors with fusion sequences, nor modified bacteria for optimizing translation and folding, offered significant advantages over solubility, instead the level of expression was increased; these results suggest, that the marked insolubility was due to the sequence characteristics, but not the induction conditions. Considering that the produced recombinant, were always with the inclusion bodies, we developed a strategy for recovering electroelution for the recovery of recombinant soluble proteins from P. falciparum. Based on the comparison of the results of prediction of solubility from bioinformatics programs and the experimental results, we concluded that the only way to know the behavior of soluble recombinant proteins of P. falciparum produced in Escherichia. coli, only is possible through experimental development.eng
dc.description.degreelevelMaestríaspa
dc.description.degreenameMagíster en Ciencias Básicas Biomédicasspa
dc.format.mimetypeapplication/pdf
dc.identifier.instnameinstname:Universidad El Bosquespa
dc.identifier.reponamereponame:Repositorio Institucional Universidad El Bosquespa
dc.identifier.repourlrepourl:https://repositorio.unbosque.edu.co
dc.identifier.urihttps://hdl.handle.net/20.500.12495/10092
dc.language.isospa
dc.publisher.facultyFacultad de Medicinaspa
dc.publisher.grantorUniversidad El Bosquespa
dc.publisher.programMaestría en Ciencias Básicas Biomédicasspa
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dc.rights.accessrightsinfo:eu-repo/semantics/closedAccess
dc.rights.accessrightshttps://purl.org/coar/access_right/c_14cb
dc.rights.localAcceso cerradospa
dc.subjectProducción de proteínasspa
dc.subjectPlasmodium falciparumspa
dc.subjectSistema procariotespa
dc.subjectGlideosomaspa
dc.subject.keywordsProtein productionspa
dc.subject.keywordsPlasmodium falciparumspa
dc.subject.keywordsProkaryotic systemspa
dc.subject.keywordsGlideosomespa
dc.subject.nlmW 50
dc.titleEvaluación de estrategias para la producción de proteínas recombinantes solubles de Plasmodium falciparum, en un sistema procariotespa
dc.title.translatedEvaluation of strategies for the production of soluble recombinant proteins of Plasmodium falciparum in a prokaryotic system.spa
dc.type.coarhttps://purl.org/coar/resource_type/c_bdcc
dc.type.coarversionhttps://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.driverinfo:eu-repo/semantics/masterThesis
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Evaluación de estrategias para la producción de proteínas recombinantes solubles de Plasmodium falciparum, en un sistema procariote
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Evaluación de estrategias para la producción de proteínas recombinantes solubles de Plasmodium falciparum, en un sistema procariote
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Maestría en Ciencias Básicas Biomédicas