Estudio de estrategias para facilitar la purificación de proteínas recombinantes en la plataforma biotecnológica basada en larvas de insectos
In this Doctoral Thesis, the proteome of lepidopteran insect larvae from the species S. frugiperda and R. nu, which constitute agronomic pests, was described with the aim of collecting valuable information to use as a biotechnological platform. Specifically, this work represents the first study of p...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2024
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_8034 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_8034.dir/8034.PDF |
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| Sumario: | In this Doctoral Thesis, the proteome of lepidopteran insect larvae from the species S. frugiperda and R. nu, which constitute agronomic pests, was described with the aim of collecting valuable information to use as a biotechnological platform. Specifically, this work represents the first study of protein characterization carried out on R. nu larvae and is particularly interesting because this lepidopteran species is geographically concentrated in our region. The adaptation of laboratory rearing of R. nu, associated with its capacity for expressing high levels of recombinant proteins and the greater complexity of these compared to other expression systems, lays the foundation for its application in large-scale biotechnological processes. Data is provided on the protein composition of the larvae and the changes that arise in it after infection by baculovirus, showing a clear predominance, after infection, of proteins that function as nutrient reservoirs (Methionine-rich storage protein, Basic JH-Suppressible protein, and Moderately methionine-rich storage protein), which are usually predominant in the larval stage of the lepidopteran because they play roles during metamorphosis, proteins related to protein translation (Elongation factor-1, Eukaryotic translation initiator), amino acid synthesis (Arginine Kinase, Glutamate dehydrogenase), and post-translational modification of proteins (HSP90, Transitional ER ATPase). The behavior of contaminant proteins present in infected larvae under different chromatographic modes was also described by studying the physicochemical characteristics of the proteins in the different fractions. It was deduced that the larvae are poor in proteins with basic isoelectric points, which translates into little adsorption to cation exchange chromatographic matrices (such as SP-Sepharose and CM-Sepharose), illustrating a possible path for rationally designing downstream processing when expressing a recombinant protein in this system.
To validate the use of the platform, a process was developed for the expression and purification of the main toxin (sphingomyelinase D) present in the venom of the brown recluse spider (Loxosceles laeta). The toxin was expressed at high yields in lepidopteran larvae despite its biological toxicity. It was established that the toxin had the correct chemical structure and was immunogenic when tested in laboratory animals at the National Institute of Biological Production (INPB). To avoid causing harm to the production animal (horse), a variant of sphingomyelinase D containing a point mutation to nullify its biological activity (dermonecrotic and hemolytic) was designed. The toxoid was used as an immunogen in place of the full venom in two horses, without causing hematological alterations in the animals. The sera were analyzed and showed neutralizing activity against the venom. These results are encouraging as they could allow for obtaining a high-purity toxoid in sufficient quantities on the insect larvae-based biotechnological platform to immunize production animals. In this way, it is expected to replace spider venom with the recombinant toxoid in the antivenom production processes at INPB, contributing a high degree of innovation to a process that has been carried out traditionally for over 100 years. It is expected to contribute to a substantial, sustainable, and safe improvement in the national manufacturing process of anti-loxoscelic antivenom. The results obtained in this Doctoral Thesis also lay the foundation for the production of other toxins present in small venomous animals for which it is difficult to obtain sufficient amounts of venom. The preliminary cost study also demonstrates the advantages of implementing processes using the insect larvae-based platform |
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