Desenvolupament d’un sistema d’entrega de fàrmacs proteics antitumorals produït en Escherichia coli basat en la proteïna majoritària de la volta eucariota

Casadevall Monells, Arnau
One of the objectives of the research group in which I joined during my TFG is the development of nuclear-directed human ribonucleases as antitumor agents. Although these variants are highly active and selective against tumor cells in vitro, their activity in vivo is limited due to pharmacokinetic issues. For this reason, different projects are being developed to improve it. Currently, a drug delivery system (DDS) is being designed that would counteract some of these drawbacks. A DDS is a nanoparticle that surrounds the drug, facilitating its delivery to the target tissue and protecting it from its elimination. Specifically, this DDS is based on taking advantage of a human proteina nanoparticle, the eukaryotic vault. Its structure is interesting due to its low immunogenicity, its in vitro stability and the possibility of producing similar structures that are empty on the inside by expressing the main protein of this structure, which is the MVP. This recombinant structure is capable of encapsulating therapeutic agents fused to the INT domain. The group is attempting to recombinantly produce MVP in E. coli to form the eukaryotic vault with a tumor penetrating peptide (TPP) to encapsulate a cytotoxic ribonuclease fused to the INT domain. As a tool to facilitate this development, in this project we designed a cargo protein that allows us to determine the functionality of this recombinant vault. This cargo consists of a fusion protein between the INT domain, GFP and glutathione Stransferase. Different purification systems have been tested for GST-GFP-INT cargo protein, both from the supernatant and from the sediment of a cell lysate, and a protocol has been established that allows it to be obtained in a large amount and purity. We show that this protein can be encapuslated within a nanoparticle formed from the refolding of MVP ​
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