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

Abstract

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