Expression and purification of mitophagy receptor BNIP3L/NIX ; Ekspresija i pročišćavanje receptora mitofagije BNIP3L/NIX

Homeostasis is one of the most important mechanisms that a living organism can have. It maintains and regulates the internal environment of organisms through numerous pathways. One of them is autophagy, a mechanism that balances the production and destruction of cellular organelles and components to preserve cellular integrity. A specific type of autophagy that degrades damaged and dysfunctional mitochondria is called mitophagy. There are a few types of mitophagy, but one of the most interesting ones is the BNIP3L/NIX-mediated mitophagy. The major mediator in this process is BNIP3L/NIX, a protein that comes in a monomeric and dimeric form. The BNIP3L/NIX is positioned on the outer mitochondrial membrane and is crucial for interactions with the autophagosomes and thus promoting mitophagy. To define and study the mechanisms behind this pathway, it would be of great benefit to produce the protein in vitro. As it is a transmembrane protein, this is very challenging. By using pGEX-4T1 plasmids, we successfully achieved the expression of GST-tagged BNIP3L/NIX within E. coli BL21 bacterial strain. Furthermore, we effectively purified the expressed protein utilizing Glutathione Sepharose beads. We have concluded that BNIP3L/NIX proteins can be induced in BL21 bacteria in a 3 hour incubation at 37°C and that they have to be extracted from the bacterial pellet rather than the supernatant. We have also concluded that the GST-NIX wt variant is the hardest to produce and purify as opposed to the mutated GST-NIX G203A and ΔTM variants. These results could potentially lead to new discoveries and research done regarding BNIP3L/NIX and its role in BNIP3L/NIX-mediated mitophagy. ; Homeostaza je jedan od najvažnijih mehanizama koje živo biće može imati. Ona održava i regulira unutarnji okoliš organizama kroz bezbroj bioloških procesa. Jedan od njih je autofagija, mehanizam koji balansira stvaranje i razgradnju staničnih organela i komponenata kako bi se održao stanični integritet. Posebna vrsta autofagije koja razgrađuje oštećene ...