|
Author
Jové Gonzalo, Marta
|
Abstract
Extracellular vesicles are in the spotlight in biomedical technologies. Their multiple biological functions awakened the research of these nanoscale vesicles as possible new technologies for medical diagnosis and treatment. More specifically, exosomes, a subtype of EVst that have specific functions in cell-cell communications and as nanocarriers for delivering material between cells, aroused the greatest interest. Nowadays, the possible roles of exosomes in biomedical applications are gold top. Despite the current advances in exosome knowledge, it’s quite recent discovery has led to several open frames. Its potential is well defined but the challenges in exosome isolation and purification leads to holding back their assessment as diagnosis tools or possible drug delivery systems. For this reason, there is an enormous need to make an effective and efficient purification and isolation methodology to obtain exosomes with no physicochemical and biological properties disruptions.
In this study, tetraspanin CD81, a transmembranal protein highly enriched protein in exosomes, was selected to introduce different tags with the aim to facilitate the exosomes isolation and purification. More precisely two locations in the Long External Loop (between leu170-Lys171 and Ser177-Gly178) were chosen as a site for tag insertion of two different tags (a 6xHis-Tag and a Strep-TagII) that facilitate the purification of CD81-modified exosomes by affinity chromatography. This modified CD81 were also fused with the fluorescent protein GFP. Hence, allowing the tracking of the modified exosomes during the purification process. To insert the different modifications in the bicistronic plasmid (eGFP-CD81-BaeI-pIRESpuro3), Golden Gate Assembly cloning was used and verified by Sanger Sequencing. The successful plasmid constructions were transfected into mammalian cells (HEK293) and the expression of the fusion proteins was analyzed by flow cytometry, fluorescent microscopy and confocal microscopy.
To sum up, the different constructs were successfully cloned into bicistronic plasmids, and the expression of the different modified CD81 was confirmed by fluorescent microscopy. The goal of this study was successfully achieved: we provided a starting point to pursue the assessment of the improved features of engineered EVs by evaluating the purification efficiency of the different CD81 modified exosomes. This future work hopefully will serve to enrich the current knowledge concerning exosomes purification and provide new frames in exosome research.
|
|
