Clonning, periplasmic expression and functional assessment of an anti_CD133 single-chain variable antibody fragment


Sabartés Mirabete, Marc


Glioblastoma Multiforme is the most common malignant primary brain tumor. Patients have a median life expectancy of 15 months despite current treatments. The inefficiency of these therapies is essentially explained by two main challenges: the presence of the blood brain barrier, which restricts access to the peritumoral area, and the existence of glioma stem cells (GSCs), which are responsible for tumor relapse. Targeted nanotherapies have opened new avenues that may enable meeting these challenges. However, targeting GSCs is challenging because surface markers, such as CD133, are also expressed in healthy cells. This is why, our group is working on the development of antibody derivatives against GSC markers that are activated only at the tumor site. We have previously produced anti-CD133 single-chain antibody fragments (scFvs) via refolding from inclusion bodies. In this final degree thesis, we expressed anti-CD133 scFvs via periplasmic expression in order to compare their binding affinity with the ones produced via refolding. We cloned anti-CD133 scFv variants with different purification tags, expressed them and purified them. scFvs were then characterized by gel electrophoresis and western blotting. Finally, we compared the binding capacity of these scFvs to the refolded scFv on CD133 positive and negative cells.



Oller Salvia, Benjamí
Lucchi, Roberta


IQS SE - Undergraduate Program in Biotechnology