Development of a fluorescence-based bacterial method for selection of aggregation inhibitors
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Utveckling av en bakteriebaserad teknlogi för selektion av aggregeringsinhibitorer. (Swedish)
Peptide aggregation ccours during the progression of many neurodegenerative diseases such as Alzhaimer's disease. For this reason, administration of aggregation inhibitors has been suggested asa viable therapeutic strategy for the prevention and treatment of such diseases. The goal of this project was to develop a system for using flow cytometry in order to select for molecules that inhibit aggregation of a specific aggregation-prone molecule. The system utilizes the fact that enhanced green fluorescent protein (EGFP) can be used as s reporter molecule for determining whether or not a given peptide is aggregating; when EGFP is expressed fused with an aggregating peptide EGFP cannot fold correctly and thus will not fluoresce, but if aggregation is inhibited EGFP can fold correctly and will fluoresce. Thus by co-expressing an aggregation-prone peptide fused with EGFP and a potential aggregation inhibitor, the effectiveness of the inhibitor will be related to the intensity of EGFP fluorescence.
For the initial development and evaluation, the aggregation prone peptide used was amyloid-ß 42 (Aß42), a peptide whose aggregation is associated with Alzheimer's disease. For the aggregation inhibitor, an Affibody nolecule known to inhibit aggregation of Aß42 was used. It was shown that co-expression of the aggregation inhibitor and Aß42 resulted in an increased EGFP fluorescence as compared to co-expression of a negative control and Aß42. This means that the system was capable of distinguishing the inhibitor from the non-inhibitor, indication that the system works in principle.
Place, publisher, year, edition, pages
Alzheimer´s disease, combinatorial protein engineering, FACS, aggregation inhibitors
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-150557OAI: oai:DiVA.org:kth-150557DiVA: diva2:744722