Background In the as of yet uncurable Parkinson´s disease aggregation of α-syn is an accelerator of pathogenesis. Oligomers of α-synuclein is considered to be neurotoxic hence blocking the endocytosis of aggregated α-syn is possibly a way of preventing pathogenesis. With a protein construct of the Receptor X (RX) previously shown to bind α-syn, it can be possible to bind soluble aggregated α-syn and decrease neuron endocytosis. Aim The aim of this study was to express, purify and trimerize two different protein constructs of RX to study the binding to α-syn monomers & oligomers and if the proteins have higher affinity to α-syn oligomers. Methods In this study two RX constructs was produced with mammalian cell transfection and purified with Strep-Tactin affinity chromatography; D1, D123mut and D123 which affinity to α-syn monomers and oligomers were studied with ELISAs. Indirect ELISAs were optimized and conducted, a competitive ELISA with D123 was tested with poor reliability. Results The results show that D1 could not be determined pure enough to examine its α-syn binding ability. D123mut was pure enough for ELISAs but did not show adequate binding to α-syn. D123 did show binding to α-syn in an indirect ELISA. Conclusion The results were not as promising as expected and did not distinctly help strengthen the theory of a recombinant RX protein as a viable drug. Although there is potential, optimization of both protein constructs and methods used is essential for future studies of RX as a therapeutic protein.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-433566 |
| Date | January 2021 |
| Creators | Simon, Isak |
| Publisher | Uppsala universitet, Institutionen för farmaceutisk biovetenskap, Uppsala University |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf, application/pdf |
| Rights | info:eu-repo/semantics/openAccess, info:eu-repo/semantics/openAccess |
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