The Expression, Purification and Characterization of Ebola Virion Protein 24 and Karyopherin Alpha 5

Detta är en Kandidat-uppsats från Uppsala universitet/Institutionen för kvinnors och barns hälsa

Författare: Marina Obaid; [2018]

Nyckelord: Ebolavirus; filovirus; VP24; KPNA5; IMAC;

Sammanfattning: Ebolavirus (EBOV) is a single stranded RNA virus that causes haemorrhagic fever in humans and other mammals. The EBOV encodes 7 proteins, NP, L, VP30, VP35, VP40, GP and VP24. VP24 is believed to be one of the EBOV proteins that causes the extreme virulence of the pathogen. The protein blocks the interaction between PY-STAT1 and KPNA, a protein that is involved in the import of PY-STAT1 into the nucleus. The nuclear import of PYSTAT1 is therefore blocked. This leads to the inhibition of IFN signalling. The purpose of this study was to express and purify VP24 and KPNA5. The proteins recombinantly expressed as a fusion tag in E. coli in lysogeny broth. Purification of VP24 was done using immobilized metal ion affinity chromatography, size exclusion chromatography and ion exchange chromatography. Characterization of the protein was analysed using circular dichroism. The results obtained from this study showed that VP24 could be purified in pH 10 buffers with little loss of protein due to aggregation and the protein was folded with an alphahelical structure. The expression and purification of KPNA5 was more complicated and further evaluation is left for future studies. The established protocol for expression and purification of VP24 and the initial work on KPNA5 will go a long way to aiding future studies on the system, thus the answer to the question regarding the extreme virulence of EBOV will be closer.

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