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Improvement of expression and isolation protocols of SARS-CoV-2 proteins under native conditions (Bachelor thesis)

Τερζενίδης, Μιχαήλ/ Terzenidis, MIchael

This thesis focuses on the optimization of expression and purification conditions under native settings for the accessory protein Orf9b of the SARS-CoV-2 virus. SARS-CoV-2 is responsible for the global COVID-19 pandemic and causes respiratory and gastrointestinal complications in mammals. Orf9b is encoded by an alternative open reading frame (ORF) within the gene of the nucleocapsid (N) protein and plays a significant role in suppressing the host antiviral response. Overexpression of Orf9b was carried out in a bacterial system, followed by purification attempts using affinity liquid chromatography techniques. Specifically, protein overexpression was achieved in E. coli BL21 cells, which had been successfully transformed with the plasmid pET-28a(+)-ORF9b-6xHis-Tag. Within the scope of this study, multiple experimental trials were conducted to determine the optimal conditions for achieving soluble protein expression and purification. Ultimately, optimization of the purification process resulted in a satisfactory outcome, yielding ≈2.5 mg of purified protein following the affinity chromatography step. Furthermore, bioinformatics and structural analyses were performed to compare Orf9b with its homologous protein from SARS-CoV-1. The crystal structure of Orf9b has been elucidated both in its isolated homodimeric form and in complex with the mitochondrial outer membrane translocase TOM70. Intriguingly, upon TOM70 engagement, Orf9b undergoes a striking conformational transition, adopting a spatial fold distinctly different from its homodimeric state, which highlights its remarkable structural plasticity. This structural plasticity of the viral protein highlights its potential metamorphic properties, enabling it to adopt alternative three-dimensional conformations depending on its environment, thereby influencing its biological functionality.