GENERATION OF MUTATIONS BY DIRECTED EVOLUTION TARGETING THE SARS-COV-2 SPIKE GENE REGİON
Project no. 24191 · Status: Kapanmış (Yayın bekleniyor) · 2022
Summary
<p>In this thesis, mutations were generated by targeting the Receptor Binding Domain of the SARS-CoV-2 spike protein. Viral RNA obtained from SARS-CoV-2 was used as the template for amplification. Error-Prone PCR conditions were established based on data derived from 48 experimental assays, and within the framework of experimental design, MgCl₂, MnCl₂, and dNTP concentrations as well as the number of PCR cycles were theoretically standardized to induce approximately 15 base pair mutations within the RBD region. A DNA polymerase lacking proofreading activity was employed throughout the amplification process.</p><p>Under these conditions, the amplified RBD fragments were recombined to generate mutant proteins, which were subsequently purified and assessed for their ACE2 binding capacities using competitive plaque assays. Several mutants exhibited increased binding compared to the wild type protein, while IgG binding assays demonstrated that specific mutations resulted in reduced antibody recognition. Three dimensional protein models were used to analyze the spatial distribution of mutations, enabling the identification of amino acid substitutions that enhanced ACE2 interaction or contributed to immune escape. </p>