EXPRESSION AND CHARACTERIZATION OF BANN-RBD SARS-COV-2 DELTA IN PICHIA PASTORIS

• Main Author: Dzul Fakhri, Muhammad
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/73189
• Institution: Institut Teknologi Bandung

Coronavirus disease 2019 (COVID-19) is a disease that damages the human respiratory tract caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID- 19 pandemic has caused more than 765 million cases based on the latest report from the World Health Organization (WHO) on the 3rd of May 2023. SARS-CoV-2 infection begins with the interaction between receptor binding domain (RBD) of the viral spike protein and the angiotensin-converting enzyme 2 (ACE2) on the cell surface. Immunization with RBD can induce an immune response to produce neutralizing antibodies, thus RBD is one of the COVID-19 vaccine candidates which are continuously being developed. Several studies have shown RBD multimers can induce a higher immune response than RBD monomers. A novel platform that can be used to assemble a multimeric RBD is ?-annulus peptide (Bann) derived from the capsid of tomato bushy stunt virus (TBSV). The aim of this study is to construct the yeast Pichia pastoris which carries the gene encoding Bann-RBD, express, and characterize the resulting Bann-RBD protein. The yeast Pichia pastoris X-33 carrying the gene encoding Bann-RBD has been constructed through homologous recombination events in the promoter region of the AOX1 gene. The PCR analysis result of recombinant yeast colonies showed a DNA band measuring ~1.25 kb which is the Bann-RBD gene. Recombination events can occur repeatedly, hence the yeast colonies that undergo multiple plasmid insertions are determined by gradual antibiotic selection. For protein expression, the Bann-RBD expression cassette is under the control of the AOX1 promoter so that its expression is induced by presence of methanol in the medium. The ?-mating factor sequence at the N-terminus of the Bann-RBD protein signals the protein to be secreted. Based on the SDS-PAGE profile of expression for 72 hours, Bann-RBD was obtained as an extracellular glycoprotein with molecular weight of ~44 and ~72 kDa. Deglycosylation of extracellular protein with endoglycosidase H resulted in a ~25 kDa protein that corresponds to the theoretical size of RBD. The optimum conditions for Bann-RBD protein expression in P. pastoris X-33-Bann-RBD are 72 hours of induction, initial OD600 of 10, and 2% methanol content. Based on the enzyme-linked immunosorbent assay (ELISA) result, the protein interacts with poly RBD IgY chicken and mono S1 IgG human antibodies. The results of this study suggest that the Bann-RBD protein can be a candidate for COVID-19 vaccine.