CONSTRUCTION OF RECOMBINANT PLASMID PHIPX4-RBDTAA-FOLDON AND EXPRESSION OF PROTEIN RBD FROM SARS-COV-2 ON HANSENULA POLYMORPHA

• Main Author: Mila Kamilah, Mutiara
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/70423
• Institution: Institut Teknologi Bandung

The Corona Virus 2019 (COVID-19) pandemic is a disease caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Mutations occur in the SARS-CoV-2 virus and the high need for global vaccination, require periodic vaccine development. One of the widely used vaccine platforms is protein subunit-based vaccines. Studies have shown that neutralizing antibodies specific to Receptor Binding Domain (RBD) can bind to either the upstream or downstream conformation of the RBD or to structures other than the ACE2 binding site, thereby blocking the binding of the RBD to ACE2. This makes the RBD an ideal antigen in the process of making protein subunit-based vaccines. Current protein S or RBD-based vaccines use insect and mammalian host cells. Using this expression system production of vaccines require high costs and a lengthy process that stays longer. Methylotrophic yeasts such as H. polymorpha can be an alternative expression system that will provide advantages such as fast production at a relatively low cost and the availability of post-translational modifications. The aim of this research was to construct H. polymorpha containing the RBD gene, expression, and characterization of the RBD protein. Plasmid construction was made using a site directed mutagenesis with pHIPX4-RBD-Foldon as the template. Glycine codon mutation into a stop codon was confirmed by using Sanger sequencing method. This change in sequence indicated that the plasmid pHIPX4-RBDTAA-Foldon which is about ~8 kb has been successfully constructed and cloned in E. coli TOP10F' cells. This plasmid still carries the RBD-Foldon coding gene, but the foldon protein is not translated into polypeptides of the foldon protein. The pHIPX4-RBDTAA-Foldon plasmid was linearized using the ScaI restriction enzyme and transformed into the H. polymorpha. Integration of the recombinant pHIPX4-RBDTAA-Foldon plasmid into the H. polymorpha chromosome occurred through homologous single crossover recombination at the 5'MOX promoter locus. Integration of the plasmid pHIPX4-RBDTAA-Foldon into the H. polymorpha NCYC495 chromosome has been successfully carried out. The presence of a ~1 kb DNA fragment in PCR colony confirmed that the RBDTAA-Foldon gene has been integrated into the H. polymorpha NCYC495 chromosome. RBD protein was expressed in H. polymorpha using BMMY medium containing 2% methanol for 72 hours with an initial cell density OD600 of 20 and 10% aeration. The RBD gene inserted into the pHIPX4 plasmid was fused with the ?-mating factor prepro signal sequence (?-MF) from Saccharomyces cerevisiae so that the protein will be secreted into the cell supernatant. The soluble protein in the media was then concentrated with Tangential Flow Ultrafiltration (TFF). SDS-PAGE analysis revealed that RBD protein has been expressed as a protein with molecular weight of ~23 kDa bands. Enzyme-Linked Immunosorbent Assay (ELISA) showed the resulted recombinant RBD can recognize mouse anti-RBD antibodies. Thus, RBD of SARS-CoV-2 produced in yeast H. polymorpha could be a potential candidate vaccine for COVID-19.