Construction of Recombinant Plasmid pHIPX4-RBDTAA-Foldon and Expression of Protein RBD From SARS-CoV-2 on Hansenula polymorpha
Mutiara Mila Kamilah (a), Fernita Puspasari (a), Ihsanawati (a), Dessy Natalia (a*)

Chemistry Department, Bandung Institute of Technology
Jalan Ganesha 10, Bandung 40132, Indonesia
*dessynatalia[at]itb.ac.id

Abstract

In 2020, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) caused a global pandemic. The development of Covid-19 vaccines that can be used globally is the most effective strategy to end the pandemic. 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 ACE2, thereby blocking the binding of the RBD to ACE2 and hence RBD is an ideal antigen for protein subunit-based vaccine. Current protein S or RBD-based vaccines use insect and mammalian host cells which require high costs and a lengthy process. Methylotropic yeasts such as H. polymorpha can be an alternative expression system as it has several advantages such as faster production at a relatively lower cost and has a post-translational modification system. The aims of this research were to construct recombinant plasmid containing RBD gene, to generate a H. polymorpha containing the RBD gene, and to produce the RBD protein. Recombinant plasmid pHIPX4-RBDTAA-Foldon has been successfully constructed and verified by restriction enzyme analysis and nucleotide sequence analysis. The resulted pHIPX4-RBDTAA-Foldon plasmid was linearized by the ScaI restriction enzyme and transformed into the H. polymorpha. Integration of the plasmid into the H. polymorpha NCYC495 chromosome has been successfully carried out as confirmed by PCR colony analysis. Subsequently, recombinant RBD has been expressed as an extracellular protein using BMMY medium containing 2% methanol as an inducer for 72 hours. 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.

Keywords: SARS-CoV-2, vaccine, Receptor Binding Domain, H. polymorpha

Topic: CHEMISTRY AND MATERIAL SCIENCES