Expression and Characterization of Bann-RBD SARS-CoV-2 Delta in Pichia pastoris Muhammad Dzul Fakhri, Ozi Jumadilla, Fernita Puspasari, Fifi Fitriyah Masduki, Ihsanawati, Dessy Natalia
Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung, Indonesia
Abstract
The recent COVID-19 disease is caused by the highly infectious pathogen SARS-CoV-2, thus the development of vaccine is urgently needed in order to prevent the spread of disease. Protein subunit vaccine based on RBD of the viral spike protein is one of the leading platform in vaccine development for COVID-19. Several studies have shown that macromolecular structure, such as multimeric protein, can induce higher protective immunity against pathogen which can counter low immunogenicity of protein subunit vaccine.. Herein, we reported the expression and characterization of multimeric protein Bann-RBD in yeast Pichia pastoris X-33. The protein is a fusion of β--annulus (Bann) peptide from tomato busy stunt virus capsid and receptor binding domain of SARS-CoV-2 Delta variant. The construct encoding the whole Bann-RBD protein is integrated into Pichia pastoris X-33 chromosome via homologous recombination in the AOX1 promotor locus. The presence of ~1.25 kb DNA band in colony PCR confirmed the insertion of Bann-RBD in chromosome. The ~2.10 kb DNA band corresponded to the intact native AOX1 gene, hence the recombinant yeast is able to utilize methanol as carbon source (Mut+ phenotype). In the expression cassette, the AOX1 promoter regulates the transcription of Bann-RBD gene under the presence of methanol. Furthermore, Bann-RBD protein has the α--mating factor signal sequence which instructs the protein to be processed in post-translational modification and secreted to the medium. The ~44 and ~72 kDa protein band in SDS-PAGE gel are the glycosylated Bann-RBD protein expressed under methanol induction. Then, deglycosylation using endoglycosidase H resulted in a ~25 kDa protein which is the theoretical molecular weight of Bann-RBD. The optimum expression conditions for Bann-RBD protein are 72 hours of 2% methanol induction with initial OD600 of 10. Interaction analysis with ELISA showed that antibodies against SARS-CoV-2 can recognize the protein. Therefore, the results of this study suggest that the Bann-RBD protein can be a potential vaccine candidate for prevention of COVID-19.
