IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Comparison of sars-cov-2-spike receptor binding domain produced in pichia pastoris and mammalian cells
Autor/es:
CRAIG, PO; ARGENTINIAN ANTICOVID CONSORTIUM
Lugar:
Virtual
Reunión:
Congreso; Argentinian AntiCovid Consortium, Craig PO. LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology (SAIB), XV Annual Meeting Argentinean Society for General Microbiology (SAMIGE); 2020
Institución organizadora:
Argentine Society for Biochemistry and Molecular Biology (SAIB) y Argentinean Society for General Microbiology (SAMIGE)
Resumen:
A few months ago, just before SARS-CoV-2 spread in Argentina, a group of researchers from different institutions and with diverse relevant expertise began a collaborative work to help fight the pandemic. Our work was aimed at producing at low cost the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, as it is useful for serological detection of infected patients, as a selection antigen to obtain neutralizing antibodies in animal models, and as a vaccine candidate. The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work, we compared the conformation of the RBD from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well-established HEK-293T mammalian cell system. Our results showed that (i) RBD produced in both systems was properly folded; (ii) mass spectrometry analysis and glycosidase digestion suggested that both forms are differentially glycosylated; (iii) both variants are conformationally stable and their stabilities depend on the ionic strength in the same way, and (iv) antibodies generated in mice injected with proteins produced in yeast recognize the protein produced in mammalian cells and vice versa. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 60 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large-scale production of serological tests for SARS-CoV-2.