COMPARISON OF SARS-COV-2-SPIKE RECEPTOR BINDING DOMAIN PRODUCED IN PICHIA PASTORIS AND MAMMALIAN CELLS

CONSORCIOARGENTINO ANTICOVID; D'ALESSIO, C (CO-CORRESPONDING AUTHOR)

Congreso; Congreso de Sociedad Argentina de Investigaciones Bioquímicas (SAIB) - Sociedad Argentina de Microbiología General (SAMIGE); 2020

A few monthsago, just before SARS-CoV-2 spread in Argentina, a group of researchers fromdifferent institutions and with diverse relevant expertises began acollaborative work to help fight pandemic. Our work was aimed at producing atlow cost the RBD domain of SARS-CoV-2 spike protein, as it is useful forserological detection of infected patients, as selection antigen to obtain neutralizingantibodies inanimal models, and as a vaccinecandidate. The yeast Pichia pastoris is a cost-effective and easily scalablesystem for recombinant protein production. In this work we compared theconformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike proteinexpressed in P. pastoris and in the well-established HEK-293T mammalian cellsystem. Our results showed that (i) RBD produced in both systems was properly folded;(ii) mass spectrometry analysis and glycosidase digestion suggested that bothforms are differentially glycosylated; (iii) both variants are conformationallystable and their stabilities depend on the ionic strength in the same way, and(iv) antibodies generated in mice injected with protein produced in yeast recognizethe 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 produceneutralizing antibodies, as well as the large scale production of serologicaltests for SARS-CoV-2.Argentinian AntiCovid Consortium (in alphabeticalorder with equal contribution of all authors): Claudia R. Arbeitman, AnalíaAmante, Gabriela Auge, Matías Blaustein, Luis Bredeston, Enrique S. Corapi,Patricio O. Craig, Leandro A. Cossio, Liliana Dain, Cecilia D?Alessio, FernandaElias, Natalia B. Fernández, Yamila Gándola, Javier Gasulla, NataliaGorojovsky, Gustavo E. Gudesblat, María G. Herrera, Lorena I. Ibañez, TommyIdrovo Hidalgo, Matías Iglesias Randon, Laura Kamenetzky, Alejandro D. Nadra,Diego G. Noseda, Carlos H. Paván, María F. Pavan, María F. Pignataro, JavierRamirez, Ernesto Roman, Lucas A. M. Ruberto, Natalia Rubinstein, Javier Santos,Francisco Velazquez Duarte, Diana E. Wetzler and Alicia M. Zelada.