Comparison of SARS-COV-2-Spike Receptor binding domain produced in Pichia Patoris and mammalian cells.

IDROVO-HIDALGO, TOMMY; ALICIA M ZELADA; ARGENTINIAN ANTICOVID CONSORTIUM

Congreso; LVI SAIB Meeting/XV SAMIGE Meeting; 2021

SAIB/SAMIGE

A few months ago, just before SARS-CoV-2 spread in Argentina, a group of researchers from different institutions and withdiverse relevant expertise began a collaborative work to help fight the pandemic. Our work was aimed at producing at lowcost the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, as it is useful for serological detection of infectedpatients, as a selection antigen to obtain neutralizing antibodies in animal models, and as a vaccine candidate. The yeast Pichiapastoris is a cost-effective and easily scalable system for recombinant protein production. In this work, we compared theconformation of the RBD from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well-established HEK-293Tmammalian cell system. Our results showed that (i) RBD produced in both systems was properly folded; (ii) mass spectrometryanalysis and glycosidase digestion suggested that both forms are differentially glycosylated; (iii) both variants areconformationally stable and their stabilities depend on the ionic strength in the same way, and (iv) antibodies generated inmice injected with proteins produced in yeast recognize the protein produced in mammalian cells and vice versa. Theproduction of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 60 mg/L of 90% pure protein, thus potentiallyallowing large scale immunizations to produce neutralizing antibodies, as well as the large-scale production of serological testsfor SARS-CoV-2.Argentinian AntiCovid Consortium (in alphabetical order with equal contribution of all authors): Claudia R. Arbeitman,Analía Amante, Gabriela Auge, Matías Blaustein, Luis Bredeston, Enrique S. Corapi, Patricio O. Craig, Leandro A. Cossio,Liliana Dain, Cecilia D’Alessio, Fernanda Elias, Natalia B. Fernández, Yamila Gándola, Javier Gasulla, Natalia Gorojovsky,Gustavo E. Gudesblat, María G. Herrera, Lorena I. Ibañez, Tommy Idrovo Hidalgo, Matías Iglesias Randon, LauraKamenetzky, Alejandro D. Nadra, Diego G. Noseda, Carlos H. Paván, María F. Pavan, María F. Pignataro, Javier Ramirez,Ernesto Roman, Lucas A. M. Ruberto, Natalia Rubinstein, Javier Santos, Francisco Velazquez Duarte, Diana E. Wetzler andAlicia M. Zelada.