TY - JOUR T1 - Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in <em>Pichia pastoris</em> and Mammalian Cells JF - bioRxiv DO - 10.1101/2020.09.17.300335 SP - 2020.09.17.300335 AU - Argentinian AntiCovid Consortium AU - Claudia R. Arbeitman AU - Gabriela Auge AU - Matías Blaustein AU - Luis Bredeston AU - Enrique S. Corapi AU - Patricio O. Craig AU - Leandro A. Cossio AU - Liliana Dain AU - Cecilia D’Alessio AU - Fernanda Elias AU - Natalia B. Fernández AU - Javier Gasulla AU - Natalia Gorojovsky AU - Gustavo E. Gudesblat AU - María G. Herrera AU - Lorena I. Ibañez AU - Tommy Idrovo AU - Matías Iglesias Randon AU - Laura Kamenetzky AU - Alejandro D. Nadra AU - Diego G. Noseda AU - Carlos H. Paván AU - María F. Pavan AU - María F. Pignataro AU - Ernesto Roman AU - Lucas A. M. Ruberto AU - Natalia Rubinstein AU - Javier Santos AU - Francisco Velazquez Duarte AU - Alicia M. Zelada Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/09/17/2020.09.17.300335.abstract N2 - 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 receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by HPLC, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 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.Competing Interest StatementThe authors have declared no competing interest. ER -