Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells
Claudia R. Arbeitman, Gabriela Auge, Matı́as Blaustein, Luis M. Bredeston, Enrique Corapi, Patricio O. Craig, Leandro A. Cossio, Liliana Daín, Cecilia D’Alessio, Fernanda Elias, Natalia Fernández, Yamila B. Gándola, Javier Gasulla, Natalia Gorojovsky, Gustavo E. Gudesblat, María Georgina Herrera, Lorena I. Ibañez, Tommy Idrovo, Matías Iglesias Rando, Laura Kamenetzky, Alejandro D. Nadra, Diego G. Noseda, Carlos H. Paván, María Florencia Pavan, María F. Pignataro, Ernesto A. Román, Lucas Ruberto, Natalia Rubinstein, Javier Santos, Francisco Velázquez, Alicia M. Zelada
Abstract
Abstract 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 severe acute respiratory syndrome coronavirus-2 (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 T m 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 high-performance liquid chromatography, 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.