Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

CLAUDIA ARBEITMAN; GABRIELA AUGE; MATÍAS BLAUSTEIN; LUIS BREDESTON; ENRIQUE CORAPI; PATRICIO CRAIG; LEANDRO COSSIO; LILIANA DAIN; CECILIA D´ALESSIO; FERNANDA ELÍAS; NATALIA FERNÁNDEZ; YAMILA GÁNDOLA; JAVIER GASULLA; NATALIA GOROJOVSKY; GUSTAVO E. GUDESBLAT; MARÍA HERRERA; LORENA IBAÑEZ; TOMMY IDROVO; MATÍAS IGLESIAS RANDO; LAURA KAMENETZKY; ALEJANDRO NADRA; DIEGO NOSEDA; CARLOS PAVÁN; MARÍA F. PIGNATARO; ERNESTO ROMÁN; LUCAS RUBERTO; NATALIA RUBINSTEIN; JAVIER SANTOS; FRACISCO VELAZQUEZ; ALICIA ZELADA

Scientific reports

NLM (Medline)

Año: 2020 vol. 10

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 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 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.