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

ARBEITMAN, CLAUDIA R.; AUGE, GABRIELA; BLAUSTEIN, MATÍAS; BREDESTON, LUIS; CORAPI, ENRIQUE S.; CRAIG, PATRICIO O.; COSSIO, LEANDRO A.; DAIN, LILIANA; DALESSIO, CECILIA; ELIAS, FERNANDA; FERNÁNDEZ, NATALIA B.; GÁNDOLA, YAMILA B.; GASULLA, JAVIER; GOROJOVSKY, NATALIA; GUDESBLAT, GUSTAVO E.; HERRERA, MARÍA G.; IBAÑEZ, LORENA I.; IDROVO, TOMMY; RANDO, MATÍAS IGLESIAS; KAMENETZKY, LAURA; NADRA, ALEJANDRO D.; NOSEDA, DIEGO G.; PAVÁN, CARLOS H.; PAVAN, MARÍA F.; PIGNATARO, MARÍA F.; ROMAN, ERNESTO; RUBERTO, LUCAS A. M.; RUBINSTEIN, NATALIA; SANTOS, JAVIER; VELAZQUEZ, FRANCISCO; ZELADA, ALICIA M.

Scientific Reports

Nature Research

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.