Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells

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

BioRxiv

Cold Spring Harbor Laboratory

Año: 2020

0362-4331

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.