Structural and Functional Comparison of SARS-CoV-2-SpikeReceptor Binding Domain Produced in​Pichia pastorisandMammalian Cells

ARGENTINIAN ANTICOVID CONSORTIUM; KAMENETZKY, L.

Scientific Reports

Cold Spring Harbor Laboratory

Año: 2020

2045-2322

The yeast ​Pichia pastorisis a cost-effectiveand easily scalable system for recombinantproteinproduction. In this work we compared the conformation of the receptorbinding domain (RBD) from SARS-CoV-2 Spikeprotein expressed in​P. pastorisandinthe well established HEK-293Tmammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, asindicated by UV-absorption, circular dichroismand tryptophan fluorescence. They also had similar stability, as indicated bytemperature-induced unfolding (observed​T​mwere 50 °C and 52 °C for RBD produced inP. pastorisandHEK-293T cells, respectively). Moreover, the stability of both variantswas similarly reduced when the ionicstrength was increased, in agreement with a computationalanalysis predicting that a set of ionic interactions may stabilize RBDstructure. Further characterization byHPLC, size-exclusion chromatography and massspectrometryrevealed a higher heterogeneity of RBD expressed in​P. pastorisrelativeto that produced in HEK-293T cells, whichdisappeared after enzymatic removal of glycans.The production of RBD in​P. pastoriswasscaled-up in a bioreactor, with yieldsabove45 mg/L of 90% pure protein, thus potentially allowing large scale immunizationsto produce neutralizing antibodies, as wellas the large scale production of serological testsfor SARS-CoV-2