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

ARBEITMAN, CLAUDIA; AUGE GABRIELA; BLAUSTEIN MATIAS; BREDESTON LUIS; CORAPI ENRIQUE; CRAIG PATRICIO; COSSIO LEANDRO; DAIN LILIANA; D'ALESSIO CECILIA; ELIAS FERNANDA; FERNÁNDEZ NATALIA; GÁNDOLA YAMILA; GASULLA JAVIER; GUDESBLAT GUSTAVO; GOROJOVSKY NATALIA; HERRERA MARIA GEORGINA; IBAÑEZ ITATI; IDROVO TOMMY; IGLESIAS RANDO MATIAS; KAMENTZKY LAURA; NADRA ALEJANDRO; NOSEDA DIEGO; PAVÁN CARLOS; PAVÁN MARIA FLORENCIA; PIGNATARO MARÍA FLORENCIA; ROMÁN ERNESTO; RUBERTO LUCAS; RUBINSTEIN NATALIA; SANTOS JAVIER; VELAZQUEZ FRANCISCO; ZELADA ALICIA

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.