Abstract
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.
Highlights
The yeast Pichia pastoris is a cost-effective and scalable system for recombinant protein production
We were able to express this protein in two different systems: P. pastoris and mammalian cells (HEK-293T), which allowed us to gain useful insights concerning receptor binding domain (RBD) conformation and stability
We attempted to express RBD in E. coli, even though an examination of its structure suggested that this system would not be suited for its expression due to the existence of 4 disulfide bonds and a non-globular shape
Summary
The yeast Pichia pastoris is a cost-effective and 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. With the proposed role of pangolins as SARS-CoV-2 intermediate hosts, CoVs from pangolins share the highest genetic similarity with this virus in the region encoding the receptor binding domain (RBD) of the Spike protein[11,14]. Due to its important role for SARS-CoV-2 entry into the host cell, Spike is the most studied protein of this virus. This transmembrane glycosylated protein is composed of 1273 amino acid assemblies as a homotrimer
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