Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced inPichia pastorisand Mammalian Cells

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 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.

A study protocol to prepare an RBD protein for vaccine against COVID-19

“…After vaccine formulation following a standard protocol, the vaccine must meet the allergen tests before application to patients following US FDA recommended vaccine evaluation and management. 65 RBD quantitation using spectrophotometry We will take a 10 μl sample of the dialyzed RBD protein and determine its concentration by measuring OD at UV 280 nm using a UV-Vis spectrophotometer following Arbeitman, et al 66 The total amount of RBD protein in the dialyzed sample will be equal to M = (OD ÷ L) x D. (Where, M = amount of RBD protein present in mg per ml in the original sample; OD = Optical density at 280 nm ; L = the length of cuvette light path in cm; D = dilution factor.) 67,68 Figure 7.…”

A study protocol to prepare an RBD protein for vaccine against COVID-19

“…After vaccine formulation following a standard protocol, the vaccine must meet the allergen tests before application to patients following US FDA recommended vaccine evaluation and management. 73,74 RBD quantitation using spectrophotometry We will take a 10 μl sample of the dialyzed RBD protein and determine its concentration by measuring OD at UV 280 nm using a UV-Vis spectrophotometer following Arbeitman, et al 75 The total amount of RBD protein in the dialyzed sample will be equal to M = (OD ÷ L) x D. (Where, M = amount of RBD protein present in mg per ml in the original sample; OD = Optical density at 280 nm ; L = the length of cuvette light path in cm; D = dilution factor.) 76,77 Evaluation of the modified RBD protein β sheet The RBD protein sequences, before (Figure 7) and after modification (Figure 8) will be tested for their alignments with known 2019-nCoV RBD protein sequence available through a computerized program, UniProt Protein Blast (UniProtKB: P00750): https://www.uniprot.org/blast/.…”

A study protocol to prepare an RBD protein for vaccine against COVID-19

“…We will take a 10 μl sample of the dialyzed RBD protein and determine its concentration by measuring OD at UV 280 nm using a UV-Vis spectrophotometer following Arbeitman, et al 49 The total amount of RBD protein in the dialyzed sample will be equal to M = (OD ÷ L) x D. (Where, M = amount of RBD protein present in mg per ml in the original sample; OD = Optical density at 280 nm ; L = the length of cuvette light path in cm; D = dilution factor.) 50,51 Evaluation of the modified RBD protein β sheet The RBD protein sequences, before (Figure 7) and after modification (Figure 8) will be tested for their alignments with known 2019-nCoV RBD protein sequence available through a computerized program, UniProt Protein Blast (UniProtKB: P00750): https://www.uniprot.org/blast/.…”