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

Reviewers: Tom Gallagher (Loyola University Chicago) | 📒📒📒 ◻️◻️ • Roslyn Bill (Aston University) | 📗📗📗📗◻️

Published onNov 25, 2020
Reviews of "Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells"
key-enterThis Pub is a Review of
Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
Description

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.

To read the original manuscript, click the link above.

Summary of Reviews: This pre-print compares S protein RBD production in Pichia pastoris compared to that produced in mammalian cells. While the study is reliable, the manuscript should be revised for accuracy and further functional testing should be performed.

Reviewer 1 (Tom Gallagher) | 📒📒📒 ◻️◻️

Reviewer 2 (Roslyn Bill) | 📗📗📗📗◻️

RR:C19 Strength of Evidence Scale Key

📕 ◻️◻️◻️◻️ = Misleading

📙📙 ◻️◻️◻️ = Not Informative

📒📒📒 ◻️◻️ = Potentially Informative

📗📗📗📗◻️ = Reliable

📘📘📘📘📘 = Strong

To read the reviews, click the links below.

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