Expression, Purification and Characterization of SARS-CoV-2 Spike RBD in ExpiCHO Cells

Overview

Journal Protein Expr Purif

Specialty Molecular Biology

Date 2022 Feb 16

PMID 35172194

Authors

Matteo De March

Michela Terdoslavich

Sulena Polez

Corrado Guarnaccia

Monica Poggianella

Alessandro Marcello

Natasa Skoko

Ario de Marco

Affiliations

Soon will be listed here.

Abstract

Reliable diagnosis is critical to identify infections of SARS-CoV-2 as well as to evaluate the immune response to virus and vaccines. Consequently, it becomes crucial the isolation of sensitive antibodies to use as immunocapture elements of diagnostic tools. The final bottleneck to achieve these results is the availability of enough antigen of good quality. We have established a robust pipeline for the production of recombinant, functional SARS-CoV-2 Spike receptor binding domain (RBD) at high yield and low cost in culture flasks. RBD was expressed in transiently transfected ExpiCHO cells at 32 °C and 5% CO and purified up to 40 mg/L. The progressive protein accumulation in the culture medium was monitored with an immunobinding assay in order to identify the optimal collection time. Successively, a two-step chromatographic protocol enabled its selective purification in the monomeric state. RBD quality assessment was positively evaluated by SDS-PAGE, Western Blotting and Mass Spectrometry, while Bio-Layer Interferometry, flow cytometer and ELISA tests confirmed its functionality. This effective protocol for the RBD production in transient eukaryotic system can be immediately extended to the production of RBD mutants.

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