Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii.

Anthony J Berndt,Stephen P Mayfield,Vanessa Heredia,Tressa N Smalley,Ashley E Sproles,Yasin Torres-Tiji,Amr Badary,Francis J Fields,Bijie Ren,Ryan Simkovsky,Andrew Webber

doi:10.1371/journal.pone.0257089

Abstract

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

Highlights

In late 2019 a novel coronavirus was identified in the Wuhan province of China with a genome sequence closely resembling that of the Severe Acute Respiratory Syndrome (SARS) coronavirus identified in 2003
A 5’ Bleomycin resistance gene (BleR) was included as part of the expression cistron to allow for selection of high expressing clones on Zeocin containing media, while a Foot-and-mouth disease virus 2A ribosomal-skip motif was placed between the BleR coding region and the Receptor Binding Domain (RBD)::mClover fusion protein, so that the RBD::mClover accumulated as a single fusion protein [23]
A separate Hygromycin resistance gene driven by a betatubulin promoter was placed 3’ of the RBD transgene to allow for double selection (Zeocin and Hygromycin), ensuring that the RBD::mClover transgene is intact in any transformant selected [32]

Summary

Introduction

In late 2019 a novel coronavirus was identified in the Wuhan province of China with a genome sequence closely resembling that of the Severe Acute Respiratory Syndrome (SARS) coronavirus identified in 2003. The respiratory disease the virus causes has since been named COVID-19 (COronaVIrus Disease 2019). Widespread use of nucleic acid tests that detect the SARS-CoV-2 RNA genome, such as RT-qPCR, have become the standard method to detect viral infection. Laboratory assays that measure antibody responses and determine seroconversion are not yet comparatively as widely available. While such serological assays are not well suited to detect acute infections, and antibody production lags days behind symptoms and infectiousness, multiple relevant applications exist for such antibody tests as they are one of the best indicators

Methods

Results

Conclusion