Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain

Abstract

The COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of the SARS-CoV-2 spike protein receptor binding domain using the CyDisCo system to create and maintain the correct disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce functional, active antigen in bacteria capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.

The nuts and bolts of SARS-CoV-2 Spike Receptor Binding Domain heterologous expression

“…Bacterial RBD production was achieved by recovering the protein from the insoluble fraction and through a careful process of refolding. Efforts to increase its solubility by using Lemo21 E. coli strain failed in agreement with previous attempts to produce this protein, or its ancestor (SARS-CoV RBD), in E. coli in its native soluble form 21,35,36 . After refolding, isolated E. coli- RBD showed a good degree of purity and was efficiently recognized by commercial antibodies.…”

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

“…Probably this is one of the reasons why RBD of SARS-CoV-2, besides its production in mammalian cells [9], has also been produced in several systems [10][11][12][13][14], including bacteria [15], despite the challenges of expressing a nonglobular protein with four disulfide bonds and the requirement of the N-glycosylation for its proper expression and folding [11].…”

Preclinical Immune Response and Safety Evaluation of the Protein Subunit Vaccine Nanocovax for COVID-19

“…The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health emergency ( 1 ). Since it was first reported in Wuhan, China, at the end of 2019, there have been over 165 million cases worldwide and nearly 3.5 million deaths as of May 2021 ( 2 ), with no obvious short-term resolution.…”