Functional Expression of the Recombinant Spike Receptor Binding Domain of SARS-CoV-2 Omicron in the Periplasm of Escherichia coli

Abstract

A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant known as Omicron has caused a rapid increase in recent global patients with coronavirus infectious disease 2019 (COVID-19). To overcome the COVID-19 Omicron variant, production of a recombinant spike receptor binding domain (RBD) is vital for developing a subunit vaccine or a neutralizing antibody. Although bacterial expression has many advantages in the production of recombinant proteins, the spike RBD expressed in a bacterial system experiences a folding problem related to disulfide bond formation. In this study, the soluble Omicron RBD was obtained by a disulfide isomerase-assisted periplasmic expression system in Escherichia coli. The Omicron RBD purified from E. coli was very well recognized by anti-SARS-CoV-2 antibodies, sotrovimab (S309), and CR3022, which were previously reported to bind to various SARS-CoV-2 variants. In addition, the kinetic parameters of the purified Omicron RBD upon binding to the human angiotensin-converting enzyme 2 (ACE2) were similar to those of the Omicron RBD produced in the mammalian expression system. These results suggest that an E. coli expression system would be suitable to produce functional and correctly folded spike RBDs of the next emerging SARS-CoV-2 variants quickly and inexpensively.

Production of neutralizing antibody fragment variants in the cytoplasm of E. coli for rapid screening: SARS-CoV-2 a case study

“…Without any further process optimization, we were able to produce 16 mg/L, 13 mg/L and 12 mg/L of purified mtDsbC-RBD fusion protein of wild type, Alpha, and Omicron variants respectively from shake-flask cultures. These purified protein yields are circa 60-fold higher than previously reported yields of wild type MBP-RBD fusion with the CyDisCo system 20 and circa 120-fold higher than periplasmically expressed MBP-RBD (Omicron BA.1) fusion 21 . The purified proteins were then subjected to electrospray ionisation mass spectrometry (ESI-MS) analysis and were found to be natively folded with all cysteines in disulfide bonds (Supplementary Table S2).…”

“…Here, we report soluble production of the RBD of SARS-CoV-2 wild type, Alpha, and Omicron variants, fused to a solubilization tag, using the CyDisCo system in the cytoplasm of E. coli. While this is not the first report to produce the RBD of SARS-CoV-2 in the cytoplasm 20 or periplasm 21 of E. coli, our use of alternative media, strain and fusion partner resulted in 60-120 × higher yields than previously reported i.e., we have a significantly more efficient production system. In addition, expression of multiple variants was shown and the redox status of the purified proteins verified.…”

Approaching Challenges Posed by SARS-CoV-2 Genetic Variants

“…These numbers highlight and support the relevance of the study of emerging SARS-CoV-2 genetic variants. The topics focused on emerging SARS-CoV-2 genetic variants and addressed in these publications include new treatment interventions (e.g., [2,3]) and vaccine formulations (e.g., [4,5]); virus detection tools (e.g., [6,7]); study of host-virus molecular interactions (e.g., [8][9][10]); virus infection, transmission, and lethality Among novel tools, the quantum mechanisms may be considered to understand SARS-CoV-2 evolution and the appearance of new genetic variants and the design of vaccine protective antigens [35]. For example, quantum genetics could be applied to study virus evolution as proposed by Baianu [36] regarded as a "multi-scale process which is initiated by underlying quantum (coupled) multi-molecular transformations of the genomic and interactomic networks, followed by specific phenotypic transformations at the level of organism and the variable biogroupoids associated with the evolution of species which are essential to the survival of the species".…”