Abstract
An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell’s receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.
Highlights
Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) [1] is responsible for the COVID-19 pandemic and continues to pose a global health threat, despite the availability of vaccines that are mRNA-based, vector-based, inactivated viruses, or DNA vaccines [2]
A solubility-enhancing peptide (SEP)-tag, C9R, consisting of three repeated blocks of three arginine residues preceded by a glycine (GR3 )3 was introduced at the C-terminus of the SARS-CoV2 receptor-binding domain (RBD) using a 2-glycine linker between
The production time in eukaryotic cells is long and the yield is moderate resulting in a high production cost, which does not meet the demands of therapeutic and clinical development
Summary
Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) [1] is responsible for the COVID-19 pandemic and continues to pose a global health threat, despite the availability of vaccines that are mRNA-based, vector-based, inactivated viruses, or DNA vaccines [2]. SARS-CoV-2 is a single-stranded, positive-sense RNA virus belonging to the coronaviridae family [4]. SARS-CoV-2 is made of four major structural proteins [4], where the homotrimeric spike protein (S protein) mediates the viral entry into the host cells through the binding of the receptor-binding domain (RBD) with ACE2 (angiotensin-converting enzyme-2), the host receptor [5,6]. The RBD of SARS-CoV-2 (isolate Wuhan-Hu-1) spans residues 319–541 of the spike protein. It is a β-sheet protein and contains four disulfide bonds and one free cysteine
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