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.

Keywords: E. coli; functional expression; omicron; spike receptor binding domain.

Figure 1

Construction of RBD_omi and MBP-RBD_omi vectors. (A) Amino acid sequence of RBD_omi used in this study. The red letters indicate cysteine residues that form disulfide bonds. (B,C) Vector maps of (B) RBD_omi and (C) MBP-RBD_omi vectors. The schematic domains are colored as follows: T7 promoter (white), PelB signal sequence (sky blue), MBP (yellow), thrombin recognition site (green), RBDomi (purple), and His6 tag (light red).

Figure 2

Expression and Ni-NTA purification of RBD_omi and MBP-RBD_omi. Purification of (A) RBD_omi, (B) RBD_omi co-expressed Dsb proteins, (C) MBP-RBD_omi, and (D) MBP-RBD_omi co-expressed Dsb proteins was analyzed by bis-tris SDS-PAGE. Black arrows indicate the band positions of the recombinant proteins. The lanes of SDS-PAGE are as follows: lane 1, protein size marker (Thermo); lane 2, cell lysate before IPTG induction; lane 3, cell lysate after IPTG induction; lane 4, periplasmic fraction obtained by osmotic shock; lane 5, Ni-NTA flow through fraction; lane 6, Ni-NTA wash fraction; lane 7 to 11, Ni-NTA elution fractions.

Figure 3

Purification of MBP-RBD_omi. (A) SDS-PAGE analysis of purified MBP-RBD_omi by Ni-NTA. The black arrow indicates the band position of the MBP-RBD_omi. The lanes of SDS-PAGE are as follows: lane 1, protein size marker (Thermo); lane 2, cell lysate before IPTG induction; lane 3, cell lysate after IPTG induction; lane 4, periplasmic fraction obtained by osmotic shock; lane 5, Ni-NTA flow through fraction; lane 6, Ni-NTA wash fraction; lane 7 to 12, Ni-NTA elution fractions. (B) Size-exclusion spectrum of MBP-RBD_omi monitored at 280 nm. The black arrow indicates the position of MBP-RBD_omi fractions. (C) SDS-PAGE analysis of purified MBP-RBD_omi by size-exclusion. The lanes of SDS-PAGE are as follows: lane 1, protein size marker (Thermo); lane 2, purified MBP-RBD_omi.

Figure 4

Purification of the soluble MBP-cleaved RBD_omi. Purification and cleavage efficiency were analyzed by bis-tris SDS-PAGE. The lanes of SDS-PAGE are as follows: lane 1, protein size marker (Thermo); lane 2, purified MBP-RBD_omi; lane 3, 50 units of thrombin; lane 4, thrombin digested MBP-RBD_omi; lane 5, Ni-NTA flow through fraction; lane 6, Ni-NTA first wash fraction; lane 7, Ni-NTA second wash fraction; lane 8, Ni-NTA elution fraction.

Figure 5

ELISA binding assay of E. coli-expressed MBP-RBD_omi and mammalian cell-expressed RBDs to (A) sotrovimab, (B) CR3022, and (C) trastuzumab. MBP and HER2 were used as controls in this study. The black, red, blue, gray, and green dots indicate the absorbances of RBD wild type, MBP-RBD_omi, mammalian cell-expressing RBD_omi, MBP, and HER2, respectively. Error bars are ±standard deviation of triplicate experiments.

Figure 6

Binding assay of (A) MBP-RBD_omi and (B) RBD_omi expressed in a mammalian system to hACE2-Fc determined by BLI. Data are shown as colored lines at different concentrations of recombinant RBDs. Red lines are the best fits of the data.