High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector

Abstract

The spike (S) protein is one of the three proteins forming the coronaviruses’ viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests–the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 –human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.

Glycosylation of Receptor Binding Domain of SARS-CoV-2 S-Protein Influences on Binding to Immobilized DNA Aptamers

“…In mammalian cells this oligosaccharide then undergoes transformation into Sia 2 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 (Sia—sialic acid, Gal—galactose), while in yeast, Man 8 GlcNAc 2 is expanded with mannose residues yielding Man 15-30 GlcNAc 2 clusters [ 19 ]. In this study we used an RBD protein with homogeneous glycosylation in CHO cell line as judged by denaturating polyacrylamide gel electrophoresis (PAGE) that provided a molecular weight about ~37 kDa [ 20 ]. Glycosylation of RBD in P. pastoris proceeded heterogeneously; ~2/3 of the protein had molecular weight ~23 kDa, and ~1/3 had molecular weight close to 40 kDa ( Figure S1 ).…”

Host genomes for the unique SARS-CoV-2 variant leaked into Antarctic soil metagenomic sequencing data

“…For example in Fukushi et al 15,16 in vitro passage of SARS-CoV on CHO cells that expressed rat ACE2 yielded a new adapted variant that encoded two new mutations in the spike protein. The same cell line was used for SARS vaccine development 17 and recently also in several SARS-CoV-2 related research studies 18,19 .…”

α-Hemolysin-Aided Oligomerization of the Spike Protein RBD Resulted in Improved Immunogenicity and Neutralization Against SARS-CoV-2 Variants

“…In addition, a flexible linker, GGGGS, was placed between mHla and RBD, as the amino acids in this linker do not contain any side chains, so it does not play much a role in protein secondary structure formation (26). The TPA signal peptide (SP) was placed at the N-terminus of the fusion protein to ensure the secretion of the recombinant protein (27), and an 8×His tag was placed at the Cterminus to facilitate purification of the target protein (Figure 1B). Both RBD and mHla-RBD were highly expressed in HEK-293F cells, and the purity of these proteins was up to 95%, as determined by SDS-PAGE after affinity chromatography and desalting (Figure 1C).…”