RT Journal Article SR Electronic T1 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 JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.11.04.368092 DO 10.1101/2020.11.04.368092 A1 Maria V. Sinegubova A1 Nadezhda A. Orlova A1 Sergey V. Kovnir A1 Lutsia K. Dayanova A1 Ivan I Vorobiev YR 2020 UL http://biorxiv.org/content/early/2020/11/11/2020.11.04.368092.abstract AB 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 similar applications.Competing Interest StatementMVS and LKD declare that they have no competing interests. SVK, NAO and IIV are inventors of the patent RU2488633, which covers the use of the p1.1 plasmid. The existence of a patent does not alter authors' adherence to sharing data and materials.CHOChinese hamster ovaryDHFRdihydrofolatereductaseEMCVencephalomyocarditis virusIRESinternal ribosome entry siteMTXmethotrexateHThypoxanthine-thymidineRBDreceptor-binding domainSARS-CoV-2Severe Acute Respiratory Syndrome Coronavirus 2ACE2angiotensin-converting enzyme 2EEF1A1Eukaryotic translation elongation factor-1 alpha genePNGase FPeptide:N-glycosidase F