Expression and purification of the receptor‐binding domain of SARS‐CoV‐2 spike protein in mammalian cells for immunological assays

The receptor‐binding domain (RBD) of the spike glycoprotein of SARS‐CoV‐2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor‐binding domain soon became the major target for COVID‐19 research and the development of diagnostic tools and new‐generation vaccines. Here, we provide a detailed protocol for high‐yield expression and one‐step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum‐free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L−1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA‐based vaccination of mice.

RNA genome [3], which is encapsulated by an envelope consisting of a membrane and four structural proteins: the Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) [4], forming the corona (crown) of the virion.Spike is the largest structural glycoprotein, functioning as a homotrimer complex on the virus' surface [5].The Spike trimer is able to interact with the host cell receptor angiotensin-converting enzyme 2 (ACE2), which is required for viral entry to the cell [4].Therefore, Spike became the major target for research and vaccine development.Spike is synthesized as a single polypeptide, which is then split into the N-terminal S1 and C-terminal S2 subunits by the furin-like protease in the Golgi apparatus [6].Each Spike contains a Cterminal transmembrane (TM) helix, which anchors the complex to the viral envelope (Fig. 1A).The largest, external part of the molecule, called the ectodomain, is required for trimer formation and virus internalization (Fig. 1A) [6].The ectodomain comprises Spike's receptor-binding domain (RBD), which directly engages the ACE2 receptor to mediate viral entry [7].Moreover, RBD is the primary target of the multiple potent neutralizing antibodies that terminate the physical interaction between RBD and ACE2, and mediate immune response and virus neutralization [8][9][10].Therefore, Spike's RBD (including its mutant forms) became a popular research and bio-industrial target to study SARS-CoV-2.The RBD was used to establish biosensors to detect viral infection and seroconversion [11,12] for research, preventive or diagnostic purposes, and to develop vaccines and therapies against viral infection and the COVID-19 disease [10,13,14].The production of recombinant RBD or its variants is cost-efficient, and the purified product is suitable for both in vitro and in vivo use.Over the years, several heterologous expression systems have been developed to produce recombinant RBD.It can be expressed in bacteria, purified from inclusion bodies, and refolded [15][16][17][18].However, bacterially produced RBD has some usage limits due to the lack of functionally relevant glycosylation, or heterotrimeric complex formation.The expression of RBD in lower eukaryotes partially solves this problem.A high yield of RBD can be achieved in baculovirus-mediated [19] or transiently transfected [20] insect cells, or in yeast systems [21].Although structurally functional, these recombinant RBDs are differently glycosylated than that of the viral RBD in humans, making them less advantageous in functional assays.The production of RBD in mammalian cell cultures, including Chinese hamster ovary (CHO) or human embryonic kidney (HEK293) cells, has overcome this problem.The receptor-binding domain is properly glycosylated in mammalian cells, easy to purify from the cell culture supernatant, reaches high quantities, and is capable of homodimer and homotrimer formation [11,[22][23][24]; hence, it is suitable for serological assays as well as in vivo experiments or diagnostic purposes.
In this research protocol, we provide a detailed, stepby-step guide for the production and one-step purification (Fig. 1) of homomeric and glycosylated (Fig. 2), as well as structurally intact (Fig. 3, Table 1), RBD in transiently transfected Expi293F human cells, which is suitable for immunological assay (Fig. 4).Up to 80 mg of RBD can be produced in 1 L of cell culture in only 4 days.The recombinant RBD purified from the clarified cell culture supernatant by immobilized metal affinity chromatography (IMAC) is extremely pure (> 98% based on SDS/PAGE analysis, Fig. 2); therefore, it does not require additional clarification steps.Moreover, it forms homodimers and homotrimers (Fig. 2); therefore, we could The secondary structure of the purified RBD protein was examined by CD spectroscopy.The CD spectrum is in good agreement with the spectrum published previously for the same protein expressed in mammalian cells [30].The specific contributions emerging from the constituent secondary structural elements determined by spectral deconvolution is presented in Table 1 and are also in good agreement with those calculated from the high-resolution coordinates of the protein of subject [29].
Table 1.Fractions of secondary structural contributions expressed as percentages, as determined by CD spectroscopy and calculated from the high-resolution structure of SARS-CoV2 spike protein RBD domain [29].
Helix (a and 3 successfully use it in immunological experiments, such as the enzyme-linked immunosorbent assay (ELISA, Fig. 4).This protocol can also be used to produce different variants of RBD.

Subcloning efficiency chemically competent DH5a
Escherichia coli cells (Invitrogen #18265017).Protein purification, buffer exchange, and concentration 1. Syringe (cellulose acetate membrane, 0.22 lm) and 250-1000 mL vacuum (PES, polyethersulfone membrane, 0.22 lm) filter units for sterile filtration.    1. Thoroughly mix the Ni Sepharose 6 Fast Flow resin (Tips &Tricks 3), take out 6 mL slurry (50% bed volume), and mix with 40 mL RBD-W buffer in a 50-mL conical tube to equilibrate the resin.2. Centrifuge the tube at 800 g, 4 °C, for 5 min in a swinging-bucket rotor with low deceleration (e.g., set the brake to 7 out of 10) to avoid mixing the resin beads due to turbulence.3. Carefully pour off and discard the buffer and resuspend the matrix in 10 mL of RBD-W buffer.Step-wash the resin in the column with 5 9 10 mL RBD-W buffer.Let the buffer flow through the resin.

Protein expression in Expi293F mammalian cells
Discard the washing buffer.9.
Buffer exchange, concentration, and long-term storage of RBD His6  14), mix gently, and test the integrity of the protein by SDS/PAGE (Fig. 2) [25].9. Filter-sterilize the purified proteins (optional).10.Prepare aliquots as desired (into prechilled microfuge tubes).11.Flash-freeze the aliquots in liquid nitrogen and store the samples at À80 °C.
Tips & Tricks  [26] and the guidelines of the Expi293 Expression System manual (https://www.thermofisher.com)should be followed.All steps must be carried out carefully and precisely under sterile conditions.Do not use antibiotics (e.g., penicillin or streptomycin) or other additives (e.g., L-glutamine and surfactants) in the cell culture medium. 5.If a 25-or 50-mm-diameter orbital shaker is used, shake the cells at 120 or 95 r.p.m., respectively.6.If cell viability is lower than 95% or cell growth is slow, repeat Steps 6-7 and perform 1 or 2 extra subculturing steps in 30 mL medium.7. It is recommended to continue subculturing in a 30 mL volume (in 125-mL flasks), too, in parallel to the expanded cells to be used for transfection.This parallel culture will serve as a reserve material for another transfection or can be used for cryopreservation of Expi293F cells (follow the guidelines of the Expi293F cells' manual).8.If smaller (e.g., for pilot experiments) or larger (e.g., for high-scale protein production) volumes are needed, use smaller or larger flasks, and perform the transfection steps according to the Expi293 Expression System manual.9.During the 4-day protein expression and secretion, Expi293F cells can reach extremely high densities; therefore, the culture may turn whitish and form cell clumps and rims over the medium.These are normal phenomena and do not affect protein production.
10.The addition of RBD-E buffer is necessary to adjust the pH of the cell culture media as well as to set the imidazole concentration to ~20 mM to decrease nonspecific binding during the IMAC purification of RBD.
The color of the medium should be pinkish.11.It is recommended to perform the purification at 4 °C.
If this is not possible, place the suspension and buffers on ice and collect the eluted samples into prechilled tubes placed on ice.The resin/medium suspension is transferred from the flask to the columns by a serological pipette or a peristaltic pump.12.Other types of buffer exchange and/or concentration can also be followed, including tangential flow filtration, dialysis, or size-exclusion chromatography [23].13.The concentration of the intact and purified RBD His6 can be measured precisely with a spectrophotometer.We use a NanoDrop OneC, however, any type of spectrophotometer that measures at 280 nm can be used.
Based on the primary sequence of the purified protein (excluding the signal peptide), the Mw and e of any RBD variant can be easily calculated with the Prot-Param online tool (https://web.expasy.org/protparam/)[27].If a spectrophotometer is not available, other types of protein concentration measurement methods may also be used [28].14.We keep the concentration of purified RBD between 0.5 and 2 mgÁmL À1 and try to avoid several cycles of freezing and thawing of the samples.When needed, we thaw the frozen stocks on ice and centrifuge (12 000 g, 4 °C, 10 min) before use in any assay.
in 2022.NP is a member of the Scientific Advisory Board of AldexChem and Bionet-Asia.

Fig. 1 .
Fig. 1.Expression and purification of recombinant receptor-binding domain (RBD) from Expi293F mammalian cell culture supernatant.(A) Schematic representation of the homotrimer Spike complex anchored to the viral envelope (left).Domain and motif architecture of Spike is shown in the right panel: SP: signal peptide (dark blue); RBD: receptorbinding domain (green); S1 subunit (gold); S2 subunit (gray); TM: transmembrane helix (light blue).The chimeric protein SP-RBD His6 consists of the signal peptide fused to the RBD domain of Spike and a C-terminal hexa histidine-tag (His6, yellow).Steps of RBD expression in transiently transfected Expi293F cells (B) and IMAC purification (C) from cell culture supernatant.ELISA, enzyme-linked immunosorbent assay; ELISpot, enzymelinked immunosorbent spot; IMAC, immobilized metal affinity chromatography.

Fig. 2 .Fig. 3 .
Fig.2.Recombinant receptor-binding domain (RBD) is glycosylated and forms homomeric complexes.Purified RBD was treated with a disuccinimidyl suberate (DSS) crosslinker and incubated at room temperature for the indicated time points (0-20 min).The reaction was quenched and samples from each time point were subjected to reducing (A) or nonreducing (B) SDS/PAGE analysis followed by Coomassie Brilliant Blue (CBB) staining (upper panels), or immunoblotting (IB, lower panels) using an anti-RBD monoclonal antibody (generated in the house).Zero (0) minutes served as the negative control (non-DSS-treated).Under nonreducing conditions non-crosslinked RBD forms homodimers and homotrimers (Panel B, 0 min, red arrows).Under reducing and nonreducing conditions, crosslinked RBD forms homodimer, homotrimer, and even multimeric high-molecular-weight complexes in a time-dependent manner.This suggests that the dimeric/trimeric forms of RBD are present in the native, purified samples.Besides noncovalent intermolecular interactions, the complexes are stabilized by disulfide bonds, as well.(C) The N-glycosylation of RBD was tested by PNGase F-treatment.Samples were incubated in the absence (À) or presence (+) of PNGase F enzyme under denaturing conditions for 30 min at room temperature and subjected to SDS/PAGE analysis followed by Coomassie Brilliant Blue staining (CBB, upper panel) and immunoblotting (IB, lower panels) with anti-RBD and anti-His6 antibodies, respectively.This shows that the electrophoretic mobility of the PNGase F-treated RBD protein changes significantly in the gel due to the removal of N-glycans, suggesting that the purified RBD protein is N-glycosylated.

Fig. 4 .
Fig.4.Receptor-binding domain (RBD)-specific IgG titers in mouse sera were determined after immunization with a spike-encoding nucleoside-modified mRNA-LNP vaccine.Mouse immunizations and ELISA were performed and nucleoside-modified mRNA-LNP vaccines were generated as described in our previous studies[31,32].Eight-week-old female BALB/c mice were immunized intramuscularly with 3 lg of full-length spike-encoding mRNA encapsulated with lipid nanoparticle (LNP).Four weeks postimmunization, sera were collected, and RBD-specific immunoglobulin (IgG) titers were determined by endpoint dilution ELISA.n = 6 mice.Error bars are SEM.Each symbol represents one animal.The limit of detection is shown as a horizontal dotted line on the graph.Statistical analysis: unpaired t-test; **P-value ≤ 0.01.

3 . 2 .
Slowly, drop-by-drop transfer the enhancer mixture to the cells (grown 18-22 h post-transfection [step 26]).Gently shake the culture during booster addition.3.3.Immediately return the flask to the 37 °C tissue culture incubator (> 80% relative humidity) with 8% CO 2 and grow the cells on a shaker at 125 rp.m. for 3 days (Tips &Tricks 9). 4. Harvesting secreted RBD (Day +4): 4.1.96-h post-transfection harvest the cells by centrifugation at 4000 g, 4 °C, for 20 min, in a swingingbucket rotor 4.2.Carefully pour the cell culture supernatant (~220-230 mL, this contains the secreted RBD His6 ) into a sterile 1-L glass flask without disturbing the pellet Discard the cell pellet.4.3.Slowly add 14 mL of RBD-E buffer into the clarified cell culture supernatant and mix evenly by gently shaking the flask (Tips &Tricks 10) 4.4.With a vacuum filter unit, filter the mixture into a sterile 1-L glass flask to remove residual cells and aggregates IMAC purification of RBD His6

4 .
Transfer the IMAC resin into the 1-L glass flask containing the cell culture supernatant from Step 4.4. 5. Incubate the flask on an orbital shaker at 60 r.p.m., for 2 h, at 4 °C.6. Fix an empty PD-10 (or similar) chromatographic column to a stand.7. Continuously transfer the resin/medium suspension from the flask to the column and let the medium flow through the resin.Discard the flow-through (Tips & Tricks 11).8.

Table 2 .
Amino acid sequence of the SP-RBD His6 protein.The signal peptide (SP) and the hexa histidine-tag (His6) are underlined.

Table 3 .
Nucleotide sequence of the SP-RBD His6 protein-encoding synthetic DNA.The signal peptide (SP) and the hexa histidine-tag (His6) are underlined.
Slowly adjust the concentration of RBD His6 to 1 mgÁmL À1 with sterile and cold PBS (Tips & Tricks 1. Sterile borosilicate glass Erlenmeyer flasks can also be used with sterile metal caps (VWR, Radnor, PA, USA #391-0951).Never use flasks that have been in contact with bacteria, because of endotoxin hazards.If reused, wash flasks and caps extensively, rinse in ultrapure water, and dry sterilize at 180 °C for 4-6 h. 2. All chemicals should be at high purity (e.g., molecular biology or cell culture grade).3. Other types of high-capacity IMAC resins can also be used, including Ni-NTA, cobalt resin, or TALON matrix from different vendors.4. Basic mammalian cell culture maintenance techniques