Expression and Purification of the RBD—A fragment containing residues 317–518 from the S glycoprotein was cloned into pSecTag2B (Invitrogen) using BamHI and EcoRI restriction sites as previously described (7 (
link), 22 (
link)). The insert was further cloned into pAcGP67-A using the forward primer 5′-ACT G
TC TAG ATG GTA CCG AGC TCG GAT CC-3′ (XbaI) and the reverse primer 5′-CAG
TAG ATC TCG AGG CTG ATC AGC G-3′ (BglII). The pAcGP67-S was co-transfected with BaculoGold linearized baculovirus DNA into SF9 cells. High titer recombinant baculovirus stock was prepared by multiple amplifications. The protein was expressed in SF9 cells, cultured in serum-free HyQ-SFX-insect medium (HyClone), and purified from conditioned medium with a HiTrap nickel-chelating column. The eluted monomeric protein was concentrated, further purified with a Superdex 75 10/300GL column equilibrated with phosphate-buffered saline plus 0.2
m NaCl, and concentrated to 5–10 mg/ml in phosphate-buffered saline plus 0.2
m NaCl.
Selection, Expression, and Purification of the High Affinity RBD-specific Fab m396 and Its Conversion to IgG1—A naïve human Fab phage display library (a total of ∼10
10 members) was constructed from peripheral blood B cells of 10 healthy donors
5and used for selection of Fabs against purified, soluble, monomeric RBD, conjugated to magnetic beads (Dynabeads M-270 Epoxy, Dynal Inc., New Hyde Park, NY) following a previously described procedure (23 (
link)). Briefly, amplified libraries of 10
12 phage-displayed Fabs were incubated with 5, 3, and 1 μg of RBD in a 500-μl volume for 2 h at room temperature during the first, second, an third rounds of biopanning, respectively. After the third round of biopanning, 95 clones were randomly picked from the infected TG1 cells, and phage enzyme-linked immunosorbent assay was used to identify clones of phage displaying Fabs with high binding affinity. Eight clones that bound to the RBD with
A450 > 1.0 were selected for further characterization. The V
H and V
L domains (V
H and V
L denote the variable domains of heavy and light chains, respectively) of these clones were sequenced. The sequences were identical for all selected clones, and the selected Fab was designated as m396. The Fab used for crystallization was purified with a HiTrap nickel-chelating column followed by a Superdex 75 10/300GL column, using phosphate-buffered saline buffer containing 0.2
m NaCl, and concentrated to 10–20 mg/ml. For its conversion to IgG1, the Fab heavy and light chains were amplified and re-cloned in the pDR12 vector (provided by D. Burton, Scripps Research Institute, La Jolla, CA) with the Fc gene fragment replaced with cDNA sequence instead of genomic DNA.
Affinity Determination by Surface Plasmon Resonance—Interactions between m396 and SCV RBD were analyzed by surface plasmon resonance technology using a BIAcore 1000 instrument (Amersham Biosciences). The SCV RBD was covalently immobilized onto a sensor chip (CM5) using carbodiimide coupling chemistry. A control reference surface was prepared for nonspecific binding and refractive index changes. For analysis of the kinetics of interactions, various concentrations of Fab or IgG m396 were injected at a flow rate of 30 μl/min using running buffer containing 150 m
m NaCl, 3 m
m EDTA, and 0.005% P-20 (pH 7.4). The association and dissociation phase data were fitted simultaneously to a 1:1 Langmuir global model by using the nonlinear data analysis program BIAevaluation 4.1. All the experiments were performed at 25 °C.
Crystallization and Structure Determination—The SCV RBD-Fab m396 complex was formed by mixing individual components in a 1:1 molar ratio and incubating overnight at 4 °C. Crystals were obtained within 2–3 weeks by sitting-drop vapor diffusion technique. The reservoir solution was composed of 15% v/v glycerol, 20% polyethylene glycol 6000, 100 m
m MES sodium at pH 6.5; crystals formed only in the drops with a 1:2 ratio for the protein and the reservoir solutions. The crystals of Fab m396 were grown with the sitting-drop vapor diffusion technique within 2 weeks. The reservoir solution was composed of 20% v/v glycerol, 16% v/v ethylene glycol, 20% w/v polyethylene glycol 6000, and 100 m
m NaCl in 30 m
m Tris-HCl (pH 8.5). Data sets up to 2.3-Ä resolution were collected at cryogenic temperature (100 K) for both the RBD-Fab complex and the unliganded Fab, each from a single crystal, at the Southeast Regional Collaborative Access Team beamline facility 22-ID of the Advanced Photon Source, Argonne National Laboratory. Data processing was carried out with the HKL2000 program suite (24 ). The structure was solved by molecular replacement with PHASER (25 (
link)), using the SCV RBD from the receptor complex (PDB code 2AJF) and four individual domains of Fab (V
H,V
L,C
H, and C
L)(C
H and C
L refer to the constant domains of heavy and light chains, respectively) from three different antibody structures (PDB codes: 1ZA6 for C
H and C
L, 1RZG for V
H, and 1W72 for V
L) as search models. The RBM (residues 430–490) of SCV RBD and most of the CDRs (complementarity-determining regions) of Fab models, which were not included in the search models, were built on the basis of difference electron density. The complex was refined with CNS (26 (
link)) at 2.3-Ä resolution. A total of 298 water molecules, a phosphate ion, and one
N-linked glucosamine moiety at Asn-330 were added at the final stage of the refinement. The final
R and
Rfree values were 19.8 and 26.1, respectively. The unliganded Fab m396 structure was solved by molecular replacement with AMoRe (27 ), using the constant domains of Fab m396 from the complex structure as the search model. The difference electron density map revealed the location of variable domains. The structure was refined using CNS (26 (
link)), and a total of 176 water molecules was added at the final stage of the refinement. The final
R and
Rfree values were 22.8 and 27.7, respectively. The O program (28 (
link)) was used for model building for both structures. Data collection, processing, and refinement statistics are summarized in Supplemental Table S1.
Prabakaran P., Gan J., Feng Y., Zhu Z., Choudhry V., Xiao X., Ji X, & Dimitrov D.S. (2006). Structure of Severe Acute Respiratory Syndrome Coronavirus Receptor-binding Domain Complexed with Neutralizing Antibody. The Journal of Biological Chemistry, 281(23), 15829-15836.
