Design of the scFv construct
The amino acid sequence of scFv was constructed from the sequence of the NIST-mAb reference material 8671 described by Formolo and coworkers (Formolo et al., 2015) (Karageorgos et al., 2017). Residues Q1 to S120 of the heavy chain (underlined) were linked to residues D1 to T108 of the light chain (italicized) using four (GGGGS) elements (Huston et al., 1988). The synthetic gene (Biobasic, Toronto, Canada) optimized for expression in E. coli was inserted in a modified pET15b vector containing ten histidines in the NdeI and BamH1 sites. After cleavage of the polyhistidine tag with thrombin, the resulting polypeptide product had 252 residues (26 kDa) with the following sequence
1- GSHMQVTLRE SGPALVKPTQ TLTLTCTFSG FSLSTAGMSV GWIRQPPGKA
51- LEWLADIWWD DKKHYNPSLK DRLTISKDTS KNQVVLKVTN MDPADTATYY 101- CARDMIFNFY FDVWGQGTTV TVSSGGGGSG GGGSGGGGSG GGGSDIQMTQ 151- SPSTLSASVG DRVTITCSAS SRVGYMHWYQ QKPGKAPKLL IYDTSKLASG 201- VPSRFSGSGS GTEFTLTISS LQPDDFATYY CFQGSGYPFT FGGGTKVEIK
251- RT
where the first four extra residues resulted from the remainder of the thrombin cleavage site (GS) and the NdeI restriction enzyme site (HM) used for cloning. In order to match the numbering of residues in the NISTmAb-scFv with the NISTmAb numbering, Q1 in the NISTmAb heavy chain corresponds to Q4, and D1 in the NISTmAb light chain corresponds to D145.
Expression and purification of scFv
Expression of labelled NISTmAb-scFv was carried out by incubating E. coli BL21(DE3) (Stratagene) harboring the pET15b10-NISTmAb-scFv plasmid in minimal media (M9) using 13C-glucose and 15N-ammonium chloride as sole source of carbon and nitrogen at 37°C. Protein expression was induced by the addition of isopropyl thio-D-galactopyranoside (IPTG) at an OD600 of 0.8. Cells were harvested 3 h post-induction by centrifugation and frozen at -80°C until purification. Expression of labelled NISTmAb-scFv in E. coli resulted in the formation of inclusion bodies. Cell pellets corresponding to a 5 L culture were resuspended in 35 mL of buffer A (10 mM TrisHCl, 100 mM sodium phosphate, 6M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0) and disrupted by sonication on ice using a 400 W Branson sonifier (ThermoFisher). After separation of cell debris, lysis was repeated once with 35 mL of buffer A and the supernatants were pooled and added to a slurry of Ni-NTA resin (Qiagen) (80 mL resin, 10 mL buffer A) and gently stirred at room temperature for 30 minutes before loading into a column. Refolding was accomplished under oxidative condition with a gradient of buffer A to B (Buffer B: 10mM TrisHCl, 100 mM sodium phosphate, pH 8.0) over 20 column volumes. The column was then washed with three column volumes of Buffer B + 60 mM imidazole pH 8.0 to remove unspecific binding. The protein was eluted off the column with Buffer B + 250 mM imidazole (pH 8.0). The efficiency of the on-column refolding was such that the procedure was repeated several times (7 up to 10 times) to extract properly folded protein by re-equilibrating the column with buffer A followed by above refolding-washing-elution protocol.
Prior to cleavage of the poly-histidine tag, the buffer was exchanged to 20 mM sodium phosphate (pH 6.0) by ultrafiltration. Cleavage was carried out at a protein concentration of 2 mg/mL using 1U of thrombin (Cytiva) per 100 µg of target protein at room temperature. While almost all starting material was cleaved after 30 minutes, the reaction was allowed to proceed overnight.
The reaction mixture was then purified on cation exchange chromatography using HiTrap SP FF columns (Cytiva) in 50 mM sodium phosphate buffer pH (6.0) with a 1M sodium chloride salt gradient. The NISTmAb-scFv eluted at around 200 mM NaCl. Protein concentration was determined by using UV spectroscopy with the theoretical extinction coefficient 18150 M− 1cm− 1 (Swissprot). NMR samples contained 0.25 mM of the uniformly isotope-labeled 13C-15N- or 15N-NISTmAb-scFv in 20 mM phosphate buffer at pH 6.0, and 5% 2H2O was used for field frequency lock. The sample temperature was kept at 313 K (40°C).
Peptide binding
The NMR titration of the peptide epitope with the NISTmAb-scFv was carried out with a 24-amino acid long polypeptide chain (NSELLSLINDMPLTNDQKKLMSNN), derived from the X-ray structure (PDBID 3ixt) epitope on the RSV virus fusion protein for motavizumab (McLellan et al., 2010).
The concentration of the stock peptide solution was 5.3 mM. A total of 10 µl of the stock solution was added to 550 µl of 1.65 mg ml− 1 (~ 63 µM) protein solution, resulting in 95 µM peptide, and a final molar ratio of peptide-to-scFv of 1:0.65 (peptide being in excess). For recording of 15N-HSQC spectra of both the peptide-free and the peptide-bound scFv, the sample temperatures were kept at 308 K (35°C).
NMR Experiments
Data were collected on Bruker NEO-600 and AVANCE IIIHD-700 MHz NMR spectrometers equipped with cryogenically cooled triple resonance inverse probes fitted with Z-axis gradients. For backbone resonance assignment, the standard double- and triple resonance experiments 2D-15N-HSQC (“hsqcetfpf3gpsi”)(Palmer et al., 1991; Kay et al., 1992; Grzesiek and Bax, 1993b; Schleucher et al., 1994), 3D-HNCO (“hncogp3d”)(Grzesiek and Bax, 1992; Schleucher et al., 1993; Kay et al., 1994), 3D-HN(CA)CO (“hncacogp3d”)(Clubb et al., 1992), 3D-CBCA(CO)NH (“cbcaconhgp3d”)(Grzesiek and Bax, 1993a; Muhandiram and Kay, 1994), 3D-HNCACB (“hncacbgp3d”)(Wittekind and Mueller, 1993), 3D-HNCA (“hncagp3d”)(Grzesiek and Bax, 1992), 3D-HN(CO)CA (“hncocagp3d”)(Grzesiek and Bax, 1992), were recorded. For assignment of the side chain 1H and 13C chemical shifts, the double- and triple resonance experiments 2D-13C-HSQC (“hsqcctetgpsisp”)(Palmer et al., 1991; Vuister and Bax, 1992), 3D-H(CC)(CO)NH (“hccconhgp3d2”) and 3D-(H)CC(CO)NH (“hccconhgp3d3”)(Montelione et al., 1992; Clowes et al., 1993; Grzesiek et al., 1993; Logan et al., 1993; Lyons and Montelione, 1993; Carlomagno et al., 1996), 3D-HA(CO)NH (“haconhgpwg3d”)(Grzesiek and Bax, 1993a; Muhandiram and Kay, 1994), 3D-HNHA (“hanhgpwg3d”)(Kuboniwa et al., 1994; Weisemann et al., 1994), 3D-HBHA(CO)NH (“hbhaconhgp3d”)(Grzesiek and Bax, 1993a; Muhandiram and Kay, 1994), 3D-HBHANH (“hbhanhgpwg3d”), 3D-(H)N(CA)NNH (“hncannhgpwg3d”) (Weisemann et al., 1993), 3D-CCHTOCSY (“hcchdigp3d2”) (Kay et al., 1993), 3D-HCCH-COSY (“hcchcogp3d”) (Kay et al., 1993), 3D-HCCH-TOCSY (“hcchdigp3d”) (Kay et al., 1993), 3D-13C-NOESY-HSQC (“noesyhsqcetgpsi3d”)(Palmer et al., 1991; Kay et al., 1992; Schleucher et al., 1994), and 3D-15N-NOESY-HSQC (“noesyhsqcf3gpsi3d”)(Palmer et al., 1991; Kay et al., 1992; Schleucher et al., 1994) were recorded. For assignment of aromatic side chain Hδ and Hε, as well as Hδ1Trp, the Yamazaki experiments, 2D-(Lee et al.)CB(CGCD)HD (“hbcbcgcdhdgp”) and 2D-(Lee et al.)CB(CGCDCE)HE (“hbcbcgcdcehegp”)(Yamazaki et al., 1993), were recorded. The names of the pulse programs for experiments selected from the standard Bruker library are written in brackets.
Data analysis and the assignment
All NMR data were processed using NMRPipe software (Delaglio et al., 1995). NMRFAM-Sparky(Goddard and Kneller; Lee et al., 2015) was employed for spectral visualization and spectral analysis. Automatic assignment routine PINE-Sparky(Lee et al., 2019) was invoked for probabilistic assignment of the backbone amide 1H, 15N, and backbone carbonyl 13C chemical shifts. Assignment of side chain resonances was done through a semi-automatic approach by initial engagement of PINE-Sparky, followed by implementation of an “inspection–verification” strategy, where PINE result for each assignment was either accepted or rejected based on a holistic approach that included inspection of complementary NOESY data.