Talon resin

After overexpression and induction, cells were pelleted and resuspended in lysis buffer (50 mM HEPES pH 7.4, 300 mM NaCl, 1 mM TCEP, 5 mM MgCl₂ and protease inhibitor cocktail). Whole cell lysate was passed through the French Pressure Cell Press (SLM-Aminco) twice at 1250 psi for lysis and centrifuged. Soluble cell lysate was incubated with pre-equilibrated TALON resin (TakaraBio) for 2 h at 4°C. After extensive washing, KRAS was eluted off the resin with elution buffer (20 mM HEPES pH 7.4, 300 mM NaCl, 1 mM TCEP, 5 mM MgCl₂ and increasing imidazole concentrations [90, 500 mM]). To cleave the MBP-tag, TEV protease was added in 1:5 ratio to eluted KRAS and was dialyzed for 3 days at 4°C in buffer while reaction proceeded (20 mM HEPES pH 7.4, 300 mM NaCl, 1 mM TCEP, 5 mM MgCl₂). Solution was reapplied to TALON resin and supernatant containing KRAS was collected. To dissociate bound nucleotide, HRAS was incubated in HEPES buffer with 10 mM EDTA and 10 M excess GMPPNP (Sigma-Aldrich) for 30 mins at 4°C. To allow rebinding, MgCl₂ was added to a final concentration of 1M and rotated for 2 h at 4°C.^{55 (link)} KRAS was further purified on a Superdex 75 10/300 GL size exclusion chromatography column (Cytiva). Main steps were checked with SDS-PAGE followed by Coomassie staining. After concentration, aliquots were flash frozen and stored at −80°C.

The nanobody A6 and a13 sequences were subcloned to pFUSE-hIgG1-Fc2 (InvivoGen, San Diego, CA, USA), which was modified to insert a 6xHis-tag at the C-terminal of the Fc domain, to produce a nanobody–human IgG1 Fc fusion antibody (Nanobody-hFc). An Fc-enhanced A6 antibody was constructed to include two mutations in the Fc domain, specifically serine (S) at residue 239 to aspartic acid (D), and isoleucine (I) at position 332 to glutamic acid (E). This mutant was named A6-hFc-SDIE. The nanobody-hFc was produced by an Expi293F transient expression system (Thermo Fisher, Walthem, MA, USA) as described above for recombinant B7-H3. Samples were purified using TALON resin (Takara Bio, Dalian, China) and polished by size exclusion chromatography (Superdex 200 10/300 GL). Purified antibodies were concentrated and buffer exchanged to PBS.

An Escherichia coli (E. coli)‐optimized synthetic gene encoding the E2 ORF from HPV‐16 was cloned into pCAL‐N‐FLAG (Agilent Technologies) containing an N‐terminal calmodulin bind site (CBS). CBS‐E2 and TAT‐CaM were expressed and purified as previously described with slight modifications.²² Briefly, CBS‐E2 was expressed in ArcticExpress (DE3) E. coli cells (Agilent Technologies) and purified by fast protein liquid chromatography using Calmodulin‐Sepharose (GE Healthcare). TAT‐CaM was expressed in BL21(DE3)pLysS E. coli cells (Agilent Technologies) and purified to near‐homogeneity by metal‐affinity chromatography using TALON resin (Takara Bio). After purification, protein constructs were dialyzed into calcium‐containing binding buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl₂, 10% glycerol pH 7.4), sterilized via syringe‐driven filtration through a 0.22 μm filter, flash frozen in liquid nitrogen and stored at −80°C until use. Samples were collected at each stage of the purification process in 2% SDS buffer and subjected to gel electrophoresis as previously described.²⁷ Elutions were further subjected to immunoblot analysis as previously described using an HPV 16 E2 monoclonal primary antibody TVG 621 (ThermoFisher) and goat anti‐mouse HRP conjugated secondary (ThermoFisher).²⁷

The RBDs of SARS-CoV-1, SARS-CoV-2, WIV1, RaTG13, SHC014, ZC45, and ZXC21 were synthesized by GenScript and cloned into vector pCMV with a preceding mu-phosphatase signal peptide and a terminal octa-histidine tag. Plasmids were transfected into 150mL suspension expi293F or HEK293F cells at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm and harvested 3 days later. Clarified supernatants were purified in batch over Talon resin (Takara) prior to buffer exchanging into 20mM Tris pH8 150mM NaCl and flash freezing.
Biolayer interferometry binding assays were performed on an Octet Red instrument at 30°C with shaking at 1,000 RPM. ARG2 biosensors were hydrated in water then activated for 300 s with an NHS-EDC solution (ForteBio) prior to amine coupling. 5–10 μg/mL of each RBD was loaded in a buffer containing 10mM pH5 sodium acetate onto ARG2 tips (ForteBio) for 600 seconds and then quenched into 1M ethanolamine for 600 seconds. A baseline in 10X kinetics buffer (ForteBio) was collected for 120 s prior to immersing the sensors in a 1:3 serial dilution of his-tagged human ACE2 (Sino Biologicals) ranging from 1,000 to 0.83nm in 10X kinetics buffer. Curve fitting was performed using a 1:1 binding model and the ForteBio data analysis software. Mean k_on, and k_off values were determined with a global fit applied to all data.

Wild-type CTX-M-14 and its variants were expressed using CTX-M-14-pET28a-TEV in E. coli BL21(DE3) cells for subsequent enzyme kinetics or X-ray crystallography experiments. E. coli containing CTX-M-14 expression plasmid were grown in an LB medium containing 25 μg/mL kanamycin. Expression of the His-tagged-CTX-M-14 protein was induced in mid-log-phase cultures with 0.2 mM IPTG at 23 °C for 20 h. The cells were pelleted and resuspended in lysis buffer (25 mM NaPO₄, 300 mM NaCl, 20 mM imidazole, pH 7.4). Sonication was used to disrupt cells, and cell debris was removed by centrifugation. Soluble fractions in the supernatant were loaded onto a column packed with Talon resin (Takara Bio), and His-tagged-CTX-M-14 was eluted with an imidazole gradient in the lysis buffer. Eluted protein was then concentrated using an Amicon® Ultra-15 Centrifugal Filter Unit (MilliporeSigma). Protein purity was visualized by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining.

Immunoprecipitations were performed as previously described [20 (link)]. GFP-binding protein (GBP; [60 (link)]) was fused to the Fc domain of human IgG (pIg-Tail; R&D Systems), tagged with His6 in pET28a (EMD Millipore), expressed in E. coli, and purified on Talon resin (Takara Bio Inc.) according to manufacturer’s instructions. GBP was bound to Protein A–coupled Sepharose, cross-linked to the resin using dimethyl pimelimidate, and rocked for 1 h at 22°C; the coupling reaction was then quenched in 0.2 M ethanolamine, pH 8.0, and rocked for 2 h at 22°C. Antibody or GBP-coated beads were washed three times with 1.5 ml of cell lysis buffer (CLB; 100 mM Tris, pH 7.2, 125 mM NaCl, 1 mM DTT, 0.1% Triton X-100, and 1X Protease Inhibitor (Roche). S2, Kc, and Kc stable line expressing inducible Cap-H2-EGFP cells were treated with RNAi for 6 days, as described above in “Cell culture and double-stranded RNAi.” On day 4, cells were transfected with inducible GFP tag only, Cap-H2-EGFP, and/or 3x-FLAG-Ubiquitin. On day 5, transfected cells were induced with 1 mM CuSO4. After 24 h, transfected cells were lysed in CLB, clarified by centrifugation, and then diluted to 2–5 mg/ml in CLB. Antibody-coated beads were mixed with lysate in 1mL total volume for 90 min at 4°C, washed three times with CLB, and then boiled in Laemmli sample buffer.

DNAs for 10 candidate affibodies selected by biopanning were synthesized and then incorporated into the bacterial expression vector, pBT7-C-His (Bioneer). Plasmid DNAs were transformed into BL21 (DE3). The cells were grown to an OD of ∼0.6–0.8 and protein expression was induced by the addition of 1 mM IPTG for 3 h. Then, cells were resuspended in lysis buffer (150 NaCl, 20 Tris-Cl, pH 8.0). Proteins were purified using affinity chromatography (Talon resin, Takara). To reduce the non-specific binding, resins were washed with 40 mM imidazole and affibody was eluted with 200 mM imidazole. After removing the remaining imidazole by dialysis overnight, affibody proteins were concentrated using centrifugal filters (Thermo).