Superdex 200 increase 10 300 gl column

Amino acids 17 to 329 (full-length), 17 to 167 (domain 1), and 168 to 329 (domain 2) of human C1QTNF4 were cloned into pET151 (Thermo Fisher Scientific). They were further cloned as GFP-C1QTNF4 fusion constructs into pET151 containing an N-terminal eGFP A206K domain followed by a short serine-glycine linker using polymerase incomplete primer extension cloning (64 ), named GFP-C1QTNF4 (aa 17–329), GFP-d1 (aa 17–167), and GFP-d2 (aa 168–329). eGFP A206K was also separately expressed in pET151. Site-directed mutagenesis for H198Q was performed using a QuikChange Lightning Kit (Agilent) according to the manufacturer’s instructions with 5’-GGGCCGCGGCAGCAACCACTCGC-3’ (Integrated DNA Technologies). Three constructs of human nucleolin, codon-optimized for E. coli (GeneArt), were cloned into pET151 with an additional C-terminal GSSDYKDDDDK tag: R1234G (aa 285–710), R123 (aa 285–561), and R4G (aa 566–710). Constructs and mutations were verified by Sanger sequencing (Eurofins Genomics). Proteins were expressed in BL21 Star DE3 E. coli (Thermo Fisher Scientific) grown in ZYP-5052 autoinduction medium (65 (link)) at 18 °C. They were purified via their hexahistidine tags with HisTrap FF Crude columns (Cytiva) and where appropriate further purified by SEC using a Superdex 200 Increase 10/300 GL column (Cytiva).

SEC was performed with an AKTA chromatography system using a Superdex 200 Increase 10/300 Gl column operated at 4 °C (both Cytiva). Frozen stock samples (∼5 mg/ml) were thawed on ice, diluted to 10 μM (~0.2 mg/ml), and incubated with 10 mM DTT for 30 min. Samples (100 μl) were injected using a 500 μl loop, and runs were performed with 0.5 ml/min flow rate using a degassed running buffer consisting of 20 mM Hepes, 100 mM NaCl, freshly added 10 mM DTT, adjusted to pH 7.4, by addition from a stock solution of 4 M NaOH (SEC buffer). Identical experiments were also performed at 3.0 mg/ml protein concentration. For integration, peak areas were defined manually and calculated by using Unicorn 7.1 (Cytiva).
For molecular mass determination of the decameric species using SEC-static light scattering, reduced WT and C172W mutant were separated as above, except at room temperature with a flow rate of 0.4 ml/min and the column connected to a Viscotek 302 to 040 Triple Detector PGC/SEC system (Malvern). Molecular weights were calculated from the acquired refractive index, right-angle, and low-angle light scattering data using Omnisec 4.7.0. For calibration, a 3 mg/ml solution of bovine serum albumin (AlbuMAX I, Gibco) was run under the same conditions.

SARS-CoV-2 RBD constructs contain residues 328–531 of the spike protein from GenBank NC_045512.2 with an N-terminal signal peptide and a C-terminal thrombin cleavage site-Twin-Strep-8×His-tag. For SPR binding assays, proteins were expressed in Expi293F cells (Thermo Fisher Scientific) at 37 °C and 8% CO2. Transfections were performed using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatants were collected three to five days after transfection and supplemented with 10x PBS to a final concentration of 2.5x PBS (342.5LmM NaCl, 6.75LmM KCl and 29.75LmM phosphates). SARS-CoV-2 RBDs were purified using a Cobalt affinity column (HisTALON Superflow column from Takara or HiTrap TALON crude column from Cytiva) followed by buffer exchange into PBS using a HiPrep 26/10 desalting column (Cytiva) or, for the Omicron BA.1 and BA.2 RBDs, a Superdex 200 Increase 10/300 GL column (Cytiva).