The pCAGGS RBD construct, encoding for the receptor‐binding domain of the SARS‐CoV‐2 Spike protein (amino acids 319–541 of the Spike protein) along with the signal peptide plus a hexahistidine tag, was provided by Dr Krammer (Mount Sinai School of Medicine, NY USA). The pLVX‐EF1alpha‐nCoV2019‐N‐2xStrep‐IRES‐Puro construct, encoding for the full‐length SARS‐CoV‐2 nucleocapsid protein (NP) fused to a double Strep‐tag at the C‐terminus, was a gift from Dr Krogan (University of California, San Francisco USA). Recombinant proteins were expressed in‐house in Expi293F human cells (Thermo Fisher, Waltham, MA, USA) by transfection of the cells with purified DNA and polyethylenimine (PEI). For secreted RBD proteins, cells were harvested 3 days post‐transfection and RBD‐containing supernatants were collected by centrifugation at 20000 g for 15 min. RBD proteins were purified in HiTrap Ni Columns in an automated fast protein liquid chromatography (FPLC; ÄKTA avant, Cytiva), concentrated through 10 kDa Amicon centrifugal filter units (Merck Millipore, Darmstadt, Germany) and resuspended in PBS. For NP, cell lysates from transfected cells were centrifuged at 20000 g for 20 min and the supernatant was loaded into a 5‐mL Strep‐Tactin XT column. The eluted nucleocapsid was concentrated through a 10 kDa Amicon centrifugal filter unit and purified with SEC (Sephadex 10/300) in PBS.
Kta avant
Manufactured by Cytiva
Sourced in United States
The ÄKTA Avant is a versatile liquid chromatography (LC) system designed for protein purification and other biomolecule separations. It features a compact and modular design, allowing for customization to meet specific laboratory needs. The system offers advanced automation and control capabilities to enhance productivity and reproducibility.
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4 protocols using kta avant
1
SARS-CoV-2 Spike and Nucleocapsid Protein Production
2
Size Exclusion Chromatography for Protein Profiling
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A Superdex 200 10/300 GL SEC column connected with ÄKTA Avant (Cytiva Marlborough, MA, USA) was prepared with a buffer containing 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM TCEP, and 5% glycerol. A sample volume of 500 μl was loaded onto the column, and the protein profile measured at 280 nm (A280) was monitored using column chromatography. Chromatography was repeated under the same conditions using a protein standard mix of 15–600 kDa (Sigma-Aldrich, cat. No. 69385-30MG) to generate a standard curve.
3
Purification of His-tagged Protein
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The bacterial culture was centrifuged at 5000× g for 30 min and the supernatant was discarded. The pellet was resuspended in lysis buffer (20 mM phosphate buffer pH 7.4, 500 mM NaCl, 1 mM PMSF, 0.1% Triton X-100). Bacterial cells were disrupted by high-pressure homogenizer (PANDA Plus 2000, GEA, Düsseldorf, Germany) through three passages at 1200 bar, keeping the cell suspension in an ice bath during the passages. All purification methods were conducted using 15 to 20 g of bacterial biomass.
IMAC: The chromatography column (XK26) was packed with 50 mL IMAC-Sepharose 6 Fast Flow resin (Cytiva, Marlborough, MA, USA), the resin was charged with Ni2+ and chromatography was performed on ÄKTA Avant (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with a buffer containing 20 mM phosphate, pH 7.4, 500 mM NaCl buffer and 10 mM imidazole. Elution was performed with increasing concentrations of imidazole (50 mM, 150 mM, and 500 mM).
Q-Sepharose: The chromatography column (XK26) was packed with 74 mL of Q-Sepharose Fast Flow (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with 50 mM Tris-HCl, pH 8.0. Elution was performed with increasing concentrations of NaCl (0 to 0.7 M).
IMAC: The chromatography column (XK26) was packed with 50 mL IMAC-Sepharose 6 Fast Flow resin (Cytiva, Marlborough, MA, USA), the resin was charged with Ni2+ and chromatography was performed on ÄKTA Avant (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with a buffer containing 20 mM phosphate, pH 7.4, 500 mM NaCl buffer and 10 mM imidazole. Elution was performed with increasing concentrations of imidazole (50 mM, 150 mM, and 500 mM).
Q-Sepharose: The chromatography column (XK26) was packed with 74 mL of Q-Sepharose Fast Flow (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with 50 mM Tris-HCl, pH 8.0. Elution was performed with increasing concentrations of NaCl (0 to 0.7 M).
4
Fc-fusion Protein Purification Optimization
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The Fc-fusion protein used in the DSP part of this study was generated using a CHO host provided by AstraZeneca, UK, Cambridge. Chromatographic experiments were carried out using an ÄKTA Avant controlled with Unicorn Software version 7.1 (Cytiva, Marlborough, MA, USA). The protein feed was purified using MabSelect Sure Protein A chromatography resin (Cytiva, Marlborough, MA, USA), subjected to a low-pH treatment, and purified further using CaptoAdhere resins (Cytiva, Marlborough, MA, USA) in flow-through mode. Screening experiments were conducted to determine the optimal conditions to purify fusion proteins on Poros 50 HS resin (Thermo Fisher Scientific, Bedford, MA) using varying conditions of the elution buffer. The elution was performed either in gradient mode from 0-0.5 M of NaCl in 20 mM of sodium citrate or step mode using 20 mM of sodium citrate (for calibration set (T1) and validation set 1 (P1)) and 50 mM of sodium citrate (for validation set 2 (P2)) with NaCl in the range 133-210 mM, at a pH range of 5.8-6.2 and a loading concentration of 10-20 g/L. The elution was fractionated into 1 mL fractions, where each fraction constituted a separate sample for spectral measurements.
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