The variable regions of the heavy and light chains of CR302217 (link) were cloned into the TGEX-HC and TGEX-LC vectors (Antibody Design Labs), respectively, according to the manufacturer’s protocol. Likewise, ACE2 (residues 1–615) was cloned into TGEX-HC. The DNA was then transfected into Expi293F cells (Thermo Fisher Scientific) by using the ExpiFectamine Transfection Kit (Thermo Fisher Scientific) following the provided protocol, and the cells were incubated in a humidified incubator at 37 °C and 8% CO2 for 5 days. The cells were then centrifuged at 5500 × g for 20 min. The supernatant media was diluted twofold in PBS and run through a 1-mL MabSelect SuRe column (Cytiva) connected to an ÄKTA start (Cytiva) and controlled by Unicorn start 1.0 software (Cytiva) according to the manufacturer’s operation manual to purify the proteins. CR3022 and ACE2-Fc were quantified by using the BCA assay (Thermo Scientific).
Kta start
Manufactured by Cytiva
Sourced in United States, United Kingdom
The ÄKTA start is a compact and easy-to-use liquid chromatography system designed for purification of biomolecules. It features intuitive software and automated methods for protein, antibody, and other biomolecule purification.
Automatically generated - may contain errors
Lab products found in correlation
Unicorn start 1, by Cytiva (2 mentions)
Expifectamine transfection kit, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Mabselect sure column, by Cytiva (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Mabselect sure, by Cytiva (1 mentions)
Q exactive hf lc ms, by Thermo Fisher Scientific (1 mentions)
Hitrap heparin hp column, by Cytiva (1 mentions)
Ni nta resin, by Bio-Rad (1 mentions)
Hitrap heparin hp, by Cytiva (1 mentions)
Emulsiflex c3, by Avestin (1 mentions)
Hitrap, by Cytiva (1 mentions)
Histrap ff, by Cytiva (1 mentions)
Ah650 swinging bucket rotor, by Thermo Fisher Scientific (1 mentions)
Strataclean resin, by Agilent Technologies (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
8 protocols using kta start
1
Cloning and Expression of CR3022 and ACE2
2
Purification of Bacterial Depolymerases
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For scl-depolymerases from P. lemoignei, the isolation of extracellular enzymes was performed by centrifugation of the crude culture medium at 2500 × g for 45 min at 4°C. The enzymes present in the supernatant fraction were concentrated by two consecutive ultrafiltration (UF) cycles using centrifugal concentrator tubes of 15 mL with a 50 kDa molecular weight cut-off regenerated cellulose membrane (Vivaspin Turbo 15 RC 50kDa, Sartorius). The concentrator tubes were filled with 10 mL of supernatant and centrifugated 5000 × g for 15 min at 4°C for the first concentration step and 7 min at 4°C for the second step.
For mcl-depolymerases from recombinant E. coli, the crude culture medium was centrifuged at 7000 × g for 30 min at 4°C. The pellet was resuspended in a lysis buffer consisting of 30 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The cells were disrupted using a French press at 1,000 bar for two cycles, keeping the mixture on ice throughout the process. The mixture was then centrifuged at 9384 × g for 15 min at 4°C to obtain the supernatant containing the depolymerase. The solution was concentrated using a 30 kDa centrifugal concentrator tubes (Amicon, Millipore). The final step of purification was performed by preparative chromatography (Äkta start, Cytiva) using a high-flow amylose resin (n°E8022S from neb, Bioconcept Ltd.).
For mcl-depolymerases from recombinant E. coli, the crude culture medium was centrifuged at 7000 × g for 30 min at 4°C. The pellet was resuspended in a lysis buffer consisting of 30 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The cells were disrupted using a French press at 1,000 bar for two cycles, keeping the mixture on ice throughout the process. The mixture was then centrifuged at 9384 × g for 15 min at 4°C to obtain the supernatant containing the depolymerase. The solution was concentrated using a 30 kDa centrifugal concentrator tubes (Amicon, Millipore). The final step of purification was performed by preparative chromatography (Äkta start, Cytiva) using a high-flow amylose resin (n°E8022S from neb, Bioconcept Ltd.).
3
Heparin Affinity Chromatography for Murine Influenza
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Mouse BAL fluid from male and female mice from the murine influenza experiments described above was pooled (2–4 mice per group) for heparin affinity chromatography. Separation of proteins was achieved using a Hi-Trap Heparin HP column (Cytiva) on an ÄKTA start (Cytiva) liquid chromatography (LC) system attached to a Frac30 (Cytiva) fraction collector. The LC method used a flow rate of 5 mL/min and was performed at 4°C. The following complex gradient was applied after loading of the sample to achieve separation: a 2.5 column volume (CV) wash at the beginning, an increase to 30% mobile phase B (2.0 M Na+) over the course of 6 CVs, an increase to 75% mobile phase B over the course of 3 CVs, and lastly a 4 CV long flush at 100% mobile phase B; 1 CV = 5 mL = 1 minute of flow. Analysis was done on UNICORN start 1.0 software (Cytiva). The heparin binding fraction was isolated, and proteomic mass spectrometry was performed on a Q Exactive HF LC-MS (Thermo Fisher Scientific) by the University of Colorado Anschutz Medical Campus Mass Spectrometry Core Facility.
4
Purification of SARS-CoV-2 Nucleocapsid Protein
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The soluble fractions of NP1-419, NP121-419, and NP250-365 were filtered with 0.45 µ filters. The filtered fraction was loaded on the FPLC column (ÄKTA start, Cytiva, Maralborough, USA) packed with Ni-NTA resin (Biorad, California, USA) pre-equilibrated with five column volumes (CVs) of 10 mM sodium phosphate buffer pH 8, 50 mM NaCl, 5 mM benzamidine, and 5 mM imidazole at a flow rate of 0.5 mL/min. Flow through was collected separately to further analyze the unbounded proteins. The column was washed with 5 CV base buffer containing 10 mM sodium phosphate, 5 mM benzamidine, 50 mM NaCl, and 5 mM imidazole. Additionally, 2 CV elution at 0.5 mL/min was completed at an imidazole gradient of 20–500 mM. The eluted fractions were collected and analyzed on 12% SDS-PAGE [18 (link),19 (link)]. The samples having the band of protein with a corresponding size to that of NP1-419, NP121-419, and NP250-365 were pooled down, respectively, and buffer was exchanged with 10 mM sodium phosphate buffer pH 8 and 150 mM NaCl buffer.
5
Expression and Purification of CtNcs2/6 Complex
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The sequences of C. thermophilum (Ct) Ncs2 (UniProt ID: G0SFJ9) and CtNcs6 (UniProt ID: G0SAK0) were codon-optimized for expression in Escherichia coli, obtained from Genscript, and cloned into pETM30 (providing a TEV cleavable N-terminal 6xHis- and GST- tag) vectors. The proteins were expressed in BL21 (DE3) pRARE in LB at 18 °C using overnight induction with 0.5 M IPTG. Bacterial pellets were resuspended in lysis buffer (50 mM Tris–HCl pH 8, 300 mM NaCl, 0.15 % TX-100, 10 mM MgSO4, 1 mM DTT, 10 mg/ml DNase, 1 mg/ml lysozyme, 10 % glycerol and a protease inhibitor cocktail) and lysed to homogeneity using a high-pressure homogenizer Emulsiflex C3 (Avestin). To form the CtNcs2/6 complex, pellets containing each construct were mixed during lysis. The proteins were purified using GSTrap (Cytiva) columns on an ÄKTAstart (Cytiva) under standard conditions. Tags were optionally cleaved with TEV protease and removed in a second GSTrap purification step. Subsequently, the proteins were purified from nucleic acid contaminants using a HiTrap Heparin HP (Cytiva) column. Finally, proteins were purified by size-exclusion chromatography on a HiLoad 26/600 Superdex 200 prep-grade column (Cytiva). Purified proteins were stored at −80 °C in storage buffer (20 mM Tris pH 8, 150 mM NaCl and 1 mM DTT).
6
Nickel Affinity Purification of Recombinant Protein
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Nickel affinity resin columns (HisTrap FF, Cytiva, Marlborough, UK) were equilibrated with 5 column volumes of a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, and 30 mM imidazole (pH 7.4)) at a flow rate of 1 mL min−1 (ÄKTA Start, Cytiva, Marlborough, UK). The filter-sterilized spent BMMY P. pastoris expression culture was then applied to the column at a rate of 1 mL min−1. The column was then washed with at least 15 column volumes of the binding buffer. The recombinant GH was then eluted using a buffer of 20 mM of sodium phosphate, 0.5 M NaCl, and 500 mM imidazole (pH 7.4). The method was adapted from [26 (link),27 (link)].
7
Nuclear Protein Extraction and Fractionation
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Nuclear extracts were prepared, dialyzed, and quantified as described in the co-IP methods section. Density sedimentation by glycerol gradient was performed and probed similar to published reports [13 (link)]. Briefly, 4.5 mL 10–30% linear glycerol gradients were prepared using an ÄKTA start (Cytiva) from density sedimentation buffer (25 mM HEPES, 0.1 mM EDTA, 12.5 mM MgCl2, 100 mM KCl, 1 mM DTT) additionally containing 30 and 10% glycerol for initial and target concentrations, respectively. Two hundred micrograms nuclear lyase was overlaid on the glycerol gradient followed by ultracentrifugation at 40,000 rpm in an AH-650 swinging bucket rotor (Thermo Fisher Scientific) for 16 h at 4 °C. Two hundred twenty-five microliters gradient fractions were collected and concentrated using StrataClean resin (Agilent). Concentrated fractions were eluted in 1.5× Laemmli buffer + 37.5 mM DTT and run on SDS-PAGE for immunoblotting.
8
Production of Recombinant Protein Antigens
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Serum samples were heat inactivated at 56°C for 5 minutes to avoid cytotoxicity and added to the cell suspension at 1:100 dilution factor. This heat treatment inactivates the complement without affecting the ability of IgE to bind to the FcεRI receptor. The transfection of the cells was performed according to the protocol described by Durocher et al., (31) . Briefly, 2µg of purified vector (pTT28-Asc l antigens) per 10 6 cells were used for transfection of suspension HEK293-6E cells using 25 kDa branched polyethylenimine (PEI) from Polysciences (Warrington, PA) in 3:1 (PEI:DNA) ratio. 24 hours after transfection, the cells were stimulated with 0.5% (w/v) Tryptone N1 of the total volume of the culture and incubated at 37°C in a humidified incubator under constant shaking for three days. The cell culture was harvested and after centrifugation the medium supernatant was collected and filtered before purification by affinity chromatography using HisTRAP-HP column in ÄKTA Start (Cytiva). Purified proteins were quantified by BCA assay (Pierce BCA Protein Assay Kit).
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