The gene encoding the FCoV N (GenBank: AY994055.1) was codon optimized for bacterial expression and synthesized by GenScript. The sequence was incorporated in pET-30a(+) backbone between NdeI and HindIII restriction sites for the expression of C-terminally His-tagged N. The Rosetta (DE3) E. coli cells transformed with the resulting plasmid were induced with 1 mM IPTG upon entering into exponential growth phase (A600 0.4). Cells were allowed to grow overnight at 16 °C and were harvested by centrifugation at 3000 rpm for 30 min at room temperature. Cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM dithiothreitol [DTT], 1% Triton-X100, 5 mM CHAPS, 0.1 mM phenyl methyl sulfonyl fluoride [PMSF]), followed by sonication on ice and clearance of the lysate by centrifugation at 4500g for 20 min. N was purified from clear lysates using AKTA pure protein purification system (GE Healthcare), as reported (30 (link)).
Akta pure protein purification system
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The AKTA pure protein purification system is a versatile and automated chromatography platform designed for the efficient purification of proteins. It employs various chromatographic techniques, including affinity, ion exchange, and size exclusion, to separate and purify target proteins from complex mixtures. The system is equipped with a range of features, such as precise flow control, UV and conductivity monitoring, and a user-friendly software interface, to ensure reliable and reproducible results.
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Lab products found in correlation
Akta pure, by Cytiva (4 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Histrap hp 5 ml, by GE Healthcare (1 mentions)
Protein g agarose resin, by Thermo Fisher Scientific (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Histrap column, by GE Healthcare (1 mentions)
Protease inhibitor mixture, by Thermo Fisher Scientific (1 mentions)
10 protocols using akta pure protein purification system
1
Codon-Optimized FCoV N Protein Expression
2
Monoclonal Antibody Purification from Mouse Ascites
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Before being inoculated
intraperitoneally with hybridoma cells, BALB/c mice were injected
with sterilized paroline (1 mL/mouse). After seven to ten days, ascites
were collected using an injection syringe and then centrifuged for
10 min at 8000 rpm. The AKTA pure protein purification system (GE
Healthcare Little Chalfont, UK) was used to obtain pure mAb. The concentration
of mAb was tested using a NanoDrop One (Thermo Fisher Scientific,
Waltham, MA, USA).
intraperitoneally with hybridoma cells, BALB/c mice were injected
with sterilized paroline (1 mL/mouse). After seven to ten days, ascites
were collected using an injection syringe and then centrifuged for
10 min at 8000 rpm. The AKTA pure protein purification system (GE
Healthcare Little Chalfont, UK) was used to obtain pure mAb. The concentration
of mAb was tested using a NanoDrop One (Thermo Fisher Scientific,
Waltham, MA, USA).
3
Purification of CCHFV nucleocapsid protein
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The protein purification was carried out as previously reported [20 (link), 22 (link), 23 (link)]. Briefly, Escherichia coli Rosetta (DE3) cells (Stratagene), transformed with plasmids expressing either wild type CCHFV N protein or stalk domain were grown in 250 ml cultures at 37°C until the OD at 600 nm reached 0.5. The protein expression was induced by the addition of 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) to the bacterial culture, followed by further incubation at 18°C for additional 20 hours. Cell pallets were re-suspended in 45 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM dithiothreitol [DTT], 0.5% Triton-X100, 5mM CHAPS, 0.1 mM phenyl methyl sulfonyl fluoride [PMSF]), followed by sonication on ice and clearance of the lysate by centrifugation at 4500xg for 20 minutes. The protein purification was carried out on AKTA pure protein purification system (GE Healthcare). The cleared cell lysates were loaded onto HisTrap NiNTA column having 5ml bed volume (Sigma), pre-equilibrated with the lysis buffer. The column was then washed with 50 ml of lysis buffer, followed by additional washing with 100 ml of wash buffer (50mM Tris, 500 mM NaCl, 0.05% Triton X-100 and 20 mM Imidazole). The bound protein was finally eluted by Imidazole gradient from 0 mM to 250 mM in the elution buffer (50mM Tris, 500 mM NaCl, 0.05% Triton X-100).
4
Purification of Tagged Proteins from E. coli
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Escherichia coli BL21 derivative Rosetta host strain, transformed with a plasmid encoding a protein of interest, were grown in LB media. Bacteria were collected by centrifugation after IPTG induction and lysed by sonication on ice (25% amplitude, 1 min total, 10 s on/off). The tagged fusion proteins were purified on His-Trap column using AKTA Pure protein purification system (GE).
5
Purification of Recombinant Proteins from E. coli
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Escherichia coli BL21, transformed with a plasmid encoding a protein of interest, were grown in LB media. Bacteria were harvested by centrifugation after IPTG induction and lysed by sonication on ice (25% amplitude, 1 min total, 10 sec on/off). The tagged fusion proteins were purified on His-Trap column using AKTA Pure protein purification system (GE).
6
Fractionation and Characterization of Fungal Secretome
7
Expression and Purification of CBD-PHY
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The expression plasmids and the protocols for expression and purification of CBD-PHY have been published in detail before. 23, 27 In brief, the WT and Y263F mutant were expressed in Escherichia coli strain BL21 (DE3). The biliverdin chromophore was incorporated in all protein lysates overnight on ice. The uniformly isotope-labeled protein was expressed in a minimal medium supplemented with 13 C-enriched glucose and 15 NH4Cl. The proteins were purified using affinity chromatography (HisTrap HP 5 ml, GE Healthcare), followed by size exclusion chromotography (Superdex 200, GE Healthcare) using A ¨kta pure protein purification system (GE Healthcare). The purified proteins were eluted in 30 mM Tris-HCl (pH 8), concentrated to 10 mg ml À1 , and flash frozen. All purifications were performed in the dark.
8
Monoclonal Antibody Production in FreeStyle 293F Cells
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KZ52 monoclonal antibody was produced via the transfection of plasmids encoding heavy and light chains into FreeStyle 293F cells using polyethylenimine. The day after transfection, cell cultures were supplemented with fresh media and with valproic acid to a final concentration of 2.2 mM. At 6 days post-transfection, supernatant was collected by spin centrifugation and filtration. IgG was isolated using Protein G agarose resin (Thermo Scientific) and subsequently purified via size exchange chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare) on an AKTA Pure Protein Purification System (GE Healthcare).
9
Expression and Purification of SNVN Nucleoprotein
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Expression of wild type SNVN NP of NP mutant lacking the RNA binding domain was carried out, as previously reported [44 (link)]. Briefly, BL21 (DE3) cells transformed with pTriEx1.1 vector harboring the NP gene, followed by induction with 1 mm isopropyl 1-thio-β-d -galactopyranoside upon entering into exponential growth phase (A600 = 0.4). Cells were allowed to grow for another 4 h at 37 °C and were harvested by centrifugation at 3000 rpm for 30 min at room temperature. Cells were resuspended in lysis buffer (20 mm HEPES, pH 8.0, 300 mm NaCl, 2 mm CHAPS, 8 m urea, 10 mm imidazole, and protease inhibitors (protease inhibitor mixture, Thermo Scientific). Cleared lysates were loaded on the 5ml HisTrap column (17-5286-01, GE healthcare) and purification was carried out on AKTA pure protein purification system (GE Healthcare) as previously reported [45 (link)]. The column was washed three times with lysis buffer containing increasing concentrations of 25 mM, 50 mM and 100 mm imidazole. The bound protein was finally eluted with lysis using an imidazole gradient from 0–250 mM. Purified protein was refolded by step dialysis in 20 mm HEPES, pH 8.0, 200 mm NaCl, 5% glycerol, and 1 mm DTT with a gradual decrease in the concentration of urea.
10
Recombinant P5CDH Protein Expression
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The P5CDH protein was obtained using pET30a. The rocA gene, encoding the P5CDH protein, was synthesized by Integrated DNA Technologies based on its sequence in the MRSA USA300. The PCR product was ligated to pET30a via In‐Fusion cloning. The recombinant plasmid was transformed into DH5α competent cells. Next, plasmids were collected from DH5α and were transformed into BL21‐competent cells. Isopropyl‐beta‐d‐thiogalactopyranoside was added to a final concentration of 1 mm for inducing the expression of P5CDH protein in BL21. The expressed protein was purified using a Ni‐NTA agarose column and an AKTA Pure Protein Purification System (GE, USA) from the harvested bacterial suspension. Then, the quality and quantity of purified recombinant P5CDH were analyzed on 10% SDS‐PAGE gel electrophoresis.
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