As reported previously51 (link)–53 (link), the IL-10/Fc fusion protein containing a human IL-10 fused at the N-terminal with a noncytolytic human IgG1 Fc was expressed by FreeStyle 293-F Cells (Gibco / Thermo Fisher Scientific) at the EPFL Protein Expression Core Facility. Supernatant of culture medium containing IL-10/Fc fusion protein was harvested by centrifugation after a 7-day culture and was filtered through a 0.22-μm membrane to obtain a clear solution. The recombinant protein was first captured with a HiTrap Protein A affinity chromatography column on an AKTA pure 25 (GE Healthcare), and eluted with an elution buffer (0.05-M sodium citrate, 0.3-M sodium chloride, pH = 3.0). The eluted protein was collected immediately in a neutralization buffer (1-M Tris–HCl, pH = 10.0) followed by concentration with membrane ultrafiltration (molecular weight cut-off 10 kDa) in a Vivaspin (GE Healthcare). The concentrated protein solution was further purified with a Superdex 200 increase size exclusion chromatography (GE Healthcare) at a flow rate of 1.0 mL/min with phosphate buffered saline (PBS) buffer on AKTA pure 25 (Extended Data Fig. 1a ,b ). The purified protein was aliquoted and stored at -80 °C before use. The purity of IL-10/Fc was confirmed with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, Extended Data Fig. 1c ).
Akta pure 25
Manufactured by GE Healthcare
Sourced in United States
The AKTA pure 25 is a versatile liquid chromatography system designed for protein purification. It features a flow rate of up to 25 mL/min and can accommodate various column sizes. The system is equipped with advanced detection and monitoring capabilities to ensure efficient and reliable purification of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Vivaspin, by GE Healthcare (2 mentions)
Hitrap protein a affinity chromatography column, by GE Healthcare (2 mentions)
Superdex 200 increase size exclusion chromatography, by GE Healthcare (2 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (2 mentions)
Histrap hp column, by GE Healthcare (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
15 ml ultrafiltration tube, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Histrap ff column, by GE Healthcare (1 mentions)
Superdex 75 increase size exclusion column, by GE Healthcare (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Expifectamine 293, by Thermo Fisher Scientific (1 mentions)
Hitrap rprotein a ff column, by GE Healthcare (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Histrap ff crude column, by GE Healthcare (1 mentions)
Hitrap q hp column, by GE Healthcare (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Pre stained protein ladder, by Thermo Fisher Scientific (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
16 protocols using akta pure 25
1
Production and Purification of IL-10/Fc Fusion Protein
2
Production and Purification of IL-10/Fc Fusion Protein
3
Automated Protein Purification Protocols
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After culture and expression, the AKTA pure25 (GE Healthcare, USA) was used for automated purification after setting up the program. The 5 mL Histrap FF purification column (GE Healthcare) was used for His-tag VHH protein purification. The 5 mL Protein A purification column (GE Healthcare) was used to purify the Fc-fused Abs. After purification, the solvent of the protein solution should be replaced with the phosphate buffer in the 15-mL ultrafiltration tube (Millipore, USA). The specific experimental procedures were according to the manufacturer’s instructions.
4
Purification of MazE-nd1 and MazF-nd1 from E. coli
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E. coli cells containing MazE-nd1 and MazF-nd1 were thawed on ice and suspended in 18 mL of binding buffer (20 mM sodium phosphate buffer (pH 8.0), 300 mM NaCl, 0.05% Triton X-100, 5 mM β-mercaptoethanol, and 40 mM imidazole). The cells were lysed by sonication and collected by centrifugation at 8900× g. The supernatant was then filtered through a 0.45-µm membrane (Millex, Darmstadt, Germany) and applied to 1 mL His-Trap FF column (GE Healthcare, Little Chalfont, UK). Non-specifically bound proteins were removed by washing with 32 column volumes (cv) of binding buffer using AKTA pure 25 (GE Healthcare). Hexa-histidine-tagged MazE-nd1 and MazF-nd1 were selectively eluted by increasing the concentration of elution buffer (20 mM sodium phosphate buffer (pH 8.0), 300 mM NaCl, 0.05% Triton X-100, 5 mM β-mercaptoethanol, and 500 mM imidazole) using the following program: flow rate, 1 mL/min; linear elution gradient, 20 cv; fraction size, 0.5 mL. Molecular weight and purity were confirmed by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis and protein concentration was determined using a Protein Assay (Bio-Rad, Hercules, CA, USA).
5
Amine-Tagged Lysostaphin in PEG Hydrogels
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Amine groups on lysostaphin were fluorescently tagged using an AlexaFluor 488 dye conjugated to a 2-kDa PEG linker functionalized with an NHS ester (Nanocs). The reaction was performed in 100 mM NaHCO3 buffer at pH of 8.3 at room temperature for 1 h with continuous mixing in the dark. Excess dye was removed from labeled protein using an AKTA Pure 25 (GE Healthcare) in combination with a Superdex 75 increase size-exclusion column (GE Healthcare) using PBS as the running buffer, at 4 °C. Labeled lysostaphin was incorporated in the hydrogel conditions tested: 4.0% wt/vol 20-kDa PEG-4MAL, 1 mM RGD, VPM and 8.0% wt/vol 10-kDa PEG-4MAL, 1 mM RGD, VPM. For the diffusion release study, hydrogels were polymerized, swollen in PBS, and incubated statically at 37 °C and 5.0% CO2. For the protease-triggered release studies, 4.0% wt/vol 20-kDa PEG-4MAL, 1 mM RGD, VPM hydrogels were swollen in PBS supplemented with 2 U/mL, 10 U/mL, or 50 U/mL collagenase type 1 (Worthington) and incubated shaking at 200 rpm, 37 °C, and 5.0% CO2. At each time point, the supernatant was sampled and read (488/530 excitation/emission) on a Synergy H4 (BioTek) plate reader. The measured fluorescence values were normalized to the fluorescence of PEG-4MAL/lysostaphin mixtures of the respective hydrogel condition.
6
Recombinant Human Antibody Production
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Total RNA was extracted from the hybridomas and reverse transcribed. The target sequences were amplified by PCR using primers targeted to the variable region of the heavy chain and the light chain of the human antibody. The sequences of the heavy and light chains were cloned into the pEHX1.1 vector and pELX2.2 vector, respectively [19 (link),23 (link),24 (link)]. A plasmid encoding both the heavy and light chains was constructed and transfected into Expi293 cells by using Expi Fectamine 293 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
At four days post-transfection, the human antibodies in the culture media were purified by using a Hi Trap rProtein A FF column (GE Healthcare, Chicago, IL, USA), and the automated chromatography system AKTA pure 25 (GE Healthcare, Chicago, IL, USA). The concentration of the purified antibodies was measured by using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
At four days post-transfection, the human antibodies in the culture media were purified by using a Hi Trap rProtein A FF column (GE Healthcare, Chicago, IL, USA), and the automated chromatography system AKTA pure 25 (GE Healthcare, Chicago, IL, USA). The concentration of the purified antibodies was measured by using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
7
Purification of Recombinant MazF Endoribonuclease
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The recombinant MazFDR0417 was purified as described previously (Miyamoto et al. 2016 , 2016 ), with slight modifications. Escherichia coli cells containing MazFDR0417 were thawed on ice and suspended in 32 mL of binding buffer (20 mmol/L sodium phosphate buffer (pH 8.0), 300 mmol/L NaCl, 5 mmol/L β‐mercaptoethanol, and 50 mmol/L imidazole). The cells were lysed by sonication and collected by centrifuging at 7000 g. The supernatant was then filtered through a 0.45‐μm membrane (Millex) and applied to a 1‐mL His‐Trap FF crude column (GE Healthcare). Nonspecifically bound proteins were removed by washing with 32 cv of binding buffer using AKTA pure 25 (GE Healthcare). Hexa‐histidine‐tagged MazFDR0417 was selectively eluted by gradually increasing the elution buffer concentration using the following program: flow rate, 1 mL/min; linear elution gradient, 20 cv; fraction size, 0.5 mL. The composition of the elution buffer was as follows: 20 mmol/L sodium phosphate buffer (pH 8.0), 300 mmol/L NaCl, 5 mmol/L β‐mercaptoethanol, and 500 mmol/L imidazole. Molecular weight and purity were confirmed by the Agilent 2200 TapeStation P200 ScreenTape Assay (Agilent Technologies). Protein concentration was determined using the Qubit Protein Assay Kit (Life Technologies).
8
Purification of g1, g1g2, g1g2g3 and g1g1 proteins
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Escherichia coli strains DH5α and BL21 (DE3) were the host strains for amplification and protein expression, respectively. pET-28a (+) was purchased from Novagen company. Subcloning of artificial genes encoding g1, g1g2, g1g2g3 and g1g1 were inserted into pET-28a_psp vectors and oligonucleotide primers were synthesized. The BamH1 and Sal1 restriction enzymes, T4 ligase and prestained protein ladder were purchased from Thermo Scientific. The Ni-NTA column, 1 mL HisTrap HP column, 1 mL HiTrap QHP column and Superdex 200 increase 10/300GL were purchased from GE Healthcare. AKTA pure 25 was purchased from GE Healthcare for protein purification. For dot blot assays, Super Enhanced ECL-chemiluminescence was purchased from sbjbio life science. All other Technologies from GBCBIOO chemicals used in the present study were of biochemical research grade.
9
Protein Size Exclusion Chromatography
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Size exclusion chromatography of protein isolates was performed using an AKTA Pure 25 protein purification system (AKTA Pure, GE Healthcare, Diegeum, Belgium) fitted with a superdex 200 increase 10/300 GL column (Cytiva, Little Chalfont, UK). Samples (40 mg/mL) were dissolved in 10 mM phosphate buffer containing 150 mM NaCl (pH 7.2) and shaken in a tube roller for 2 h, which was then centrifuged at 10,000× g for 15 min. The supernatant was then filtered through a 0.2 µm polyether sulphone filter. A 0.5 mL aliquot of the sample was injected into the column, and the phosphate buffer (10 mM, 150 mM NaCl) was passed through the column at a constant flow rate of 0.5 mL/min. UV absorbances were collected for 280 nm. Molecular weight standard 15–670 kDa (Sigma Aldrich, Melbourne, Australia) was used for calibration. A calibration curve was constructed by plotting elution time against the log molecular weight of the protein standard. The linear equation thus obtained was to calculate the molecular weight for known elution times.
10
Recombinant Influenza Antigen Production
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