pPICZα vector containing XEG1-His34 (link) or empty vector was transformed into Pichia pastoris strains X33 (Muts+) or KM71 (Muts). P. pastoris was cultured overnight in the YPD medium at 30 °C and subsequently grown in the BMGY (Buffered Glycerol-Complex Medium) and BMMY (Buffered Methanol-Complex Medium) (pH = 6.5) for protein expression. The recombinant protein XEG1-His or EV was purified from the supernatant of the P. pastoris culture harboring pPICZα-XEG1-His or pPICZα using the AKTA™ avant 25 (GE Healthcare) through the HisTrap™ FF Ni Sepharose Columns (5 ML, 17525501; GE Healthcare) and HiTrap™ Desalting Prepacked Columns (5 ML, 29048684; GE Healthcare).
Akta avant 25
Manufactured by GE Healthcare
Sourced in Sweden
The AKTA avant 25 is a versatile liquid chromatography system designed for laboratory use. It is capable of performing various chromatographic techniques, including size exclusion, ion exchange, and affinity chromatography. The system offers automated operation, precise flow control, and robust performance to support a wide range of purification applications.
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Lab products found in correlation
Akta avant, by Cytiva (9 mentions)
Hitrap, by Cytiva (2 mentions)
Histrap ff ni sepharose columns, by GE Healthcare (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Cr22giii, by Hitachi (1 mentions)
R15a rotor, by Hitachi (1 mentions)
Zorbax 300sb c18, by Agilent Technologies (1 mentions)
Hiprep, by Cytiva (1 mentions)
Toyopearl af rprotein a hc 650f, by Tosoh (1 mentions)
Clarisolve 20ms, by Merck Group (1 mentions)
Vibra cell, by Sonics (1 mentions)
Histrap hp, by GE Healthcare (1 mentions)
Akta pure 25, by GE Healthcare (1 mentions)
Akta pure, by Cytiva (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
11 protocols using akta avant 25
1
Recombinant Protein Expression in P. pastoris
2
Expression and Purification of EF-Tu
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BL21(DE3) pLysS cells were transformed with pET21a-tufA and grown in 6 l of LB medium containing 100 μg/ml ampicillin, 20 μg/ml chloramphenicol, and 5% (w/v) glucose at 37°C until the OD600 reached 0.4. The expression of EF-Tu was induced with 0.5 mM IPTG and the cells were incubated at 37°C for additional 3 h. The cells were harvested and resuspended in 100 ml of buffer A [20 mM Tris–HCl (pH 7.6), 20 mM imidazole, 300 mM NaCl, 10 μM GTP, 1 mM β-mercaptoethanol] with 0.1 mg/ml PMSF. The cells were sonicated on ice for 10 min and the lysate was centrifuged (13 000 rpm, 4°C, for 15 min, CR22GIII equipped with an R15A rotor, Hitachi Koki). The supernatant was filtered thorough Minisart prefilter-GF and Minisart 0.45 μm syringe filters (Sartorius) and then applied to a 5 ml HisTrap HP column at 4°C (GE Healthcare) equipped on an AKTA avant 25 (GE Healthcare). The column was washed with 20 column volumes of buffer A, and the protein was eluted with a linear gradient from buffer A to buffer B [20 mM Tris–HCl (pH 7.6), 250 mM imidazole, 150 mM NaCl, 50 mM KCl, 10 μM GTP, 1 mM β-mercaptoethanol] over 20 column volumes. The fractions containing the protein were combined and dialyzed in 2 l of buffer C [10 mM Tris–HCl (pH 7.6), 50 mM KCl, 10 μM GTP, 1 mM DTT] with 3.5K MWCO membrane. The concentration of EF-Tu was measured by protein assay kit.
3
Purification of Anti-Obesity Peptide
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To separate and purify the fraction possessing anti-obesity efficacy obtained by UF into a single-peptide fragment, the ultrafiltrate was separated and purified step by step using three FPLC columns (Akta Avant 25, GE Healthcare, Uppsala, Sweden). Ion-exchange chromatography (IEC) was performed using Mono-Q 5/50 GL (5*50 mm, 10 µm), using binding buffer (50 mM phosphate buffer (pH 7)) and elution buffer (1 M NaCl‒phosphate buffer, pH 7). The elution buffer concentration was increased from 0% to 100%, the column volume value was 20, flow rate 2 mL/min, and absorbance 280 nm. After IEC, HiPrep 26/10 desalting column binding buffer (50 mM phosphate buffer (pH 7)) was used at a flow rate of 10 mL/min to remove the salt in the sample. Next, reversed-phase, preparative fractionation was used to separate and purify the fractions with anti-obesity efficacy from the IEC eluate. Separation and purification were performed using an Agilent Zorbax 300SB-C18 PrepHT (7 μm, 21.2 × 250 mm) column with acetonitrile (ACN) (gradient from 100% to 0%) and water as the mobile phase at a flow rate of 3 mL/min and absorbance of 280 nm. The active phase fraction from the reversed-phase preparative analysis was analyzed at 280 nm at a flow rate of 0.5 mL/min at 45% ACN with an Agilent Zorbax 300SB-C18 (5 μm, 4.6 × 250 mm) reversed-phase column.
4
Purification of Monoclonal Antibody
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Cell culture harvest was clarified by a two-stage depth filtration train of Clarisolve 20MS and F0HC (MilliporeSigma, Burlington, MA) followed by sterile filtration using a Sartopore 0.8/0.2 μm filter (Sartorius Stedim, Goettingen, Germany), operated in tandem. All chromatography was performed on an AKTA AVANT 25 (GE Healthcare). Clarified harvest was loaded onto a Protein A column (TOYOPEARL AF-rProtein A HC-650F, Tosoh Bioscience, King of Prussia, PA) and eluted with 100 mM glycine–HCl, pH 3.5. The material was then conditioned to 185 mM sodium chloride using 5 M NaCl stock solution (Lonza, Walkersville, MD) and titrated to pH 9.0 using 2 M glycine-OH, pH 10.5. The conditioned material was then purified via TOYOPEARL NH2-750F anion-exchange chromatography (Tosoh Biosciences), operated in flow-through mode using 50 mM Tris–HCl pH 8.0, 200 mM NaCl as the column equilibration and load chase buffer.
5
Recombinant Enzyme Purification by IMAC
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Pure enzymes were performed by metal-affinity chromatography. Firstly, When the optical density (OD) at 600 nm reached 0.6–0.8 (about 3–4 h), the cultures were cooled on ice, and gene expression was induced by the addition of 0.5 mM (0.4 mM) IPTG, and the mixture was cultured at 25 °C for an additional 12 h [5 (link)]. The biomass was harvested by centrifugation for 10 min at 12000 rpm and 4 °C. The resulting cell pellets were washed, and resuspended in PBS buffer (300 mM NaCl and 50 mM phosphate buffer at pH 8.0). Then the resuspended cells were incubated on ice for 20 min and lysed by sonication (Sonics Vibra - Cell) (300 W, 2 s, 1 s, Crushing time: 10 min). The crude extract was centrifuged at 12000 rpm for 30 min at 4 °C to remove cellular debris. As a control, E. coli BL21(DE3) cells harboring plasmid pET-28a (+) were treated similarly [21 (link)]. The crude extracts were used for activity determination and purification. Then the soluble portion was loaded on a Histrap™ HP chelated column connected to an AKTA avant 25 (GE Healthcare). The bound enzyme was eluted with 250 mM imidazole in 20 mM phosphate buffer (pH 7.0). Enzyme solutions were desalted and stored at 4 °C until further use [22 (link)].
6
Anion Exchange Chromatography Protocol for Protein Purification
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A GE AKTA Avant 25 (GE Healthcare Life Sciences) system was used to set up the anion exchange chromatography capture step. The column used was a 1 mL GE CaptoQ HiTrap and the buffers as follows; equilibration buffer 50 mM sodium acetate pH 6.0 (Buffer A), elution buffer 1 M sodium chloride in 50 mM sodium acetate pH 6.0 (Buffer B) at a flowrate of 1 mL/min. Concentrated HCCF was diluted 1:4 into equilibration buffer to adjust conductivity and favor binding of target material to resin. A 30 mL volume was then loaded onto the column followed by a 14 CV wash (100% buffer A), 40 CV linear gradient elution (0‐35% buffer B), 5 CV strip (100% Buffer B) and regeneration (10 CV 0.5 M NaOH, 5 CV Buffer A, 10 CV 20% EtOH). 2 mL fractions of column wash and eluate were collected and absorbance at 280 nm monitored throughout the run. The eluate was immediately frozen at −20°C until further analysis.
7
Plasma IgG Virus Filtration Kinetics
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Plasma IgG solution filtration experiments were conducted to determine the pressure dependence of flux for the virus filter. After washing and equilibration with 20 mM sodium acetate, 100 mM NaCl, pH 5.0 buffer, plasma IgG solution (5, 10, or 15 mg/ml) in 20 mM sodium acetate, 100 mM NaCl buffer at pH 5.0 was filtered at constant flow rates corresponding to 10, 20, 50, or 100 LMH on a 0.0003 m2 Planova BioEX filter to a target throughput of 100 L/m2. Filtration was conducted using AKTA avant 25 (GE Healthcare). After prefiltering the sample solution with a 0.2 µm microfilter (Minisart RC 25 mm, Sartorius), the solution was filled into Superloop 150 ml (GE Healthcare), and the solution was loaded to the virus filter using the system pump.
8
PEG-S-Fab Purification by FPLC
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PEG-S-Fab was purified using an AKTA™ avant25 fast protein liquid chromatography purification system (GE Healthcare Bio-Sciences Corp.) and a Superdex 10/300 GL column at a flow rate of 0.8 mL/min. The column was first equilibrated with two column volumes (CVs) of distilled water and two CVs of PBS before applying the samples. All the collected fractions were analyzed by Coomassie blue and barium iodide complex staining after SDS-PAGE under reducing conditions. The fractions of the purified PEG-S-Fab were pooled together for further studies.
9
Purification of TecG, TseG, and TsiG
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The full‐length open reading frames (ORFs) of TecG, TseG, TsiG were cloned into the pET28a and pET41a vectors, and were transformed into E. coli BL21 (DE3). Subsequently, bacteria were inoculated on 20 mL LB medium at 37°C and 180 rpm for 12 h. Following this, 1% of the bacterial volume was transferred to 200 mL LB medium and cultured at 37°C and 180 rpm for 2–3 h until OD600 reached 0.6. At this point, isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) was added to the culture medium at a final concentration of 0.1 mM at 16°C and 180 rpm with shaking overnight. The bacteria were centrifuged at 5000 g at 4°C for 30 min and resuspended in Buffer A or phosphate‐buffered saline (PBS). After ultrasonic crushing, the supernatant was harvested at 4°C at 10,000 g for 20 min, and the protein was purified using the AKTA avant 25 (GE Healthcare). The expression of proteins was evaluated using SDS‐PAGE.
10
AKTA-Driven Chromatography and Filtration
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The AKTA pure 25 or the AKTA avant 25 (GE Healthcare) was used to control the filtration and/or chromatography column processes. AKTA pure 25 was used for all studies except the plasma IgG filtration. The chromatography column(s) were connected to the column valve and the virus filter was connected to the outlet valve. For the integrated mAb process, the filter was positioned after the flow restrictor. AKTA avant 25 was used for plasma IgG filtration by connecting the filter to the column valve. The flow restrictor was removed to ensure that the pressure monitor displays filtration pressure, and pressure was recorded using the PreC pressure monitor on the AKTA avant 25.
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