dUTPQ (Figure S3B ) was prepared by conjugating Amino-11-dUTP (Lumiprobe) to BHQ-10 succinimidyl ester (Biosearch Technologies). In a typical reaction, 2 μL 50 mM Amino-11-dUTP and 2.5 μL 50 mM BHQ-10 succinimidyl ester were mixed with 45.5 μL 100 mM sodium tetraborate, pH 8.5, and the reaction was allowed to proceed overnight at room temperature in the dark. The mixture was separated on a HiTrapQ column (GE Healthcare) using an AKTA pure chromatography system (GE Healthcare). The mixture was diluted 10-fold in solvent A (10% acetonitrile in water) and loaded onto the column, followed by a gradient of 0-100% solvent B (3 M LiCl) over 20 column volumes. The elution profile of the mixture showed a single additional peak with absorbance at 516 nm relative to the elution profiles of Amino-11-dUTP and BHQ-10 succinimidyl ester alone. Fractions corresponding to this peak were pooled and added to 30 volumes ice-cold acetone to precipitate dUTPQ, which was collected by centrifugation, washed with cold acetone, and resuspended in 10 mM Tris, pH 8.0.
Akta pure chromatography system
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The AKTA Pure chromatography system is a versatile and automated platform designed for protein purification. It facilitates the separation, isolation, and purification of biomolecules, such as proteins, from complex mixtures. The system is equipped with advanced features that enable precise control and monitoring of the purification process.
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Lab products found in correlation
β lactose, by Merck Group (3 mentions)
Consumer packable glass column, by GE Healthcare (2 mentions)
α lactose agarose affinity resin, by Merck Group (2 mentions)
Peptone, by Solarbio (2 mentions)
Sds page, by Solarbio (2 mentions)
Both of the e coli bl21 de3 and expression vector pet28a, by Thermo Fisher Scientific (2 mentions)
Pd 10 desalting column, by GE Healthcare (2 mentions)
Ni nta column, by GE Healthcare (2 mentions)
Coomassie brilliant blue g250, by Solarbio (2 mentions)
Glycine, by Solarbio (2 mentions)
Hitrap q column, by GE Healthcare (1 mentions)
Ultraflex maldi tof tof mass spectrometer, by Bruker (1 mentions)
Blue dextran, by GE Healthcare (1 mentions)
Superdex s200 increase 10 300 gl, by GE Healthcare (1 mentions)
Histrap 5 ml column, by GE Healthcare (1 mentions)
Superdex 200 16 60 column, by GE Healthcare (1 mentions)
Ja25.50 rotor, by Beckman Coulter (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Emulsiflex c5, by Avestin (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
25 protocols using akta pure chromatography system
1
Synthesis and Purification of Quenched dUTP Conjugate
2
Fmoc-based Peptide Synthesis and Purification
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The peptides were synthesized by the N–9–fluorenylmethyloxycarbonyl (Fmoc) strategy using a Liberty Blue automated microwave peptide synthesizer (CEM, Stallings, NC, USA), as previously described [12 (link)]. The purification of the peptides was accomplished by liquid chromatography using an AKTA pure chromatography system (GE Healthcare, Chicago, Illinois, USA) with purity >95%. The sequence and degree of purity (>95%) were confirmed by mass spectrometry by an ULTRAFLEX MALDI–TOF/TOF mass spectrometer (Bruker, Fremont, CA, USA).
3
Gel Filtration Chromatography Calibration
4
Immunoglobulin G Isolation by Affinity Chromatography
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Immunoglobulins G were isolated from blood serum by affinity chromatography on AKTA Pure chromatography system GE Healthcare on a Protein G Sepharose column (Polosukhina et al., 2004 (link); Smirnova et al., 2021 (link)). Serum diluted 1:3 with buffer A (50 mM Tris–HCl, pH 7.5, 150 mM NaCl) was loaded onto the Protein G Sepharose column. Proteins that did not interact with the sorbent were washed with 8 ml of the same buffer until the absorption at 280 nm completely disappeared. Nonspecifically adsorbed proteins and lipids were eluted with 3 ml of buffer A containing 1% Triton X-100 and 0.3 M NaCl. Then the column was washed with 15 ml of buffer A. IgGs were eluted with 0.1 M glycine-HCl, pH 2.6. The isolated antibody fractions were neutralized with 1 M Tris–HCl, pH 8.8. After isolation, all IgG samples were subjected to homogeneity analysis using 5–18% SDS-PAGE and Coomassie staining.
5
Lactose-Agarose Affinity Chromatography
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Affinity of IDO-Gal3 for lactose was determined using affinity chromatography in an AKTA Pure chromatography system (GE Life Sciences) equipped with consumer-packable glass column (GE Life Sciences) packed with α-lactose agarose affinity resin (Sigma-Aldrich). Proteins were eluted with a linear gradient of β-lactose (Sigma-Aldrich) in phosphate buffer.
6
Purification of His-Tagged CENP-V Protein
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CENP-V was fused to an N-terminal His-Tag and/or monomeric GFP, expressed from a baculovirus vector in SF9 insect cells (Expression Systems, cat# 94-001 F, https://expressionsystems.com/product/insect-cells/ ). Three days after infection, cells were harvested and lysed in buffer A (20 mM Hepes, 1 M NaCl, 20 mM imidazole, 1 mM DTT, pH 7.5) supplemented with EDTA-free Protease Inhibitor Cocktail (Roche) using an Emulsiflex C5 (Avestin). The lysate was cleared by centrifugation (18.000 g, JA-25.50 rotor (Beckman Coulter), 60 min, 4 °C). Soluble material was applied to a HisTrap 5 ml column (GE Healthcare) and washed with 10 column volumes (CV) buffer A, 5 CV ATP-buffer (5 mM ATP in 2xPBS 10 mM MgCl2) and with buffer A containing 40 mM imidazole. Finally, the protein was eluted by applying the elution buffer (20 mM Hepes, 1 M NaCl, 300 mM imidazole, 1 mM DTT, pH 7.5). The eluted fraction was concentrated by centrifugation using an Amicon concentrator (10 kDa cutoff), and subjected to size exclusion chromatography using a Superdex-200 16/60 column (GE Healthcare) equilibrated with 20 mM Hepes, 300 mM NaCl, pH 7.0 at room temperature, using an Akta Pure chromatography system (GE Healthcare). Pooled samples were concentrated by centrifugation in Amicon tubes and snap frozen in liquid nitrogen.
7
Recombinant IgG4 Production for Zika Research
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The variable genes encoding clone 44 were cloned into a modified expression vector containing the human IgG4 construct, and transfected into Expi293F cells (Invitrogen) as described previously (26 ). IgG was purified by affinity chromatography using MabSelect columns with the AKTA Pure chromatography system (GE Healthcare, Chicago, IL, USA) following the manufacturer's protocol. An in-house discovered Ab specific to Zika virus envelope domain III (27 (link)) was also produced in human IgG4 format and used as an isotype control.
8
Recombinant Protein Expression and Purification
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Synthetic genes encoding the 99 amino acid protein named
PRS17 and its active-site mutant PRS17-D25N
(DNA2.0, Menlo Park, CA) were cloned in pJ414 vector flanked by Nde1
and BamHI sites and transformed into Escherichia coli BL-21 (DE3, Stratagene). DNA sequencing
and electrospray ionization mass spectrometry were used to verify
the recombinant construct. Protein expression, purification, and refolding
were carried out as described previously.55 (link),56 (link) In brief, cells were expressed in Luria-Bertani medium and induced
with 2 mM isopropyl β-d -1-thiogalactopyranoside. The
harvested cells were lysed by the addition of lysozyme (100 μg/mL)
and sonication at 4°C. The pelleted inclusion bodies were washed
and solubilized in 50 mM Tris–HCL, pH 8.0, 8 M guanidine hydrochloride,
5 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM dithiothreitol.
The protein was purified over a Superdex-75 column (HiLoad 2.6 cm
× 60 cm, GE HealthCare) under denaturing conditions. The peak
fractions were further purified by reverse-phase high-performance
liquid chromatography on a SOURCE 15RPC ST 4.6/100 column using AKTA
pure chromatography system (GE HealthCare). The purified protein was
refolded by extensive dialysis against 30 mM formic acid and concentrated
to the desired level using Amicon Ultra concentrators.
PRS17 and its active-site mutant PRS17-D25N
(DNA2.0, Menlo Park, CA) were cloned in pJ414 vector flanked by Nde1
and BamHI sites and transformed into Escherichia coli BL-21 (DE3, Stratagene). DNA sequencing
and electrospray ionization mass spectrometry were used to verify
the recombinant construct. Protein expression, purification, and refolding
were carried out as described previously.55 (link),56 (link) In brief, cells were expressed in Luria-Bertani medium and induced
with 2 mM isopropyl β-
harvested cells were lysed by the addition of lysozyme (100 μg/mL)
and sonication at 4°C. The pelleted inclusion bodies were washed
and solubilized in 50 mM Tris–HCL, pH 8.0, 8 M guanidine hydrochloride,
5 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM dithiothreitol.
The protein was purified over a Superdex-75 column (HiLoad 2.6 cm
× 60 cm, GE HealthCare) under denaturing conditions. The peak
fractions were further purified by reverse-phase high-performance
liquid chromatography on a SOURCE 15RPC ST 4.6/100 column using AKTA
pure chromatography system (GE HealthCare). The purified protein was
refolded by extensive dialysis against 30 mM formic acid and concentrated
to the desired level using Amicon Ultra concentrators.
9
Recombinant Vip3Aa Protein Expression and Purification
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The Vip3Aa protein was expressed in E. coli BL21(DE3) at 25 °C for 48 h in autoinduction terrific broth medium. Bacterial cells were collected by centrifugation and resuspended in lysis buffer (20 mM Tris–HCl pH 8.0 and 300 mM NaCl). After the cells were lysed by a high-pressure cell crusher (Union-Biotech), the supernatant was collected, run through Ni-NTA agarose resin (Qiagen), and washed with 20 mM Tris–HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0. The small ubiquitin-like motif tag was removed with homemade His-tagged ULPI protease at room temperature (20–30 °C) for 2 h and proteins were then eluted with lysis buffer. The proteins were further purified by HiTrap Q HP ion-exchange chromatography and Superdex 200 Increase 10/300 Gl gel filtration chromatography using an AKTApure chromatography system (GE Healthcare Life Sciences). Proteins corresponding to the molecular weight of the tetramerized Vip3Aa were used for subsequent biochemical and bioassay analyses. The expression and purification steps of other Vip3Aa truncation variants and mutations were similar to those of Vip3Aa. The concentration of proteins was quantified by a NanoPhotometer N60 (Implen). The molar absorption coefficient of each protein was predicted by Protean (DNASTAR) based on the amino acid composition of the protein.
10
Protein-oligonucleotide interaction analysis
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All samples were run and absorbance data (280 and 254 nm) recorded on an Akta Pure chromatography system (GE Healthcare Lifesciences). Samples (90 μg DM15; 23 μg DM15 + 7.4 μg RPS6 oligonucleotide (1:1 molar ratio) pre-incubated on ice for 20 min; 7.4 μg RPS6 oligonucleotide; or molecular weight standards (BioRad)) were analysed with a HiLoad 16/600 Superdex 75 pg column (GE Healthcare Lifesciences) in 50 mM Tris–HCl, pH 8, 200 mM NaCl, 1 mM DTT, 2% glycerol.
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