mAbs were produced via transient transfection of HEK Expi293 cells (Thermo Fisher Scientific) following the same procedure as described above for CyRPA. mAb was purified from culture supernatants via protein G purification using a 5 ml protein G column (GE Healthcare) in TBS. mAb was eluted in 1.6 ml fractions in glycine (200 mM, pH 2.0) into Tris buffer (1 M, pH 9.0). Fractions were pooled and concentrated to 2 ml before SEC using an S200 16/600 column (GE Healthcare). Fabs were produced through papain digestion using the Fab Preparation kit (Thermo Fisher Scientific) following the manufacturer’s instructions.
S200 16 600 column
Manufactured by GE Healthcare
The S200 16/600 column is a size exclusion chromatography column designed for the separation and purification of biomolecules. The column has a bed volume of 120 mL and an internal diameter of 16 mm, making it suitable for a variety of laboratory-scale applications. The column is constructed with materials that are compatible with a wide range of buffers and solvents, allowing for versatile use in different sample preparation and purification workflows.
Automatically generated - may contain errors
Lab products found in correlation
Kta pure, by GE Healthcare (3 mentions)
Expi293 hek cells, by Thermo Fisher Scientific (2 mentions)
Captureselect c tag affinity resin, by Thermo Fisher Scientific (2 mentions)
Fab preparation kit, by Thermo Fisher Scientific (1 mentions)
Kta explorer fplc, by GE Healthcare (1 mentions)
Dylight 488, by Thermo Fisher Scientific (1 mentions)
Expifectamine, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Monolith nt 115, by NanoTemper (1 mentions)
Mo affinity analysis software, by NanoTemper (1 mentions)
Gibson assembly method, by New England Biolabs (1 mentions)
Kta pure system, by GE Healthcare (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
7 protocols using s200 16 600 column
1
Monoclonal Antibody Production and Purification
2
Fab Fragment Fluorescent Labeling
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The purified Fab fragment (3 mg/mL) was labeled with a 10-fold molar access of DyLight 488 (Thermo Fisher Scientific, 20341) for 2 hours at room temperature in the dark. Excess label was removed on S200 16/600 column (28-9893-35, GE Healthcare) in PBS using ÄKTA-Explorer FPLC (GE Healthcare).
3
Recombinant CyRPA Protein Production
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The CyRPA construct used was based on the CyRPA sequence from the 3D7 clone of P. falciparum encompassing amino acids 29–362. A mammalian secretion peptide, MEFQTQVLMSLLLCMSGAAA, was added upstream of residue 29 to enable secretion from a mammalian expression system, along with a four-glycine linker followed by an EPEA tag (C-tag) on the C-terminus44 (link). The following mutations were introduced to remove three potential N-linked glycosylation sites: S147A, T324A, and T340A.
HEK Expi293 cells (Thermo Fisher Scientific) were transfected following the manufacturer’s protocol using expifectamine (Thermo Fisher Scientific), including the addition of enhancer 1 and enhancer 2 (Thermo Fisher Scientific) 18 h post-transfection. Supernatants were harvested 4 days after initial transfection via centrifugation.
CyRPA was first purified through C-tag affinity purification using a 10 ml column packed with CaptureSelect C-tag affinity resin (Thermo Fisher Scientific). Harvested supernatants were run over the column at 5 ml/min and then washed for 10 column volumes (CV) with TBS (150 mM NaCl, 20 mM Tris pH 7.4); CyRPA was eluted using 2 M MgCl2. Fractions were pooled and concentrated to 2 ml before size exclusion chromatography (SEC) using an S200 16/600 column (GE Healthcare) on an Äkta pure (GE Healthcare) into TBS.
HEK Expi293 cells (Thermo Fisher Scientific) were transfected following the manufacturer’s protocol using expifectamine (Thermo Fisher Scientific), including the addition of enhancer 1 and enhancer 2 (Thermo Fisher Scientific) 18 h post-transfection. Supernatants were harvested 4 days after initial transfection via centrifugation.
CyRPA was first purified through C-tag affinity purification using a 10 ml column packed with CaptureSelect C-tag affinity resin (Thermo Fisher Scientific). Harvested supernatants were run over the column at 5 ml/min and then washed for 10 column volumes (CV) with TBS (150 mM NaCl, 20 mM Tris pH 7.4); CyRPA was eluted using 2 M MgCl2. Fractions were pooled and concentrated to 2 ml before size exclusion chromatography (SEC) using an S200 16/600 column (GE Healthcare) on an Äkta pure (GE Healthcare) into TBS.
4
Assembling and Purifying the RCR Complex
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After purification of RH5, CyRPA and RIPR, assembled RCR complex was produced by mixing equimolar concentrations of each protein and incubating for 60 min at room temperature. The assembled complex was then purified by SEC using an S200 16/600 column and Äkta pure (GE Healthcare) into TBS.
For storage, all protein samples were flash-frozen in liquid nitrogen in 1 ml aliquots.
For storage, all protein samples were flash-frozen in liquid nitrogen in 1 ml aliquots.
5
Quantifying Hexon-hLF Binding Affinity
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Microscale thermophoresis (MST) analysis was carried out to determine the binding affinity between hexon and hLF using Monolith NT.115 (Nanotemper Technologies). Hexon was labeled with AlexaFluor594 (ThermoFisher Scientific) according to the manufacturer’s instructions. Excess dye was removed by SEC on S200 16/600 column (GE Healthcare) equilibrated with buffer C. MST experiments were performed in buffer D containing 0.05% (vol/vol) Tween 20. A range of 16 different concentrations of hLF or hLF mutants was generated by serial dilution (1:1) while the same amount of labeled hexon was added to each dilution. Following 5-min incubation at 25°C, the samples were taken up in capillaries for MST measurements that were performed at 25°C at 50% LED power, and 40% MST power. The MST curves were fitted using MO.Affinity analysis software (Nanotemper Technologies).
6
Recombinant Human Lactoferrin Production
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A commercially synthesized DNA fragment (Genewiz) encoding hLF (UniProt: Q19KS2) was cloned into the pHLsec vector (46 (link)) using the Gibson assembly method (New England Biolabs). Eight hLF mutants were produced by introducing desired point mutations during the synthesis of DNA fragments (GenScript). The plasmids encoding the wild-type (WT) LF, and its mutants were transfected and transiently expressed in Expi293F cells (ThermoFisher Scientific) following the manufacturer’s protocol.
LF and its mutants were purified from the culture supernatant obtained after centrifugation. After dialysis against 20 mM HEPES pH 7.2, 30 mM NaCl (buffer A), the supernatant was loaded onto a Mono S cation exchange chromatography column (GE Healthcare) equilibrated with buffer A using an ÄKTA Pure system (GE Healthcare). LF and its mutants were eluted by a linear NaCl gradient using buffer A and 20 mM HEPES pH 7.2, 1 M NaCl (buffer B) and subsequently analyzed by SDS-PAGE. Desired fractions were incubated with 0.5 mM FeCl3 in the presence of 200 mM NaHCO3 for 1 h at room temperature and further purified SEC on a S200 16/600 column (GE Healthcare) equilibrated with 20 mM HEPES pH 7.2, 150 mM NaCl (buffer C). The purified LF was concentrated using Amicon Ultra centrifugal filters (Merck Millipore) and stored at −80°C until further use.
LF and its mutants were purified from the culture supernatant obtained after centrifugation. After dialysis against 20 mM HEPES pH 7.2, 30 mM NaCl (buffer A), the supernatant was loaded onto a Mono S cation exchange chromatography column (GE Healthcare) equilibrated with buffer A using an ÄKTA Pure system (GE Healthcare). LF and its mutants were eluted by a linear NaCl gradient using buffer A and 20 mM HEPES pH 7.2, 1 M NaCl (buffer B) and subsequently analyzed by SDS-PAGE. Desired fractions were incubated with 0.5 mM FeCl3 in the presence of 200 mM NaHCO3 for 1 h at room temperature and further purified SEC on a S200 16/600 column (GE Healthcare) equilibrated with 20 mM HEPES pH 7.2, 150 mM NaCl (buffer C). The purified LF was concentrated using Amicon Ultra centrifugal filters (Merck Millipore) and stored at −80°C until further use.
7
Recombinant RH5 Production in Drosophila S2
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Production of full-length recombinant RH5 in Drosophila S2 cells was as previously published42 (link). The RH5 sequence was based on the 3D7 clone of P. falciparum with four mutations to remove putative glycosylation sites (T40A, T216A, T286A, and T299A) and a C-terminal C-tag (EPEA). Cells were harvested in 2 L batches and then subjected to tangential flow filtration (Millipore Pellicon 3) with a 10 kDa cut-off. Subsequently, RH5 was purified using the same two-step purification as CyRPA: affinity purification using a 10 ml CaptureSelect C-tag affinity resin (Thermo Fisher Scientific) and SEC using an S200 16/600 column and Äkta pure (GE Healthcare) in into TBS.
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