Hitrap imac hp column

The plasmid encoding the P. falciparum rhoptry antigen PfRAMA-bio-his (Addgene, plasmid #50737) was a gift from Gavin Wright (Wellcome Sanger Institute, Cambridge, UK). The PfRAMA sequence was based on the 3D7 strain and comprised amino acids Y17-S840, where any predicted N-linked glycosylations were removed by substituting alanine for serine/threonine at these sites. Additionally, the vector included a 25 kDa C-terminal tag-region comprising ratCD4 (d3 + 4), a biotinylation sequence and a hexahistidine (His6) tag^{21 (link)}.
The recombinant protein was expressed by Expi293F cells (Gibco), by transiently transfecting the PfRAMA-bio-his plasmid following the manufacturer’s instructions. The secreted protein was harvested three days post transfection and purified by immobilized metal affinity chromatography (IMAC) using a 5 mL HiTrap IMAC HP column charged with 0.1 M NiSO₄ × 6 H₂O on the ÄKTAxpress system (both GE Healthcare). PfRAMA was eluted in 500 mM imidazole in sodium phosphate buffer pH 7.4, immediately followed by buffer-exchanging into PBS using Amicon ultra centrifugal concentrators (Millipore). The quality of PfRAMA was evaluated by SDS-PAGE and western blotting using respectively InstantBlue staining (Expedeon) or an anti-his (C-term)-HRP conjugated antibody (Miltenyi Biotec) for detection.

For the production of recombinant proteins, the stably transfected HEK293-EBNA cells were grown to about 80% confluence and were transferred to expression medium without FBS. Conditioned medium containing secreted proteins was collected every 3 days and replaced with fresh expression media for 12 days. Conditioned medium was centrifuged at 2000xg for 10 min to pellet the cells before storing at −20°C. After thawing, recombinant proteins were purified from conditioned media by HPLC, on a HiTRAP Imac HP column (GE Healthcare, 17-0920-03), eluted with imidazol and desalted on silica column with PBS (GE Healthcare, HiPrep 26 /10Desalting) or on a Vivaspin column (Vivaspin Sartorius). The absolute concentration of soluble Agrins was estimated on a coomassie blue stained gel by comparison with a known amount of a commercial purified Agrin.

Solubilized MbxA was refolded by dropwise dilution from 6 M to 400 mM urea with IMAC buffer (50 mM Tris pH 7.8, 400 mM NaCl, 10 mM CaCl₂) with constant stirring at room temperature. All subsequent steps were performed at 4 °C. Refolded MbxA was loaded on a 5 ml Ni²⁺ loaded HiTrap IMAC HP column (GE Healthcare) and eluted with a linear 0–75 mM histidine gradient with elution buffer (50 mM Tris pH 7.8, 400 mM NaCl, 10 mM CaCl_2, 75 mM histidine). Elution fractions containing MbxA were pooled and concentrated with Amicon Ultra-15 Centrifugal Filter Units (50 kDa NMWL, Merck Millipore). Histidine was removed with a PD10 desalting column (GE Healthcare) at room temperature and the protein eluted with cold IMAC buffer. Activity of MbxA was tested on Columbia agar with 5% sheep blood (Oxoid).

The gene encoding N-HGbRL (residues 1–133; GenBank: ACD83144) was amplified by PCR using primers containing the NdeI and BamHI restriction sites, with a His-tag sequence inserted before the stop codon. The PCR product was cloned into the pET-3a expression vector (Novagen), and the resulting vector was transformed into Rosetta 2(DE3)pLysS (Novagen). The cells were grown in LB medium containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Expression was induced with 50 μM isopropyl-β-D-thiogalactopyranoside, supplemented with 100 mg/l FeSO₄•7H₂O and 17 mg/l δ-aminolevulinic acid. After overnight incubation at 37 °C, the cells were harvested by centrifugation and resuspended in 50 mM Tris-HCl, 200 mM NaCl, pH 8.0, sonicated and centrifuged to remove cell debris. The supernatant was then applied to a Ni²⁺-charged HiTrap IMAC HP column (GE Healthcare), and N-HGbRL was eluted with a gradient of up to 1.0 M imidazole. Fractions containing the protein were pooled and further purified by size-exclusion chromatography on a Superdex 200 column (GE Healthcare).

The recombinant His-tagged FhCyLS-2 was purified from the concentrated and desalted expression medium by Ni-affinity chromatography on a HiTrap IMAC HP column (GE Healthcare) using the manufacturer’s protocol for elution with imidazole at pH 8. It was followed by size-exclusion chromatography, using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare) equilibrated with 50 mM Tris-HCl, 200 mM NaCl, pH 8. The purified protein was concentrated and buffer-exchanged into 10 mM Tris-HCl, 10 mM NaCl, pH 8, using an Amicon Ultra-3K centrifugal unit (Millipore). Purification was monitored by Laemmli SDS-PAGE on 15% polyacrylamide gels stained by Coomassie Brilliant Blue R-250. Edman sequencing of purified recombinant FhCyLS-2 demonstrated the N-terminal sequence starting with IRG, which indicated a proteolytic trimming and removal of the N-terminal dipeptide AG during protein expression.