Ni sepharose excel

The DNA sequence encoding EBV gL (residues 24 to 137; UniProt ID G3CKR4) linked to EBV gH (residues 19 to 678; UniProt ID G3CKS5; C-terminal 6× His tag) with a (G4S)3 linker and the DNA sequence encoding EBV gB (residues 25 to ∼683, UniProt ID R4R670) were cloned into pcDNA3.1. A CD5 signal peptide DNA sequence was inserted into the N terminus of genes of interest. Antibodies against EBV gHgL, E1D1 (45 (link)), and AMMO1 (60 (link)) and recombinant gHgL and gB were expressed in 293F cells. The supernatant was collected and purified by Ni⁺ affinity chromatography (Ni Sepharose Excel; Cytiva). For gHgL, the eluate was concentrated and purified by size exclusion chromatography (SEC) (Superdex 200 Increase 10/300 GL; Cytiva) running at 50 mM NaCl and 50 mM HEPES, pH 7.5; fractions were collected and applied to anion-exchange chromatography (HiTrap Capto Q; 5 mL; Cytiva) and eluted by linear ion-strength increment; fractions were concentrated and purified by SEC (Superdex 200 Increase 10/300 GL) running at 50 mM HEPES and 150 mM NaCl, pH 7.5. For gB, the eluate was concentrated and purified by SEC (Superdex 200 Increase 10/300 GL; Cytiva) running at 50 mM NaCl and 150 mM HEPES, pH 7.5. For E1D1 and AMMO1, the supernatant was captured by protein A beads (L00210; GenScript) followed by SEC (Superdex 200 Increase 10/300 GL; Cytiva) running in PBS.

The gene encoding scFv16 was synthesized (GeneArt, Regensburg, Germany) and subcloned into a modified pFastBac vector, with the resulting construct encoding the GP67 secretion signal sequence at the N-terminus, and a His₈ tag, followed by a TEV cleavage site at the C-terminus^{38 (link)}. His₈-tagged scFv16 was expressed and secreted by Sf9 insect cells, as previously reported^{38 (link)}. Sf9 cells were collected by centrifugation at 5000 × g for 10 min, and the secreta-containing supernatant was combined with 5 mM CaCl₂, 1 mM NiCl₂, 20 mM HEPES, pH 8.0, and 150 mM NaCl. The supernatant was mixed with Ni Sepharose excel (Cytiva) and stirred for 1 h at 4 °C. The bound resin was washed with buffer containing 20 mM HEPES, pH 8.0, 500 mM NaCl, and 20 mM imidazole, and further washed with 10 column volumes of buffer containing 20 mM HEPES, pH 8.0, 500 mM NaCl, and 20 mM imidazole. The protein was then eluted with 20 mM Tris, pH 8.0, 500 mM NaCl, and 400 mM imidazole, and the eluted fraction was concentrated and loaded onto a Superdex 200 10/300 Increase size-exclusion column, equilibrated in buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl. Peak fractions were pooled, concentrated to 5 mg ml^-1 with a centrifugal filter device (10-kDa MW cut-off; MilliporeSigma, Burlington, MA, USA), and frozen in liquid nitrogen.

Eukaryotic expression system was used to purify human EphA2 and HCMV gH/gL. Briefly, expression plasmid was mixed with Polyethyleneimine Linear (PEI) MW40000 (40816ES, Yeasen, China) at a molar ratio of 1:3 in 293F medium (UP1000, Union, China), and the mixture was transfected to Expi293F (A14527, ThermoFisher, Massachusetts). Then the cells were cultured at 37°C with 5% CO2 and 120 rpm shaking for 6 days, and the supernatant was harvested by centrifuge, filtered with 0.45 um filtering membrane, and applied to gravity column loaded with Ni Sepharose Excel (17371202, Cytiva, Washington, DC). The column was washed by PBS with 30 mM imidazole and eluted by PBS with 500 mM imidazole. The elution was concentrated by ultracentrifuge and further purified by size exclusion chromatography (SEC) using Superdex 200 increase 10/300GL (28990944, Cytiva, Washington, DC) on an AKTA Pure25M (GE healthcare, Massachusetts).

Using the NovaCHOice transfection kit (Merck Millipore), the recombinant MUC2-C expression vector was transiently transfected into CHO-S cells growing in 800 ml flasks and FreeStyle CHO growth medium (Gibco) without serum. The extracellular recombinantly expressed protein was collected 72 h after the transfection by centrifuging the cell media at 500 × g and 6000 × g and discarding the cell pellet. To obtain the high-mannose recombinant MUC2-C the expression was done in presence of 1 μg/ml kifunensine (Toronto Research Chemicals).
The recombinant protein was purified from the supernatant by Immobilized metal affinity chromatography (IMAC) method using Ni-Sepharose® Excel (Cytiva) in a loaded gravity column (BioRad). The loaded resin was washed with 300 mM NaCl, 20 mM HEPES pH 7.4 and 40 mM imidazole, and the protein was eluted with 300 mM NaCl, 20 mM HEPES pH 7.4 and 300 mM imidazole. For the high-mannose MUC2-C the elution was dialyzed over-night against 150 mM NaCl, 20 mM MES, pH 5.5, and treated with EndoH (Roche) glycosidase at 4 °C. The products were then purified by Size Exclusion Chromatography (SEC) using an Äkta HPLC system (GE Healthcare) with a HiLoad 16/600 Superdex 200 column (GE Healthcare) in 150 mM NaCl, 20 mM HEPES, pH 7.4.