The sequences encoding for E-301 and E-301-LOF containing Salmonella typhimurium sialidase were cloned into the mammalian expression vector pCEP4 (Thermo Fisher Scientific). Human embryonic kidney 293T cells were then transiently transfected with the DNA constructs using the Expi293 system and following standard protocols according to the manufacturer’s instructions (Thermo Fisher Scientific). Purification of E-301 and E-301 LOF was performed directly from transfection harvests using a HiTrap Protein A affinity column (GE Healthcare) and eluted with 1 M arginine (pH 3.9). Anion-exchange chromatography was used as a secondary purification method, and the final product was dialyzed into PBS (pH 7.4). The biochemical characterization of E-301 and E-301 LOF, including purity, HER2 binding affinity, and enzymatic activity, was conducted as described previously (25 ).
Hitrap protein a affinity column
Manufactured by GE Healthcare
The HiTrap Protein A affinity column is a laboratory equipment used for the purification of antibodies and other proteins containing the Protein A binding site. It is a pre-packed, ready-to-use column that utilizes Protein A ligand immobilized on agarose beads to selectively capture and isolate the target proteins from complex mixtures.
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3 protocols using hitrap protein a affinity column
1
Purification and Characterization of E-301 Sialidase
2
Purification and Characterization of E-301 Sialidase
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The sequences encoding for E-301 and E-301-LOF containing Salmonella typhimurium sialidase were cloned into the mammalian expression vector pCEP4 (Thermo Fisher Scientific). Human embryonic kidney 293T cells were then transiently transfected with the DNA constructs using the Expi293 system and following standard protocols according to the manufacturer’s instructions (Thermo Fisher Scientific). Purification of E-301 and E-301 LOF was performed directly from transfection harvests using a HiTrap Protein A affinity column (GE Healthcare) and eluted with 1 M arginine (pH 3.9). Anion-exchange chromatography was used as a secondary purification method, and the final product was dialyzed into PBS (pH 7.4). The biochemical characterization of E-301 and E-301 LOF, including purity, HER2 binding affinity, and enzymatic activity, was conducted as described previously (25 ).
3
Recombinant E-301 and E-301-LOF Purification
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The DNA encoding for E-301 and E-301-LOF were cloned into the mammalian expression vector pCEP4 vector (Thermo FisherScientific). HEK 293 cells were then transiently transfected with the DNA constructs using Expi293 Expression system and standard protocols according to the manufacturer's instructions (Thermo FisherScientific). Purification of E-301 and E-301-LOF was performed directly from transfection harvests using a HiTrap Protein A affinity column (GE Healthcare) and eluted with 1M arginine pH 3.9. Anion-exchange chromatography was used as a secondary purification method and the final product was dialyzed into 1X PBS 7.4. The biochemical characterization of E-301 and E-301-LOF, including purity, Her2 binding affinity, and enzymatic activity, was conducted as described previously (WO2019136167).
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