Mixed mode affinity chromatography carrier
A mixed-mode, affinity technology, used in carrier binding/immobilization of peptides, chemical instruments and methods, ion exchange of chelates, etc. Effect
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Embodiment 1
[0644] 1. Fabrication of Carriers for Mixed-Mode Affinity Chromatography
[0645] (1) Preparation of wet gel
[0646] Cross-linked agarose-based substrate "Sepharose 4Fast Flow" (manufactured by GE Healthcare) ("SEPHAROSE" is a registered trademark) was repeatedly suspended and washed with pure water on a glass filter to obtain Substrate slurry. Pure water was removed from the substrate slurry by suction filtration to obtain a wet gel.
[0647] (2) Preparation of base material before coating - introduction of epoxy group
[0648] 50 g of the obtained wet gel, 76 mL of pure water, and 25 g of 2-chloromethyloxirane were put into a 300-mL three-necked flask, and stirring of the contents of the flask was started in a 45° C. warm bath. Stirring was performed until the temperature in the flask became 45°C. In a 45°C warm bath, 20.4 g of a 50% (w / w) sodium hydroxide aqueous solution was dropped into the flask over 2 hours while maintaining the temperature in the flask at about 45...
Embodiment 2
[0675] 1. Fabrication of Carriers for Mixed-Mode Affinity Chromatography
[0676] In "Preparation of Cyclic Peptide Immobilization Carboxylation Carrier-Introduction of Cyclic Peptide", except for the point of using the cyclic peptide B (SEQ ID NO. 14) represented by the following formula (B) instead of the above-mentioned cyclic peptide A, it is carried out in the same manner as The cyclic peptide immobilized carboxylation carrier was prepared in the same manner as Example 1.
[0677] Lys-Lys-Lys-Asp-Xaa5-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa15-Thr...(B)
[0678] Wherein, in formula (B), both Xaa5 and Xaa15 are amino acid residues derived from L-homocysteine, and Xaa5 and Xaa15 form a disulfide between the thiol groups of the side chain of L-homocysteine bond for cross-linking.
[0679] 2. Determination of Immobilized Amount of Cyclic Peptide
[0680] The cyclic peptide-immobilized amount (affinity ligand-immobilized amount) of the obtained cyclic peptide-immobilized car...
Embodiment 3
[0686] 1. Fabrication of Carriers for Mixed-Mode Affinity Chromatography
[0687] In "Preparation of Carboxylated Carrier - Introduction of Cation Exchange Group", a carboxylated carrier was prepared in the same manner as in Example 1 except that the amount of sodium chloroacetate was changed to 6.1 g.
[0688] In addition, in "Preparation of cyclic peptide-immobilized carboxylation carrier-introduction of cyclic peptide", in addition to the point of using the carboxylation carrier prepared above and instead of the above-mentioned cyclic peptide A, a cyclic peptide represented by the following formula (C) is used C (SEQ ID NO: 15) Except for this point, a cyclic peptide-immobilized carboxylation carrier was prepared in the same manner as in Example 1.
[0689] Lys-Lys-Lys-Asp-Xaa5-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa15-Thr...(C)
[0690] Wherein, in the formula (C), Xaa5 is an amino acid residue derived from N-ε-chloroacetyl-L-lysine, Xaa15 is an amino acid residue derived...
PUM
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