Solid-phase chelator material, method for producing thereof and use thereof for the purification of proteins
A chelating agent and chelating technology, applied in the preparation method of peptides, ion exchange of chelates, separation methods, etc., can solve the problems of reduced protein binding ability and scattered metal ions
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Embodiment 1
[0240] Example 1: Synthesis of a new chelating agent by preparing diethylenetriamine agarose, the reaction of EDTA and ethylenediamine and the covalent coupling of EDTA-EDA-EDTA and agarose
[0241] Synthesis of diethylenetriamine agarose
[0242] 10 ml of agarose pellets (WorkBeads 40SEC, BioWorks Sweden AB, Uppsala) were resuspended in 10 ml of 1 M caustic soda solution and incubated for 2 hours on a thermoshaker. Then 5 ml of epichlorohydrin were added and the suspension was heated at 30° C. for 4 hours. The suspension was filtered with suction and washed six times with dd water. The agarose was then resuspended in 20 ml of 5% diethylenetriamine solution (pH 10.5) and incubated at 65°C for 20 hours. Agarose was aspirated dry, washed four times with dd water and four times with phosphate-buffered saline, and then stored in anhydrous dimethylformamide. Synthesis of EDTA-EDA-EDTA
[0243] 853 mg of EDTA dianhydride (3.33 mmol) were dissolved in 15 ml of dimethylformamide a...
Embodiment 2
[0246] Example 2: Synthesis of a novel chelating agent by preparing pentaethylenehexamine agarose, reacting EDTA with ethylenediamine and covalently coupling EDTA-EDA-EDTA to agarose
[0247] Synthesis of Pentaethylene Hexamine Agarose
[0248] 10 ml of agarose pellets (WorkBeads 40SEC, BioWorks Sweden AB, Uppsala) were resuspended in 10 ml of 1 M caustic soda solution and incubated for 2 hours on a thermoshaker. Then 5 ml of epichlorohydrin were added and the suspension was heated at 30° C. for 4 hours. The suspension was filtered with suction and washed six times with dd water. The agarose was then resuspended in 20 ml of 5% pentaethylenehexamine (pH 10.5) and incubated at 65°C for 20 hours. Agarose was aspirated dry, washed four times with dd water and four times with phosphate-buffered saline, and then stored in anhydrous dimethylformamide. Synthesis of EDTA-EDA-EDTA
[0249] 853 mg of EDTA dianhydride (3.33 mmol) were dissolved in 15 ml of dimethylformamide and a solu...
Embodiment 3
[0252] Example 3: Formation of Dimeric EDTA Chains by Sequential Coupling of EDTA, Ethylenediamine and EDTA
[0253] Synthesis of diethylenetriamine agarose
[0254] 10 ml of agarose pellets (WorkBeads 40SEC, BioWorks Sweden AB, Uppsala) were resuspended in 10 ml of 1 M caustic soda solution and incubated for 2 hours on a thermoshaker. Then 5 ml of epichlorohydrin were added and the suspension was heated at 30° C. for 4 hours. The suspension was filtered with suction and washed six times with dd water. The agarose was then resuspended in 20 ml of 5% N-diethylenetriamine solution (pH 10.5) and incubated at 65°C for 20 hours. Agarose was aspirated dry, washed four times with dd water and four times with phosphate-buffered saline, and then stored in anhydrous dimethylformamide.
[0255] EDTA-agarose (NH 2 )Synthesis
[0256] 10 ml of amino agarose was resuspended in 10 ml of dimethylformamide, and a solution mixed with 1500 mg of 4,4'-ethylenebis(2,6-morpholinedione) and 10 ...
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