Affinity separation polymer of lactoferrin and affinity purification method of lactoferrin
A technology for lactoferrin and separation materials, applied in the field of affinity separation materials for lactoferrin, can solve the problems of limited industrial application, high production cost, and high cost, and achieve large-scale purification, low production cost, and high production efficiency. Effect
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example 1
[0017] 1. Preparation of separation materials
[0018] Take NH 2 -Sepharose (500ml), with 1M NaCl of 5 times of volumes successively, after fully washing with 10 times of volumes of distilled water, drain and transfer to a reaction vessel, add acetone-dissolved trichlorotriazazine (100g) in an ice-water bath, stir with saturated NaHCO 3 Adjust the pH between 6 and 7, take it out after 3 hours of reaction, and wash with 3×10 times the volume of water / acetone (0:1, 1:3, 1:1, 3:1, 1:0) successively to obtain 1-Amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (450ml).
[0019] Weigh Beta-alanine (100g) and dissolve it with deionized water (300ml), mix with 1-amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (300ml), and wash with saturated NaHCO 3 Keep the pH between 6.5 and 8.0, place at 50°C and stir for 24 hours. After reaction finishes, with 1M NaCl of 3 times of volumes successively, fully wash with 10 times of volumes of distilled water, obtain 1-amino-Sepharose-3-Beta-alanine...
example 2
[0023] Take transgenic milk samples frozen at -70°C, melt them in a water bath at 40°C, centrifuge at 4500rpm for 30min at 4°C, take the middle layer of skim milk, adjust the pH value to 4.6 with 1N HCl, centrifuge at 12000rpm at 4°C for 20min, take the supernatant, and Adjust the pH to 7.0 with 1N NaOH, adjust the conductivity to 5.6ms / cm with saturated NaCl solution, and load the sample to 10 times the volume of Tris-HCl buffer (50mM Tris, pH7.0, conductivity 5.6ms / cm) In the chromatographic column (1×5cm) that the 1-aminoSepharose-3-Beta-alanine-5-chloro-2,4,6-triazoxide affinity separation material prepared by Example 1 step 1 is equilibrated , followed by washing with Tris-HCl buffer (50mM Tris, pH7.0, conductivity 5.6ms / cm) until the light absorption at 280nm reaches the baseline, glycine-hydrochloric acid buffer (0.1MGly, pH2.4) to elute the bound lactoferrin After collection, the purity of recombinant human lactoferrin was analyzed by SDS-PAGE to be 85.1%.
example 3
[0025] Take transgenic milk samples frozen at -70°C, melt them in a water bath at 40°C, centrifuge at 4500rpm for 30min at 4°C, take the middle layer of skim milk, adjust the pH value to 4.6 with 1N HCl, centrifuge at 12000rpm at 4°C for 20min, take the supernatant, and Adjust the pH to 7.5 with 1N NaOH, adjust the conductivity to 6.0ms / cm with saturated NaCl solution, and load the sample to 10 times the volume of Tris-HCl buffer (50mM Tris, pH7.5, conductivity 6.0ms / cm) In the chromatographic column (1×5cm) that the 1-aminoSepharose-3-Beta-alanine-5-chloro-2,4,6-triazoxide affinity separation material prepared by Example 1 step 1 is equilibrated , followed by washing with Tris-HCl buffer (50mM Tris, pH 7.5, conductivity 6.0ms / cm) until the light absorption at 280nm reaches the baseline, glycine-hydrochloric acid buffer (0.1M Gly, pH 2.4) to elute the bound milk As for ferritin, the purity of recombinant human lactoferrin analyzed by SDS-PAGE after collection was 82%.
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