A kind of affinity peptide and its application
A kind of affinity peptide and affinity technology, applied in the direction of peptides, specific peptides, peptide preparation methods, etc., can solve the problem of unsatisfactory separation methods and media, inability to effectively separate κ-type antibodies and λ-type antibodies, and easy inactivation and other problems, to achieve the effect of cheap and easy to obtain quality control, facilitate medium preparation, and facilitate quality control
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Embodiment 1
[0019] Example 1: Synthesis of Polypeptides
[0020] 1) Activated resin: Weigh 1000 mg of Fmoc Cys-wang Resin, soak it in N,N-dimethylformamide (DMF) for 30 minutes to swell.
[0021] 2) Deprotection: remove DMF by pressure filtration, add a DMF solution containing 20% piperidine for 15min boiling reaction with nitrogen, and remove by pressure filtration; wash the resin three times with isopropanol and three times with DMF, and then use the ninhydrin method to detect the resin Fmoc removal condition.
[0022] 3) Condensation reaction: Weigh 1.4 mmol of FMOC-Tyr, take O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU), 1-hydroxybenzotriazepine The DMF mixed solution of oxazole (HOBt) and N,N-diisopropylethylamine (DIEA) was used as the reaction solution, and the reaction was boiled with nitrogen at room temperature for 2 hours; after the reaction, the resin was washed with isopropanol three times, and then with DMF. Three times, the ninhydrin method was ...
Embodiment 2
[0026] Example 2: Preparation of Affinity Media
[0027] 1) Dissolve 10 mg of antibody affinity peptide (amino acid sequence shown in SEQ ID No. 1) in 10 mL of 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) buffer, pH 7.4.
[0028] 2) Take 1 mL of sulfhydryl-activated Sephadex.
[0029] 3) The compounds in steps 1) and 2) were mixed and reacted for 12 hours.
[0030] 4) After the reaction is completed, the gel particles are washed with Hepes buffer to obtain an affinity medium.
Embodiment 3
[0031] Example 3: Affinity peptide separation and purification of kappa chain antibody
[0032] 1) Prepare 10 mmol / L Hepes buffer, pH 7.0, NaCl content 0.15 M, and set flow rate 1 mL / min.
[0033] 2) Using the liquid prepared in step 1) as the mobile phase, run the AKTA protein purification system.
[0034] 3) Take 1 mL of the affinity medium and 1 mL of the Protein L medium in Example 2 to pack the separation column respectively.
[0035] 4) 1 mL of antibody solution was loaded respectively, and 3 mL of effluent was collected.
[0036] 5) Prepare 0.1M HCl-Gly buffer at pH 2 as eluent.
[0037] 6) Load 3 mL of eluent respectively, and collect 3 mL of eluent.
[0038] 7) Measure the content and purity of kappa chain antibody in the effluent and eluate before adsorption and after adsorption, respectively, and calculate the total extraction rate. The results are shown in Table 1 below.
[0039] Table 1. Comparison of Adsorption Properties between Affinity Peptide Media and Pr...
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