Method for fast purifying cysteine protease inhibitor
A cysteine protease and inhibitor technology, which is applied in protease inhibitors, chemical instruments and methods, animal/human peptides, etc., can solve the problems of low sample activity recovery rate, unsatisfactory purification effect, cumbersome operation, etc., and achieve It is convenient for large-scale extraction and purification, and the effect of convenient sample source
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Embodiment 1
[0034] The activation of the nylon membrane and the modification of the bonded chitosan, the specific steps are as follows:
[0035] (1) The nylon membrane was first hydrolyzed in 1M HCl at 25°C for 24 hours, and then activated with formaldehyde. Ten hydrolyzed nylon membranes were immersed in 20ml of formaldehyde solution (>36.5wt.%), added with 0.2ml of phosphoric acid (85wt.%), reacted at 60°C for 7h, and washed with hot water at 40-50°C for several times.
[0036] (2) The above-mentioned formaldehyde-activated nylon membrane is immersed in 10ml of chitosan solution (dissolved with 1vol.% acetic acid) with a mass fraction of 1.5%, reacted at room temperature for 1h, then transferred to an 80°C oven, took it out after 1h, and used 1vol .% acetic acid and deionized water to wash away unreacted chitosan. The content of chitosan on the nylon film can be tested according to the ninhydrin method.
Embodiment 2
[0038] The preparation of the nylon affinity membrane with papain as ligand, the specific steps are as follows:
[0039] The above-mentioned modified nylon membrane was immersed in 21ml 0.05M Tris-HCl buffer solution (containing 0.05M L-cysteine and 0.02M EDTA, pH8.0), kept in a 35°C water bath for 30min, and then added 9ml (10mg / ml ) papain solution (pH8.0) and 0.6ml (mass fraction is 25%) glutaraldehyde solution, cooling after continuing constant temperature oscillation reaction 8h, with deionized water and 0.05M Tris-HCl buffer solution (pH8.0) successively Wash thoroughly until the washing solution is colorless, and store in 0.05M Tris-HCl buffer (pH 8.0).
Embodiment 3
[0041] The condition optimization of the nylon affinity membrane with papain as ligand, the specific steps are as follows:
[0042] (1) Determination of the concentration of the crosslinking agent glutaraldehyde. Weigh 5 groups of 0.35g (dry mass, the same below) chitosan-modified nylon membranes, add 21ml Tris-HCl buffer solution (containing 0.05M L-cysteine and 0.02M EDTA, pH8.0), 35°C Incubate in a water bath for 30 minutes, add 9ml (10mg / ml) papain solution (pH8.0), add 0.1ml, 0.3ml, 0.6ml, 0.9ml, 1.2ml of glutaraldehyde solution with a mass fraction of 25%, and put it in a water bath at 35°C Shake the reaction for 8h. Determination of enzyme activity. It was found that when the concentration of glutaraldehyde was 0.5%, the enzyme activity reached the highest.
[0043] (2) Determination of the reaction pH value. Weigh 0.35 g of modified nylon membranes from each of the 6 groups, add buffer solution and enzyme solution with pH values of 5.0, 6.0, 7.0, 8.0, 9.0, and ...
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