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Method for purifying and recombining glutamine transaminase
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A technology of glutamine and transaminase, applied in the field of purification, can solve the problems of low purity, low scope of application and low recovery rate of transglutaminase, and achieve the effect of reducing purification steps, increasing application scope and improving recovery rate
Inactive Publication Date: 2019-04-09
TAIXING YIMING BIOLOGICAL PRODS
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[0003] Most of the methods for purifying transglutaminase in the prior art are separated by ion exchange chromatography, which has a low recovery rate and the concentration of the enzyme will be diluted, resulting in low purity of transglutaminase and a low scope of application
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Embodiment 1
[0023] Embodiment 1, a method for purifying recombinant transglutaminase comprises the following steps;
[0024] Step 1. When recombining glutamyl transaminase, add a histidine tag to the C-terminus of the recombinant glutamyl transaminase;
[0025] Step 2, making the target protein transglutaminase bound to the filler in the nickel ion affinity column, the protein binding capacity of the filler is 42mg protein / ml filler;
[0026] Step 3, use solution A to wash the solution obtained in step 2 to the baseline balance, remove the non-target protein in the filler, the solution A is a balance solution, and its composition is 20mM, pH=7.4 KH2PO4-K2HPO4 buffer, 100mM NaCl, 20mM imidazole;
[0027] Step 4. Use B solution to elute the target protein of the solution obtained in step 3, and collect the eluate corresponding to the UV280 peak. The B solution is the eluent, and its composition is 20mM, pH=7.4 KH2PO4-K2HPO4 Buffer, 100mM NaCl, 300mM imidazole;
[0034] Embodiment 2, a method for purifying recombinant transglutaminase, comprising the following steps;
[0035] Step 1. When recombining glutamyl transaminase, add a histidine tag to the C-terminus of the recombinant glutamyl transaminase;
[0036] Step 2, making the target protein transglutaminase bound to the filler in the nickel ion affinity column, the protein binding capacity of the filler is 40mg protein / ml filler;
[0037] Step 3, use solution A to wash the solution obtained in step 2 to the baseline balance, remove the non-target protein in the filler, the solution A is a balance solution, its composition is 20mM, pH=7.4 KH2PO4-K2HPO4 buffer, 100mM NaCl, 20mM imidazole;
[0038] Step 4. Use B solution to elute the target protein of the solution obtained in step 3, and collect the eluate corresponding to the peak of UV280. The B solution is the eluent, and its composition is 20mM, pH=7.4 KH2PO4-K2HPO4 Buffer, 100mM NaCl, 300mM imidazole;
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Abstract
The invention relates to the technical field of purification, in particular to a method for purifying and recombining glutaminetransaminase. The method comprises following steps: step one, during recombining of glutaminetransaminase, a histidine tag is added at a C terminal of the recombined glutaminetransaminase; step two, a target proteinglutamine transaminase is combined on packing of a nickelion affinity column; step three, a solution obtained in step two is washed by a solution A until a baseline is balanced, and non-target protein in the packing is removed; step four, target proteinof a solution obtained in step three is eluted by a solution B; step five, sodiumchloride and imidazole contained in protein eluent obtained in step four are removed with a dialysis method. Glutamine transaminase activated in vitro is purified by a nickelion exchange column, for an eluting enzyme solution, saline ions are removed with the dialysis method, treatment conditions are mild, little protein loss is caused, recovery rate of glutamine transaminase can be increased, and purity of the recovered enzyme solution is improved.
Description
technical field [0001] The invention relates to the technical field of purification, in particular to a method for purifying recombinant glutamine transaminase. Background technique [0002] Transglutaminase, also known as transglutaminase (TGase), is a monomeric protein with an active center and a molecular weight of about 38,000 composed of 331 amino groups, which can catalyze intramolecular and intermolecular covalent crosslinking of protein polypeptides , so as to improve the structure and function of protein, and have significant effects on the properties of protein such as: foaming, emulsifying, emulsifying stability, thermal stability, water retention and gel ability, etc., thereby improving the flavor, taste, texture and quality of food Appearance, etc. Traditional meat processing technology usually adds a lot of salt and phosphoric acid to improve its water holding capacity, consistency and texture. Recently, foods with less salt and less phosphoric acid have been w...
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