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Purifying method of shrimp tropomyosin

A technology of tropomyosin and purification methods, which is applied in the direction of animal/human peptides, peptide sources, peptides, etc., can solve the problems of difficult sample recovery, expensive consumables, and low sample purity, which is conducive to large-scale production and reduces production The effect of high cost and protein purity

Inactive Publication Date: 2015-01-07
TIANJIN UNIV OF COMMERCE
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0008] 1. The purity of TM protein extracted and separated from animal organisms is not high, and the purity of samples obtained by molecular sieve, ion exchange chromatography or HPLC is less than 95% by using the existing purification process route;
[0009] 2. In the existing purification process, the equipment used in the HPLC purification technology is expensive, the consumables are expensive (whether it is the purification column material or the elution solvent), the preparation scale is small, and the sample is not easy to recover; the target protein of the ion exchange chromatography exists in the washing During peak removal, the purity of the obtained sample is not high, and molecular sieve chromatography is needed to increase the purity to more than 90%, but the processing capacity of this purification process is not high. Simple, because the processing volume is too small, it is not conducive to large-scale processing of purified samples

Method used

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  • Purifying method of shrimp tropomyosin
  • Purifying method of shrimp tropomyosin
  • Purifying method of shrimp tropomyosin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Preparation of crude shrimp tropomyosin:

[0032] ①Take 100g of Litopenaeus vannamei with heads, shells and guts removed, add 1000mL of 0.9% sodium chloride solution, and homogenate; 7.5. In Tris-HCl buffer solution with a concentration of 20mmol / L, wherein the Tris-HCl buffer solution with a concentration of 20mmol / L contains 50mmol / L KCl. After centrifugation at 4000×g, the supernatant was discarded and the precipitate was collected. The precipitate was redissolved in Tris-HCl buffer solution with a pH value of 7.5 and a concentration of 20 mmol / L, and the precipitate was collected by centrifugation, and repeated 3 times.

[0033] ②Add 400 times the volume of cold acetone 200mL pre-cooled at -20°C overnight to the above precipitate, seal the seal, mix thoroughly with a magnetic stirrer at 0°C for 30min, centrifuge at 4000×g for 10min, collect the precipitate, and repeat After the "mixing-centrifugation" step twice, the precipitate was transferred to a clean filt...

Embodiment 2

[0044] (1) Preparation of crude shrimp tropomyosin:

[0045] ①Take 100g of Litopenaeus vannamei with heads, shells and guts removed, add 1000mL of 0.9% sodium chloride solution, homogenate, add 1000mL at a concentration of 20mmol / L, 1:10 (m / v), pH value is 7.5 in Tris-HCl buffer solution containing 50mmol / L KCl, centrifuged at 4000×g, discard the supernatant, redissolve the precipitate in the above buffer solution, collect the precipitate by centrifugation, repeat 3 times.

[0046] ②Add 400 times the volume of cold acetone 200mL pre-cooled at -20°C overnight to the above precipitate, seal the seal, mix thoroughly with a magnetic stirrer at 0°C for 30min, centrifuge at 4000×g for 10min, collect the precipitate, and repeat After the steps of "mixing-centrifugation" twice, the precipitate was transferred to a clean filter paper, and air-dried at room temperature to obtain shrimp fibrin acetone powder, about 10 g.

[0047] ③Take 5 g of shrimp fibrin acetone powder, add 75 mL of d...

Embodiment 3

[0056] (1) Preparation of crude shrimp tropomyosin:

[0057] ①Take 100g of Litopenaeus vannamei with head, shell and gut removed, add 1000mL of sodium chloride solution with a concentration of 0.9%, homogenate, add 1000mL with a concentration of 20mmol / v at 1:10(m / v) L. The pH value is 7.5 in Tris-HCl buffer solution containing 50mmol / L KCl. Centrifuge at 4000×g, discard the supernatant, redissolve the precipitate in the above buffer solution, collect the precipitate by centrifugation, and repeat 3 times.

[0058] ②Add 400 times the volume of cold acetone 200mL pre-cooled at -20°C overnight to the above precipitation, seal the seal, and mix thoroughly with a magnetic stirrer at 0°C for 30min. Centrifuge at 4000×g for 10 min to collect the precipitate, repeat the “mixing-centrifugation” step twice, then transfer the precipitate to a clean filter paper, and air-dry at room temperature to obtain shrimp fibrin acetone powder, about There are 10g.

[0059] ③Take 5 g of shrimp fib...

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Abstract

The invention discloses a purifying method of shrimp tropomyosin and particularly discloses a purifying method which is high in purity and beneficial to large-scale production. The purifying method comprises the following steps: dissolving a shrimp tropomyosin crude product into equilibration buffer with a pH value of 3.2-3.6 to obtain a sample liquid; adding the sample liquid into an anionic chromatography column, eluting by use of equilibration buffer with the pH value of 3.2-3.6, collecting the liquid corresponding to a column penetrating peak; regulating the pH value of the liquid corresponding to the column penetrating peak to 5.8-6.0, adding the liquid into a cationic hromatography column, eluting by use of equilibration buffer with the pH value of 5.8-6.0, collecting the liquid corresponding to the column penetrating peak, performing dialysis and desalination, and drying to obtain the high-purity shrimp tropomyosin. According to the purifying method disclosed by the invention, a target protein exists in the column penetrating column by combining anion exchange chromatography with cation exchange chromatography, so that the activity of the protein can be kept very well, and the purity of the obtained protein is high; by virtue of two-step ion-exchange column chromatography, the purifying method is great in treatment capacity, simple to operate and beneficial to large-scale production.

Description

technical field [0001] The invention relates to the technical field of protein extraction, in particular to a method for purifying shrimp tropomyosin. Background technique [0002] 30%-40% of the people in the world suffer from food allergies, among which the allergies caused by crustaceans such as shrimps and crabs and their products are particularly prominent. Tropomyosin (TM) in the muscle tissue of these animals is the most important and most sensitizing allergen protein. Preparation of TM protein products with higher purity is of great significance for studying the structure and allergenicity of the protein, and how to reduce its allergenicity. at the same time. High-purity TM protein also has very important application value for the detection and detection of allergens and as a standard protein. [0003] The current process of isolating and purifying TM protein from shrimp is generally divided into three stages: [0004] The first stage is to prepare dry shrimp fib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/18
CPCC07K14/43509
Inventor 吴子健薛璐胡志和李洋
Owner TIANJIN UNIV OF COMMERCE
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