A kind of purification method of whey protein

Abstract

The invention provides a purification method for whey protein. The purification method comprises: taking mammal milk as a raw material, performing pretreatment to obtain whey, and performing hydrophobic chromatographic separation, so as to obtain purified alpha-whey alhumin and beta-lactoglobulin. The invention also provides a method for obtaining purified recombinant human alpha-whey alhumin and animal endogenous alpha-whey alhumin and beta-lactoglobulin. The method comprises: when whey protein contains recombinant human alpha-whey alhumin, firstly performing anion exchange chromatography before hydrophobic chromatographic separation, so as to separate recombinant human alpha-whey alhumin from endogenous alpha-whey alhumin and beta-lactoglobulin, and then performing hydrophobic chromatographic separation, so as to obtain purified recombinant human alpha-whey alhumin and animal endogenous alpha-whey alhumin and beta-lactoglobulin.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying recombinant human α-lactalbumin from transgenic milk, and can also purify animal endogenous α-lactalbumin and β-lactoglobulin. Background technique [0002] α-lactalbumin (LA) is an important functional protein specifically expressed in the mammary glands of most mammals. In addition to meeting the essential amino acids required for the growth and development of young animals, it also has important physiological functions such as inhibiting tumor growth and improving sleep. Function. α-Lactalbumin has a high content of essential amino acids, especially tryptophan, which is involved in nervous system activities such as mood regulation. In addition, α-lactalbumin can stimulate the metabolism of mucous membranes and effectively prevent gastrointestinal infections in infants and young children. Significantly, α-lactalbumin can act on tumor cells, cause apoptosis ...

AI Technical Summary

Problems solved by technology

[0005] At present, the methods for separation and purification of α-lactalbumin and β-lactoglobulin mainly use chromatographic separation, membrane separation and salt precipitation, but the principle of membrane separation is based on the molecular weight of the protein. Coarse separation, thus resulting in a decrease in the purity of human α-lactalbumin and a corresponding decrease in the recovery rate; and precipitation of the protein by salts will lead to denaturation of the protein and a decrease in the yield; separation methods using chromatography mainly include hydrophobic interaction chromatography , ion exchange chromatography, gel filtration chromatography, etc.

Among them, ion exchange chromatography is the most commonly used chromatographic technology, which has low cost, acid and alkali resistance, large sample volume and is easy to scale up for industrial production; gel filtration chromatography is generally located in the fine purification stage of the protein purification process, and has high resolution. , high protein purity, etc., but due to the high cost of this chromatography technology, it is not suitable for industrial scale-up production; hydrophobic interaction chromatography needs to be equipped with a high-concentration salt solution to dilute the protein solution, so protein precipitation may occur. The loss of protein, but due to the different physicochemical properties of different proteins, the ability to tolerate high concentration salt solution is also different

[0006] It is found in relevant patent literatures related to the separation and purification of α-lactalbumin and β-lactoglobulin from milk and large-scale preparation. Heating to 60°C-95°C for 10min-60min and then cooling pretreatment to obtain serum rich in β-lactoglobulin and α-lactalbumin, and then separated by anion chromatography to obtain human α-lactalbumin, bovine α- Whey albumin and bovine β-lactoglobulin, because this method adds inorganic compounds (calcium chloride) in the pretreatment stage and heats the milk sample for a long time, it is easy to cause denaturation of whey protein and loss of activity, so it is difficult to apply in industrial production

Chinese patent application number 200910072780.X discloses a method for preparing whey protein powder containing low β-lactoglobulin. This method has cumbersome steps and low yield, and is difficult to apply to large-scale production

U.S. Patent US6312755 describes the separation of α-lactalbumin by treating whey at an acidic pH value, and this method is not suitable for large-scale production

U.S. Patent No. 5,008,376 describes the separation of α-lactalbumin by membrane separation technology with different pore sizes. First, an ultrafiltration membrane with a pore size of 100 kD is selected to pass the protein solution rich in α-lactalbumin through the membrane, and then a small pore size of 10 kD is selected. The ultrafiltration membrane enriches α-lactalbumin, but the purity of α-lactalbumin obtained by this method is low

[0007] In order to develop products with α-lactalbumin and β-lactoglobulin as the main functional components, it is necessary to extract recombinant human α-lactalbumin, animal endogenous α-lactalbumin and β-lactoglobulin from the milk of transgenic animals, respectively. Isolation and purification, but because the molecular characteristics of bovine α-lactalbumin and human α-lactalbumin are very similar, for example, bovine α-lactalbumin has 76% amino acid sequence homology and 88% structural similarity The molecular weights are 14,078Da and 14,186Da respectively, the isoelectric point pI4.2–4.6, and milk contains a large amount of bovine endogenous protein, which makes it difficult to purify recombinant human α-lactalbumin from milk, and according to The above methods have certain limitations, such as low yield, low purity, high cost and cannot form large-scale production

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