Method for extracting a protein from milk

Abstract

The invention relates to a method for extracting a protein from milk, having at least one hydrophobic pocket and a negative charge to the natural pH of milk, that comprises the following steps: a) skimming and delipidation of the milk; b) passing the delipidated and skimmed fraction containing the protein on a chromatographic substrate on which is grafted a ligand having both a hydrophobic characteristic and an ionic characteristic in pH conditions enabling the protein to be retained on the substrate, the pH being higher than 4.6; c) elution of the protein; d) purification of the eluted fraction by removing the milk proteins from the eluted fraction; and e) recovering the protein.

What is claimed is: 
     
         1 . A process for the extraction of a protein present in milk, wherein said protein has at least one hydrophobic patch and a negative charge at the natural pH of milk, comprising:
 a) skimming and defatting said milk,   b) transferring said defatted and skimmed fraction containing said protein to a chromatographic support onto which is grafted a ligand that is simultaneously hydrophobic and ionic, under pH conditions allowing said protein to be captured on said support, said pH being higher than 4.6,   c) eluting said protein,   d) purifying said eluted fraction by removing milk proteins from said eluted fraction, and   e) recovering said protein.   
     
     
         2 . The process according to  claim 1 , wherein after said skimming and defatting step (step a) and prior to step b), a milk clarification step takes place. 
     
     
         3 . The process according to  claim 2 , wherein said clarification step takes place by adding a chelating agent at a concentration such that, after mixing with said milk, the micellar structure of said milk disappears, yielding clarified milk (caseins in solution or lactoserum). 
     
     
         4 . The process according to  claim 1 , wherein after said skimming and defatting step (step a) and prior to step b), casein subunit clusters are precipitated. 
     
     
         5 . The process according to  claim 1 , wherein said ligand that is simultaneously hydrophobic and ionic is 4-mercapto-ethyl-pyridine. 
     
     
         6 . The process according to  claim 1 , wherein step c) is performed using (a) a mixture of urea, concentration of which is between 1.2 and 8 M, and glycine, concentration of which is between 25 mM and 50 mM, (b) aqueous solutions of acidic pH comprised between 4 and 6, (c) aqueous mixtures comprising two or three components selected from the group consisting of sodium phosphate, final concentration of which is between 5 mM and 50 mM, and ethylene glycol; sodium citrate, final concentration of which is between 5 mM and 50 mM, and ethylene glycol; sodium phosphate, final concentration of which is between 5 mM and 50 mM, and ethylene glycol; TRIS/NaCl and a calcium salt, final concentration of which is between 1 mM and 10 mM, and ethylene glycol; and sodium caprylate, final concentration of which is between 10 mM and 100 mM, and ethylene glycol; (d) an aqueous mixture, the conductivity of which, linked to the presence of compounds, is lower than 3 mS/cm, or (e) water. 
     
     
         7 . The process according to  claim 1 , wherein step d) takes place through anion-exchange chromatography. 
     
     
         8 . The process according to  claim 7 , wherein after said anion-exchange chromatography step, said elution of said protein takes place using a 1 mM to 50 mM calcium ion solution. 
     
     
         9 . The process according to  claim 1 , wherein said protein is a recombinant protein. 
     
     
         10 . The process according to  claim 9 , wherein said recombinant protein is a coagulation protein. 
     
     
         11 . The process according to  claim 10 , wherein said coagulation protein is factor VII and/or activated FVII (factor VIIa). 
     
     
         12 . The process according to  claim 1 , wherein skimming and defatting is performed simultaneously with a glass filter having a positive zeta potential. 
     
     
         13 . The process according to  claim 1 , wherein skimming is performed with an organic solvent, wherein said organic solvent is trichloroacetic acid. 
     
     
         14 . A process for the extraction of a protein present in milk comprising:
 a) skimming and defatting milk with a glass filter having a positive zeta potential to yield a skimmed fraction comprising said protein;   b) binding said protein in said skimmed fraction on a support comprising a ligand; and   c) eluting said protein.   
     
     
         15 . The process according to  claim 14 , wherein said ligand is 4-mercapto-ethyl-pyridine or mercapto-benzimidazole sulfonic acid. 
     
     
         16 . The process according to  claim 14 , wherein after said step of eluting, said protein is purified by affinity chromatography, hydrophobic chromatography, cation-exchange chromatography, anion-exchange chromatography, or size-exclusion chromatography. 
     
     
         17 . The process according to  claim 14 , wherein said protein is selected from the group consisting of factor II, activated factor II, factor VII, activated factor VII, factor IX, activated factor IX, factor X, activated factor X, C protein, activated C protein, S protein, Z protein, and mixtures thereof. 
     
     
         18 . A process for the extraction of a protein present in milk comprising:
 a) transferring a plasmid comprising a promoter and a gene for a protein into an embryo of a non-human mammal;   b) introducing said embryo into a female mammal of the same species that will give birth to a transgenic mammal with said gene;   c) inducing lactation of said transgenic mammal to generate milk comprising said protein;   d) skimming and defatting said milk with a glass filter having a positive zeta potential to generate skimmed milk comprising said protein;   e) transferring said skimmed milk to a chromatographic support comprising a ligand that is simultaneously hydrophobic and ionic; and   f) eluting said protein from said chromatographic support.   
     
     
         19 . The process according to  claim 18 , wherein said promoter is selected from the group consisting of a whey acidic protein promoter, a casein promoter, and a β-lactoglobulin promoter. 
     
     
         20 . The process according to  claim 18 , further comprising fluidising said milk by adding an aqueous solvent to said milk prior to said step of skimming and defatting. 
     
     
         21 . A process for the extraction of a protein present in milk comprising:
 a) transferring milk containing said protein to a chromatographic support to which is bound a ligand that is simultaneously hydrophobic and ionic; and   b) eluting said protein from said chromatographic support.   
     
     
         22 . The process according to  claim 21 , further comprising skimming and defatting said milk prior to transferring said milk to said chromatographic support. 
     
     
         23 . The process according to  claim 21 , wherein eluting is performed by transferring a solution based on copper, zinc, or manganese salts over said chromatographic support.