| 摘要 |
#CMT# #/CMT# Extraction of protein having at least one hydrophobic pocket and a negative charge at the natural pH of the milk, present in milk comprises skimming and defatting the milk, passing the defatted and skimmed fraction containing the protein on a chromatography medium on which a ligand having both hydrophobic and ionic character is grafted under pH conditions that retains the protein on the medium, eluting the protein, purifying the eluted fraction by removing the milk proteins from the eluted fraction and recovering the protein. #CMT# : #/CMT# An independent claim is included for the use of a positive zeta potential filter glass for simultaneously skimming and defatting the milk. #CMT#USE : #/CMT# The method is useful to extract proteins present in milk (claimed), where the milk is fresh or frozen obtained from a transgenic mammal e.g. sheep, goats, rabbits (preferred), mice, rats or sow. #CMT#ADVANTAGE : #/CMT# The method is easy to implement as it contains only few steps and does not necessarily need to clarify the milk before starting the extraction process. #CMT#ORGANIC CHEMISTRY : #/CMT# Preferred Process: The process further comprises clarifying the milk after the skimming and defatting step and before the step of passing the fraction on a chromatographic medium. The clarification of the milk is carried out by adding a chelating agent at a concentration after mixing the agent with the milk and the micelle structure of the milk disappears to give clarified milk (casein as solution or whey). The method further comprises precipitating the aggregates of subunits of the casein after the skimming and defatting step and before the step of passing the fraction on a chromatographic medium. The step of purification of the fraction is carried out by anion exchange chromatography, where the elution of the protein is carried out by using a solution of calcium ions at 5 mM. Preferred Components: The ligand is 4-mercapto-ethyl-pyridine. The protein is a recombinant protein, a coagulated protein, Factor VII or activated Factor VII (Factor VIIa). #CMT#EXAMPLE : #/CMT# A sequence of BamH1-Hind III (fragment 6.3 Kb) gene WAP was introduced between the sequences of Bam H1 and Hind III to obtain a plasmid p1. During cloning, the Bam H1 site was deleted and replaced by Cla I contained in the vector p1, which capable of receiving a foreign gene depending on a promoter WAP 6.3 Kb. The introduction of foreign gene can be done by Sal I poly linker. The inserts containing the promoter and foreign genes can be isolated from plasmid after cutting the two Not 1 sites present at the ends of the poly linker of plasmid p-poly III-I. A plasmid p2, obtained from plasmid pi, contains the gene promoter of the rabbit WAP (6.3 Kb) and the human gene FVII. The fragment used to obtain transgenic rabbits was introduced between the two Not 1 sites. A site Hind III was introduced in the sequence head of the gene (leader) by mutagenesis that serves as a site of cloning. The transgenic rabbits were obtained by the microinjection technique. The non-skimmed and frozen milk containing protein (150 g/l) and fat (15% cream) obtained from the transgenic rabbits was liquefied and skimmed by: mixing a volume of the milk with 2 volumes of aqueous solvent and filtering the mixture using a positive zeta potential glass fiber filter, Ultipor GF plus (RTM: Filter) to obtain a perfectly liquid and defatted raw material suitable for chromatography, where the solvent was aqueous phosphate solution with low ionic strength (less than 100 mM) with or without the addition of sodium chloride or the solvent contains a chelating agent (e.g. trisodium citrate or ethylene diamine tetraacetic acid) in a concentration, which after mixing with milk, that causes disappearance of the micelle structure of the milk to obtain clarified milk (casein as solution or whey) (e.g. a final concentration of 0.253 M sodium citrate and pH 8.0 provides the perfect clarification of milk). The skimmed and defatted fraction (4 ml) was subjected to affinity chromatography, where the Factor VII-transgenic protein in the milk was stabilized on gel (2 ml), MEP-Hypercel (RTM: Sorbent). The elution step was carried out by using ethylene glycol as solvent with 30 mM phosphate buffer at pH 8 to obtain the Factor VII protein. |
