title: A novel method for separation of caseins from milk by phosphates precipitation.
abstract: Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze-thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.
authors.affiliation: a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Miaoli , Taiwan , Republic of China.
authors.forename: Chon-Ho
authors.initials: CH
authors.lastname: Yen
authors.forename: Yin-Shen
authors.initials: YS
authors.lastname: Lin
authors.forename: Ching-Fu
authors.initials: CF
authors.lastname: Tu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001671:Biochemistry / Q000379:methods*; D002021:Buffers; D002364:Caseins / Q000302:isolation & purification*; D011232:Chemical Precipitation; D005164:Factor IX / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D006629:Hirudins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine / Q000235:genetics; D000067816:Whey Proteins
chemical_list: D002021:Buffers; D002364:Caseins; D006629:Hirudins; D008894:Milk Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D000067816:Whey Proteins; D005164:Factor IX
keywords: casein; chromatography; phosphate; precipitation; purification; transgenic pigs