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.

Structural and functional characterization of recombinant human growth hormone isolated from transgenic pig milk

“…However, because zinc ion induces hGH dimerization [41], this process could not be utilized in our study. Moreover, experiments using calcium phosphate particles (CAP) [42], citric acid [43], and sodium phosphate [44] were explored for casein removal from the milk of transgenic animals; however, casein was not completely removed. After several trials and various experiments, we established the pretreatment process outlined in Fig 2B. Nevertheless, there were substantial impurities remaining after the process, including rhGH fragments digested by tryptic peptidases [45][46][47].…”

The Promise and Challenges of Determining Recombinant Bovine Growth Hormone in Milk

“…This justifies that such strategy cannot be retained in the present case. As an alternative, a study reported the use of a phosphate buffer (1M; pH 6.0) to eliminate most of the caseins instead of using hydrochloric acid in the objective of extracting recombinant human factor IX (rhFIX) and recombinant hirudin (rH) in milk, which allowed a whey protein fraction containing more than 90% recovery for both recombinant hormones [72]. This approach could be tested to overcome the challenge associated to the extraction of rbGH from the micellar network formed by caseins.…”

Quantitative Estimation of Casein in Different Milk Samples

“…It is tasteless, odorless with isoelectric pH 4.6 [14,15]. In milk, calcium phosphate and casein form colloidal particles called casein micelles [7,16]. The richest profile of amino acids present in the casein shows that it is a valuable source of energy for the development of the brain and the strengthening of muscles and bones [17].…”