Protein CaCl2 precipitation

Following denaturation, the whey proteins coprecipitate with the caseins on acidification to pH 4.6 or addition of CaCl2 at 90°C, to yield a range of products known as casein-whey protein co-precipitates (Figure 4.43). The main attraction of such products is an increase in yield of about 15%, but the products also have interesting functional properties. However, they have not been commercially successful. 

Proteins were precipitated with ammonium sulphate (30%-50% ), desalted and loaded on Octyl-Sepharose CL-4B column (11 x 1.3 cm) equilibrated with the 0.03 M MBS buffer pH 6.0 with 120mM CaC. Unbound proteins were eluted with 0.01 M MBS pH 6.0 with 50 mM CaCl2. The PLD activity was found in the fractions eluted with 0.2 mM BDTA. Active fractions were pooled, concentrated on Centricon 30 and applied on the native PAGB (8%) according to Wang (3). Finally the active PLD protein was electroeluted from polyacrylamide gel. 

Table 15.4 Crystallization conditions for DNA-protein complexes using other precipitants (NaCI, Na Formate, CFI3COONFI4, NFI4OFI, sodium citrate, CaCl2, CFl3C00Na, potassium phosphate. )... 

Addition of CaCl2 to about 0.2 M causes aggregation of the casein such that it can be readily removed by low-speed centrifugation. If calcium is added at 90°C, the casein forms coarse aggregates which precipitate readily. This principle is used in the commercial production of some casein co-precipitates in which the whey proteins, denatured on heating milk at 90°C for 10 min, co-precipitate with the casein. Such products have a very high ash content. 

Jensenius et al. (1981) reported two methods which circumvent many of the earlier problems for the isolation of IgY. The yolk, of which 50% is non-aqueous, is separated from the egg white and its membrane is cut open. The yolk (20 ml) is diluted with 80 ml TBS (10 mM Tris-HCl buffer, pH 7.4, and 140 mM NaCl), and may be stored for several months in the cold in the presence of 0.1% NaNj. Any precipitate formed is removed by centrifugation and 6.0 ml 10% (w/v) dextran sulphate (Pharmacia) in TBS is added, under mixing. Excess of dextran sulphate is precipitated with 15 ml 1 M CaCl2. The precipitate is extracted with 50 ml TBS to recover any protein carried with the precipitate. IgY is then recovered from the combined supernatants by the method given in Section... 

Fig. 1.50. Solubility of p-casein, partially dephospho-rylated by phosphoprotein phosphatase Precipitation pH 7.1 2.5 mg/ml protein 10 mmol/L CaCl2 35 °C 1 h. (according to Yoshikawa et al., 1974)...

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