Reconstituted micelle formation using reduced, carboxymethylated bovine kappa-casein and human beta-casein.

In milk, kappa-casein, a mixture of disulfide-bonded polymers, stabilizes and regulates the size of the unique colloidal complex of protein, Ca2+ and inorganic phosphate (Pi) termed the casein (CN) micelle. However, reduced, carboxymethylated bovine kappa-CN (RCM-kappa) forms fibrils at 37 degrees C and its micelle-forming ability is in question. Here, the doubly- and quadruply-phosphorylated human beta-CN forms and 1:1 (wt:wt) mixtures were combined with RCM-kappa at different beta/kappa weight ratios. Turbidity (OD(400 nm)) and a lack of precipitation up to 37 degrees C were used as an index of micelle formation. Studies were with 0, 5 and 10 mM Ca2+ and 4 and 8 mM Pi. The RCM-kappa does form concentration-dependent micelles. Also, beta-CN phosphorylation level influences micelle formation. Complexes were low-temperature reversible and RCM-kappa fibrils were seen. There appears to be equilibrium between fibrillar and soluble forms since the solution still stabilized after fibril removal. The RCM-kappa stabilized better than native bovine kappa-CN.

A folding pattern that is stable to thermal cycling is achieved by long term storage of recombinant human beta-casein with four extra N-terminals amino-acid residues at -20 degrees C.

Studies have followed the turbidity (OD400 nm) of beta-casein (CN) as temperature (T) increased from 4 to 37 degrees C. Native non-phosphorylated beta-CN showed a turbidity increase above 25 degrees C and precipitated at about 22 degrees C in 5mM Ca+2. These patterns were reproducible upon T-cycling while those of recombinant beta-CN proteins are not. Here, a wild-type recombinant that was thermally stable after being frozen in solution and stored at -20 degrees C for a prolonged period of time was denatured with guanidine HCl and refolded by dialysis against buffer. This protein was again not stable to T-cycling. A recombinant mutant with four extra N-terminal amino acids was very stable to T-cycling, both with and without 5mM Ca+2. However, it was still much different than the native protein. These results indicate that there are probably many energy minima for this protein and emphasize the possibility of "chaperon-like" conditions for proper folding of human beta-CN.