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

Studies have followed the turbidity (OD_{400 nm}) of β-casein (CN) as temperature (T) increased from 4 to 37 °C. Native non-phosphorylated β-CN showed a turbidity increase above 25 °C and precipitated at about 22 °C in 5 mM Ca⁺². These patterns were reproducible upon T-cycling while those of recombinant β-CN proteins are not. Here, a wild-type recombinant that was thermally stable after being frozen in solution and stored at -20 °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 5 mM 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 β-CN.