Comparison of native and recombinant non-phosphorylated human β-casein: further evidence for a unique β-casein folding pattern

Recombinant wild-type non-phosphorylated human β-casein was obtained from Escherichia coli. Turbidity vs. temperature (T) without Ca2+ showed wild-type self-association like native except for irreversibility upon T-cycling with the original pattern re-established after concentrated urea/dialysis. With Ca2+, wild-type was more native-like. Intrinsic Trp fluorescence spectra were similar but with lowered intensity for the wild-type protein. Changes in extrinsic ANS fluorescence from 4 to 37 °C showed less exposure of hydrophobic surface for wild-type than native. Trp to ANS fluorescence resonance energy transfer was higher for wild-type than native at 4 °C but 2- to 3-fold lower at 37 °C. The native protein must be directed by the environment and/or a chaperone to fold into a unique, somewhat flexible, conformation, unaltered by urea during purification. Wild-type protein, with many native properties, does not spontaneously fold to the native conformation, even after solubilization with urea. T-cycling gives a stable conformation that is different from the native.