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.
Sood
Satish M
SM
Department of Biochemistry and Microbiology, Biochemistry Division School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA. ssood@llu.edu
Booth
Cassie
C
Jhawar
Harbir
H
Slattery
Charles W
CW
D000596:Amino Acids / Q000737:chemistry*; D002364:Caseins / Q000737:chemistry* / Q000235:genetics; D004355:Drug Stability; D005615:Freezing; D006801:Humans; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D000596:Amino Acids; D002364:Caseins; D011994:Recombinant Proteins