Complete refolding of bovine beta-lactoglobulin requires disulfide bond formation under strict conditions.
beta-Lactoglobulin (beta-LG) denatured with 6 M guanidine hydrochloride (GdnHCl) containing a reducing agent and subsequently dialysed against phosphate-buffered saline (PBS) resulted in incomplete refolding of this protein despite the fact that the biological activity for retinol-binding was recovered to almost the same degree as that of the native molecule [Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., Kaminogawa, S. J., Biol. Chem. 268 (1993) 22414-22419]. The enzyme probe method, evaluation of hydrophilicity values, in-gel mobility on SDS-PAGE, and evaluation of disulfide bonds with the Ellman method showed exposure of the hydrophobic region(s) and incorrect disulfide bond formation in such dialyzed beta-LG molecules. We reveal in this present work that complete refolding could be attained by diluting denatured beta-LG with PBS containing a reducing agent, before slow reoxidation of the sulfhydryl groups upon dialysis for gradient removal of the reducing agent in 6 steps. Complete renaturation was confirmed by analyzing the retinol-binding activity, CD spectra, intrinsic fluorescence, binding ability of monoclonal antibodies (mAbs), and SDS-PAGE. Step-by-step disulfide bond formation was considered to be critical for the complete refolding of denatured beta-LG. Our method can contribute to establish a procedure for complete refolding of useful recombinant proteins in vitro without such biological aids as chaperones.
Hattori
Makoto
M
Institute of Symbiotic Science and Technology, Division of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu-City, Tokyo 183-8509, Japan. makoto@cc.tuat.ac.jp
Hiramatsu
Kazuhiko
K
Kurata
Takashi
T
Nishiura
Mika
M
Takahashi
Koji
K
Ametani
Akio
A
Kaminogawa
Shuichi
S
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D019791:Guanidine; D007782:Lactoglobulins / Q000378:metabolism* / Q000502:physiology; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation / Q000502:physiology; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007782:Lactoglobulins; D004228:Dithionitrobenzoic Acid; D019791:Guanidine