Refolding of SDS- and thermally denatured MM-creatine kinase using cyclodextrins

We have tried to refold thermally-denatured MM-CK using detergent and cyclodextrins as protein folding assistants. This procedure, named artificial chaperone-assisted refolding, has been extensively tested to refold carbonic anhydrase B. Here, we describe a study which shows that this procedure can be applied to refold a dimeric multidomain protein : MM-creatine kinase. The pair SDS/hydroxy-propyl beta-cyclodextrin was used in this sequential refolding method. In the first step, the protein was denatured by SDS which is able to strongly inhibit aggregation. In the second step, hydroxy-propyl beta-cyclodextrin, an efficient SDS-stripping agent, is added and the denatured enzyme can regain its native structure as shown by the 75% reactivation. In conclusion, this study suggests that this procedure can be widely used to refold monomeric, as well as oligomeric, multidomain proteins.