REFOLDING OF SDS-CREATINE 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 art ificial chaperone-assisted refolding, has been extensively tested to r efold 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 w as used in this sequential refolding method. In the first step, the pr otein was denatured by SDS which is able to strongly inhibit aggregati on. 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, thi s study suggests that this procedure can be widely used to refold mono meric, as well as oligomeric, multidomain proteins. (C) 1996 Academic Press, Inc.