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.
Couthon
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Laboratoire Biomembranes et Enzymes Associés, UPRESA 5013, Villeurbanne, France.
Clottes
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Vial
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C
D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000818:Animals; D001704:Biopolymers; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D003505:Cyclodextrins / Q000737:chemistry*; D004793:Enzyme Reactivators; D006358:Hot Temperature; D007527:Isoenzymes; D017510:Protein Folding; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D047392:beta-Cyclodextrins
D001704:Biopolymers; D003505:Cyclodextrins; D004793:Enzyme Reactivators; D007527:Isoenzymes; D047392:beta-Cyclodextrins; D000073738:2-Hydroxypropyl-beta-cyclodextrin; D012967:Sodium Dodecyl Sulfate; D003402:Creatine Kinase