2School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-0170
* To whom correspondence should be addressed.
Received September 10, 1997
A new two-step procedure of protein refolding in vitro, proposed by Rozema and Gellman and named artificial chaperone-assisted refolding, is discussed. The new approach has been inspired by the two-step mechanism of the GroE system. In the first step, the protein is captured by a detergent under conditions that would normally lead to irreversible protein aggregation (heating or denaturant removal). In the second step, removal of detergent from the protein--detergent complex is triggered by addition of a cyclodextrin which is capable of forming inclusion complexes with detergent, allowing the protein to refold. The protein refolded with artificial chaperones (detergent and cyclodextrin) may be purified via a two-step protocol. After refolding was complete, the solution was passed through a 0.22-µm filter, to remove aggregated protein, and then through a M = 10 kD cutoff filter. The second filtration was intended to allow the low-molecular-weight artificial chaperones to pass, but to retain the refolded enzyme. The application of the above procedure for refolding of carbonic anhydrase B from human erythrocytes, hen egg white lysozyme, pig heart citrate synthase, and creatine kinase from rabbit skeletal muscles (MM isoenzyme) is discussed.
KEY WORDS: refolding, artificial chaperones, carbonic anhydrase B, lysozyme, citrate synthase, creatine kinase, detergents, cyclodextrins