Artificial chaperone-assisted refolding of proteins.

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-micro(m) 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.