外文资料Artificial Chaperone-Assisted Refolding of Denatured-Reduced Lysozyme.pdfVIP

15760 Biochemistry 1996, 35, 1576015771 Artificial Chaperone-Assisted Refolding of Denatured-Reduced Lysozyme: Modulation of the Competition between Renaturation and Aggregation† David Rozema‡ and Samuel H. Gellman* Department of Chemistry, Uni ersity of Wisconsin, Madison, Wisconsin 53706 Recei ed July 5, 1996; Reised Manuscript Receied September 18, 1996 ABSTRACT : Conditions that promote renaturation of an unfolded protein also promote protein aggregation, in many cases, because these competing intramolecular and intermolecular processes are driven by similar networks of noncovalent interactions. The GroEL/GroES system and related biological chaperones facilitate the renaturation of substrate proteins by minimizing the aggregation pathway. We have devised a two- step method in which small molecules, “artificial chaperones,” facilitate protein refolding from a chemically denatured state. In the first step, the protein is captured by a detergent as guanidinium chloride is diluted to a non-denaturing concentration; formation of a proteindetergent complex prevents both protein aggregation and proper refolding. In the second step, a cyclodextrin strips detergent from the protein, allowing the protein to refold. Here we describe the first application of this method to a protein that must form disulfides in the native state. Lysozyme (hen egg white) can be refolded from the Gdm-denatured, DTT-reduced state in good yields at final protein concentrations as high as 1 mg/mL with the artificial chaperone method. Several mechanistic aspects of artificial chaperone-assisted refolding have been probed, and a detailed mechanism for the