Artificial chaperone-assisted refolding of citrate synthase.
The power of genetic engineering methods, along with increasing genomic information, makes heterologous expression of proteins an extremely important biochemical tool. Unfortunately, proteins obtained in this way often are not in their native form, and folding becomes a crucial step in protein production. We have recently developed a strategy that promotes the folding of chemically denatured proteins via the sequential addition of low molecular weight "artificial chaperones." Here we describe in detail the application of this method to porcine heart citrate synthase. Refolding yields of as high as 65% have been achieved. Mechanistic studies indicate that there are significant differences between artificial chaperone-assisted refolding of citrate synthase and artificial chaperone-assisted refolding of two other proteins that have been examined, carbonic anhydrase B (Rozema, D., and Gellman, S. H. (1996) J. Biol. Chem. 271, 3478-3487) and lysozyme (Rozema, D., and Gellman, S. H. (1996) Biochemistry 35, 15760-15771). The differences among these three test proteins reveal the range of procedural variation that must be considered in the application of the artificial chaperone method to new proteins.
Daugherty
D L
DL
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Rozema
D
D
Hanson
P E
PE
Gellman
S H
SH
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000378:metabolism; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology*; D002942:Circular Dichroism; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D018832:Molecular Chaperones / Q000502:physiology*; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013552:Swine; D047392:beta-Cyclodextrins
D002593:Cetrimonium Compounds; D003505:Cyclodextrins; D003902:Detergents; D018832:Molecular Chaperones; D047392:beta-Cyclodextrins; D002950:Citrate (si)-Synthase; D009113:Muramidase; D002256:Carbonic Anhydrases; D019791:Guanidine; C031215:betadex; D000077286:Cetrimonium