Artificial chaperone-assisted refolding of chemically denatured alpha-amylase.
It is now well established that alpha-cyclodextrin (alpha-CD) is a valuable folding agent in refolding processes of several denatured enzyme solutions. The refolding of Gu-HCl denatured alpha-amylase in the dilution-additive mode revealed that alpha-CD enhanced the refolding yield by 20-30% depending upon alpha-CD concentration. However, the refolding efficiency of the Gu-HCl denatured alpha-amylase through the artificial chaperone-assisted method indicated that alpha-CD enhanced the activity recovery of denatured alpha-amylase by almost 50% and also increased the reactivation rate constant relative to the unassisted control sample. The higher refolding efficiency should be due to different mechanism played by alpha-CD in this technique. In addition, our data indicated that higher refolding yields are obtained when the residual Gu-HCl concentration is low in the refolding environment and when the capture agent is removed not in a stepwise manner from the protein-detergent complexes in the stripping step of the whole process. Collectively, the results of this investigation expand the range of procedural variations used to refold different denatured proteins through artificial chaperone-assisted method.
Yazdanparast
Razieh
R
Institute of Biochemistry and Biophysics, P.O. Box 13145-1384, the University of Tehran, Tehran, Iran. yazdan@ibb.ut.ac.ir
Khodagholi
Fariba
F
Khodarahmi
Reza
R
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D002942:Circular Dichroism; D003902:Detergents; D004793:Enzyme Reactivators / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D000516:alpha-Amylases / Q000737:chemistry*; D047391:alpha-Cyclodextrins / Q000737:chemistry*
D002593:Cetrimonium Compounds; D003902:Detergents; D004793:Enzyme Reactivators; D018832:Molecular Chaperones; D047391:alpha-Cyclodextrins; D000516:alpha-Amylases; D019791:Guanidine; C032613:alpha-cyclodextrin; D000077286:Cetrimonium