Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase.
Insoluble beta-cyclodextrin (beta-CD) copolymers have been used for the refolding of thermally and/or chemically denatured carbonic anhydrase with refolding yield of 40% using 300 mg of the copolymer/ml refolding solution containing 0.042 mg/ml protein. In an attempt to enhance the refolding yield with lower quantities of the copolymer, a new beta-CD-rich copolymer with higher beta-CD content was synthesized. Regarding the need for rapid stripping of the detergent molecules from the detergent-protein complexes formed in the capture step of the technique (artificial chaperone-assisted refolding), experimental variables (e.g. copolymer and the protein contents) were optimized to improve the refolding yields along with depressing the aggregate formation. In addition, comparative studies using different ionic detergents and the copolymer were conducted to get a more comprehensive understanding of the detergent's tail length in the stripping step of the process. Our results indicated that under the optimal developed refolding environment, the denatured CA was refolded with a yield of 75% using only 5mg of the copolymer/1.2 ml refolding solution containing 0.0286 mg/ml protein. Taking into account the recycling potential of the copolymer, the new resin, with significant cost-cutting capability, is a suitable candidate for industrial applications.
Yazdanparast
Razieh
R
Institute of Biochemistry and Biophysics, PO Box 13145-1384, The University of Tehran, Tehran, Iran. yazdan@ibb.ut.ac.ir
Khodarahmi
Reza
R
D000327:Adsorption / Q000187:drug effects; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology; D004811:Epichlorohydrin / Q000138:chemical synthesis / Q000737:chemistry*; D005453:Fluorescence; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012995:Solubility / Q000187:drug effects; D013055:Spectrophotometry, Infrared; D047392:beta-Cyclodextrins / Q000138:chemical synthesis / Q000737:chemistry*
D003902:Detergents; D011108:Polymers; D047392:beta-Cyclodextrins; C102558:cyclodextrin-epichlorohydrin polymer; D004811:Epichlorohydrin; D002256:Carbonic Anhydrases