Operational regimes for a simplified one-step artificial chaperone refolding method.
The "artificial chaperone method" for protein refolding developed by Rozema et al. (Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117 (8), 2373-2374) involves the sequential dilution of denatured protein into a buffer containing detergent (cetyltrimethylammonium bromide, CTAB) and then into a refolding buffer containing cyclodextrin (CD). In this paper a simplified one-step artificial chaperone method is reported, whereby CTAB is added directly to the denatured solution, which is then diluted directly into a refolding buffer containing beta-cyclodextrin (beta-CD). This new method can be applied at high protein concentrations, resulting in smaller processing volumes and a more concentrated protein solution following refolding. The increase in achievable protein concentration results from the enhanced solubility of CTAB at elevated temperatures in concentrated denaturant. The refolding yields obtained for the new method were significantly higher than for control experiments lacking additives and were comparable to the yields obtained with the classical two-step approach. A study of the effect of beta-CD and CTAB concentrations on refolding yield suggested two operational regimes: slow stripping (beta-CD/CTAB approximately 1), most suited for higher protein concentrations, and fast stripping (beta-CD/CTAB approximately 2.7), best suited for lower protein concentrations. An increased chaotrope concentration resulted in higher refolding yields and an enlarged operational regime.
Lanckriet
Heikki
H
Department of Chemical Engineering, University of Cambridge, UK.
Middelberg
Anton P J
AP
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D002479:Inclusion Bodies / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011506:Proteins / Q000737:chemistry; D047392:beta-Cyclodextrins / Q000737:chemistry*
D002593:Cetrimonium Compounds; D018832:Molecular Chaperones; D011506:Proteins; D047392:beta-Cyclodextrins; D009113:Muramidase; C031215:betadex; D000077286:Cetrimonium