Abstract
Polyethylene glycol (PEG) inhibited aggregation during refolding of bovine carbonic anhydrase B (CAB) through the formation of a nonassociating PEG-intermediate complex. Stoichiometric concentrations of PEG were required for complete recovery of active protein during refolding at aggregating conditions. For example, a PEG (Mr = 3350) to CAB molar ratio ([PEG]/[CAB]) of 2 was sufficient to inhibit aggregation during refolding at 1.0 mg/ml (33.3 microM) protein and 0.5 M guanidine hydrochloride. In addition, the PEG concentration required for enhancement was dependent upon the molecular weight and only molecular weights between 1000 and 8000 were effective in inhibiting aggregation. In the presence of PEG, the rate of refolding was the same as that observed for refolding without the formation of associated species. Refolding in the presence of PEG resulted in the rapid formation of a PEG complex with the molten globule first intermediate, and this PEG-intermediate complex did not aggregate. The CAB refolding kinetics in the presence of PEG were determined and used to develop a model of the PEG enhanced refolding pathway. The mathematical model was validated by independent activity measurements of CAB refolding. This model predicted that PEG enhanced refolding of CAB occurred by a specific interaction of PEG with the molten globule first intermediate to form a nonassociating complex which continued to fold at the same rate as the first intermediate. The predicted pathway and binding properties of PEG indicate that PEG enhanced refolding may be analogous to chaperonin mediated protein folding.
Highlights
From the Biotechnology Process Engineering Center, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139, $Pharmaceutical Research and Development, Genentech Inc., South San Francisco, California 94080, and the §Department of Biology, University of Maryland Baltimore County, Baltimore, Mabland 21228
Polyethylene glycol (PEG) enhanced refolding of carbonic anhydrase B (CAB) occurred by a specific interaction of PEG with themolten globulefirst intermediate to form a nonassociating complex which continued to foldat thesame rate as the firstintermediate
To determinethe effect of PEGonthe CAB refolding pathway, experimentswere first performed a t final conditions which resulted in the transientassociation of the first intermediate (1.0 M GdnHCland 0.50 mg/ml (16.7 p ~ C)AB)
Summary
In the presence of PEG, the rateof refolding was the same as that observed for refolding without intermediate and PEG has revealed that PEG binds weakly tothe firstintermediate at equilibrium in 2 M guanidine hydrochloride (GdnHCl) (Cleland and Randolph, 1992). These studiesalso showed that atleast one PEG binding site existed on the molten globule firstintermediate, butthe reaction stoichiometry for refolding conditions was not determined. PEG enhanced refolding of CAB occurred by a specific interaction of PEG with themolten globulefirst intermediate to form a nonassociating complex which continued to foldat thesame rate as the firstintermediate.
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