Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis

Abstract

Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.

Control of aggregation in protein refolding: A variety of surfactants promote renaturation of carbonic anhydrase II

“…Our results can usefully be discussed in terms of the reaction scheme shown in Figure 8, based on the work of Cleland et al (1992) and several other laboratories (Wong & Tanford, 1973;Ikai et al, 1978;Stein & Henkens, 1978;Dolgikh et al, 1984;Cleland & Wang, 1990a;Semisotnov et al, 1990). Although some of our results are not fully consistent with this scheme, it provides a very useful reference framework.…”

“…2A) show clearly that, within the dead time of mixing, the maximum apparent turbidity is reached, and that this decays monotonically over the time course of the experiment. This result is somewhat surprising in light of light-scattering results (Cleland & Wang, 1990a), which show large particles increasing with time-albeit at much lower protein and denaturant concentrations. Putting aside for the moment the results with n-hexanol additive, we see -30% decrease (initial to final observation time) in the turbidity at 4 mg/mL CAII, and about the same percentage decrease for the 2-mg/mL solution.…”

“…In this study we employ the extensively studied refolding of carbonic anhydrase (CAII) in dilute guanidinium chloride (GdmC1) solutions (Yazgan & Henkens, 1972;Wong & Tanford, 1973;Ikai et al, 1978;Cleland & Wang 1990a, 1990bSemisotnov et al, 1990;Cleland et al, 1992).' These studies have shown the existence of two intermediates between denatured and native protein.…”

Peracetylated Bovine Carbonic Anhydrase (BCA-Ac18) Is Kinetically More Stable than Native BCA to Sodium Dodecyl Sulfate

“…It aggregates during renaturation when denatured with heat 26 or acid, 27 or when incubated in intermediate concentrations (1-3 M) of guanidinium chloride (GuHCl). [28][29][30] For example, Hammarström et al observed low (<10%) recovery of activity of human carbonic anhydrase (HCAII) when it was renatured by dilution from 2 to 0.3 M GuHCl, but high (>80%) recovery of activity when diluted from 6 M GuHCl to 0.3 M. 30 McCoy et al 26 also observed aggregation of BCA in refolding of the protein, denatured in a solution of 0.1% SDS and renatured with dialysis. 31 We believe that irreversible aggregation interferes with refolding of BCA (and BCA-Ac 18 ) in intermediate concentrations of SDS and prevents equilibration (eq 1).…”

Refolding of recombinant porcine growth hormone in a reducing environment limits in vitro aggregate formation

“…Various refolding protocols of differing complexity have been attempted to overcome these difficulties [3,[5][6][7]. However, there appear to be no simple generally applicable means of obtaining high yields of recombinant biologically active disulphide bonded proteins.…”