Solid-phase Assisted Refolding of Carbonic Anhydrase Using β-Cyclodextrin-Polyurethane Polymer

Abstract

Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of denatured proteins. Independent refolding of several structurally diverse proteins by this approach has provided promising results regarding significant suppression of aggregation along with enhanced refolding yields. However, from the industrial point of view, some modifications seem to be essential for making the technique more efficient. In that regard and with a cost-cutting goal we designed, for the first time, a beta-cyclodextrin-polyurethane polymer to replace the soluble beta-cyclodextrin as the stripping agent for refolding of carbonic anhydrase. Our results indicated that under the optimally developed refolding environment, the denatured carbonic anhydrase was refolded with a yield of 75% using 15 mg/mL of the beta-cyclodextrin-polyurethane polymer, a yield near to stripping by soluble beta-CD. This new stripping approach seems to constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble beta-cyclodextrin. However, further-improvements in solid-phase artificial chaperone assisted technique are demanded either through synthesizing better stripping agents or by optimizing and defining better refolding environments.

Synthesis and characterization of β-cyclodextrin-conjugated magnetic nanoparticles and their uses as solid-phase artificial chaperones in refolding of carbonic anhydrase bovine

“…It was previously observed that the refolding yield decreases with an increase in the concentration of the protein to be refolded [23,25,44]. When refolding was performed at high protein concentrations, inactive aggregates formed by intermolecular interaction were the predominant products.…”

“…As shown in Fig. 11, at low CD-APES-MNPs concentrations, most of the released protein molecules remained in their nonnative states with exposed hydrophobic areas attracting the ANS molecules leading to high fluorescence emission intensities [25]. However, at higher appropriate concentrations of CD-APES-MNPs (40 mg/ml), more efficient refolding has taken place which is evident by a decrease in ANS fluorescence.…”

“…The decrease in refolding efficiency at higher than optimal concentrations of the CD-APESMNPs is possibly due to the fact that the solid-phase ACA approach faces difficulties due to mass transfer limitation (e.g., slow detergent diffusion to the immobilized CD) and/or the low substitution content of b-CD in the CD-bearing solid phase [23,30]. Alternatively, there is the possibility of an ''inappropriate'' removal rate of the detergent molecules from the detergent-protein complexes [25]. Under such conditions, the stripped and unfolded protein molecules will form intermolecular hydrophobic interactions which will finally lead to aggregation and/or misfolding.…”

“…This view could be evaluated through analyzing the microenvironment of the refolded and the partially refolded intermediates by using the ANS fluorescence probe [43]. Use of too much striping agent might affect the detergent-stripping rates which in turn will lead to the exposure of hydrophobic surfaces of the stripped and the unfolded intermediates which are quite suited for intermolecular interactions and aggregate formations [25]. The lower yield obtained using higher amounts of magnetic nanoparticles values might also possibly be due to nonspecific binding of the folded or refolded enzymes to these nanoparticles.…”

“…A few insoluble solid adsorbents, such as b-CD-epichlorohydrin copolymer [22][23][24], b-CD-acrylamide copolymer beads [24], b-CDpolyurethane polymer [25], b-CD-bonded silica particles [26], were previously used as host molecules for detergents in artificial chaperone-assisted (ACA) refolding of various denatured proteins. Although there were some previous works carried out on solidphase-assisted protein refolding, no or limited work on nanosized magnetic particle-assisted protein refolding has been published to the best of our knowledge.…”