Molecularly imprinted poly β-cyclodextrin polymer: Application in protein refolding

Regarding our previous report on refolding of alkaline phosphatase [Yazdanparast and Khodagholi, 2005 Arch. Biochem. Biophys] it was found that in spite of the anti-aggregatory effect of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitteronic detergent, the recovered activity was almost the same as the recovered activity obtained through the unassisted approach. The low recovery yield is probably due to the bulky groups of the detergent that interfere with its entrance into the small cavity of the stripping agent, cyclodextrin, implying that the stripping of detergent molecules from the detergent–protein complexes plays a major role in successful refolding processes. To improve the efficiency of CHAPS stripping, we evaluated, for the first time, the stripping potential of a molecular imprinting polymer designed to replace β-CD. In this approach, CHAPS was used as the template and the refolding of GuHCl denatured alkaline phosphatase was studied. Our results indicated that under the optimally developed refolding environment and similar to stripping by soluble β-CD, a refolding yield of 79% was obtained for denatured alkaline phosphatase using 20 mg/ml of the molecularly imprinted poly (β-CD) polymer. The major advantage of the new stripping agent, besides of its recycling option and ease of separation from the finished product, is its high potential of preventing aggregate formation. Based on these results, it seems that the new stripping strategy can constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble β-cyclodextrin.     

Beta-cyclodextrin-bonded silica assists alkaline phosphatase and carbonic anhydrase refolding in a solid phase assisted refolding approach

The processes of protein refolding by artificial chaperones suffer from tedious steps of purifications which will finally affect the production costs. Replacement of the soluble stripping agent with immobilized beta-cyclodextrin or beta-cyclodextrin polymer beads might elevate some of these problems. Regarding this fact, we synthesized and evaluated various cyclodextrin-bonded silica particles to evaluate the refolding yields of denatured alkaline phosphatase and carbonic anhydrase. Our results indicated that refolding of denatured alkaline phosphatase raised from 30%, in the absence of chaperone, to about 65% in the presence of 70 mg/ml of the beta-cyclodextrin-bonded silica gel and to 74% in the concomitant presence of the new stripping agent and MgSO_4, a yield near to stripping by soluble beta-cyclodextrin. The refolding yield of carbonic anhydrase in the presence of beta-CD-bounded silica gel resin was significantly lower than the value obtained in the presence of soluble beta-CD (76% vs 54%). These data indicate that refolding of proteins by the silica gel immobilized beta-CD resin can be achieved though with lower yields. Regarding the high cost of downstream purification steps associated with soluble beta-CD, application of insoluble stripping agent might provide an alternative approach to cut down the industrial costs.     

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

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.     

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.     

Protein refolding assisted by molecular tube based α-cyclodextrin as an artificial chaperone

In this study, we evaluated, for the first time, the application of molecular tube based alpha-cyclodextrin for improving the refolding yields of two different enzymes: carbonic anhydrase and alkaline phosphatase. Our results indicate that under the optimal developed refolding environments, the denatured carbonic anhydrase and alkaline phosphatase were refolded with a yield of 51 and 61% using 15 and 5 mg/ml of the molecular tube, respectively. Regardless of lower refolding yields compared with liquid-phase artificial chaperone assisted approach, the new technique (solid-phase artificial chaperone assisted refolding) benefits from easier and faster separation of the refolded product from the refolding environment, recycling of the stripping agent, and finally, significantly less environmental effect at the industrial levels. However, further improvements in solid-phase artificial chaperone assisted technique are needed either through synthesizing better stripping agents or by optimizing and defining better refolding environments.     

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.     

Alkaline phosphatase refolding assisted by sequential use of oppositely charged detergents: a new artificial chaperone system

A novel artificial chaperone system, based on combination of oppositely charged detergents, was elaborated to refold soluble alkaline phosphatase. Upon dilution of urea-denatured alkaline phosphatase to a nondenaturing urea concentration in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used detergents with variable charges and tail lengths to initiate and complete the refolding process. The results obtained from various analyses (fluorescence, UV, circular dichroism, surface tension, turbidity measurements and activity assays) indicated that the extent of refolding assistance was different due to detergents structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic interactions among the capturing and stripping detergents used in this investigation. Collectively it is expected that protein refolding process can be achieved easier, cheaper and more efficient, using the new technique reported here.     

The glycophosphatidylinositol anchor oppositely affects unfolding and refolding of alkaline phosphatase

Regarding the world wide success of artificial chaperone-assisted protein refolding technique and based on its well worked-out mechanism, it is anticipated that the lipid moieties of glycosylphosphatidylinositol (GPI) group, which is present in some membrane proteins, might interfere with the capturing step of the technique. To find an answer, we evaluated the chemical denaturation and also the refolding behavior of insoluble and soluble alkaline phosohatase (ALP), with or without GPI group, respectively. The results indicated that the presence of GPI in the enzyme increased the stability of the protein against chemical denaturation while it decreased its refolding yield by the artificial chaperone refolding technique. The lower refolding yield, compared to soluble ALP (sALP), might be due to a less efficient stripping step caused by new interactions imparted to the refolding elements of the system especially those among the hydrophobic tails of GPI and the capturing agent of the technique. These new interactions will interrupt the kinetics of detergent stripping from the captured molecules by the stripping agent (i.e., cyclodextrins). This situation will lead to higher intermolecular hydrophobic interactions among the refolding protein intermediates leading to their higher misfolding and aggregation.     

Comparative studies of the artificial chaperone-assisted refolding of thermally denatured bovine carbonic anhydrase using different capturing ionic detergents and beta-cyclodextrin

Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of some of the denatured proteins. Since identical concentrations of various detergents do not induce similar variations in the protein structures, we arranged to evaluate the artificial chaperoning capabilities of several ionic detergents as a function of charge, structure, and the hydrophobic tail length of the detergent. Our results indicate that carbonic anhydrase can be refolded from its denatured state via artificial chaperone strategy using both anionic and cationic detergents. However, the extent of refolding assistance (kinetic and refolding yield) were different due to protein and detergent net charges, detergent concentrations, and the length of hydrophobic portion of each detergent. These observed differences were attributed to physical properties of CA-detergent complexes and/or to the kinetics of detergent stripping by beta-cyclodextrin from the protein-detergent complexes which is apparently dependent on the detergent-beta-CD association constants and the nature of the partially stripped complexes.     

Evaluation of Chaperone-like Activity of Alginate: Microcapsule and Water-soluble Forms

To evaluate the chaperone-like activity of alginate stabilization and refolding of alkaline phosphatase (ALP) was investigated in the presence of alginate through two different approaches, the soluble form and microcapsule assisted methods. It was found that in the presence of microcapsules, ALP can be stabilized to a higher degree compared with the water-soluble form, whereas the denatured ALP is refolded with a higher yield through latter method. Lower refolding yields of alginate beads compared with its soluble form may be the result of lower refolding rate of ALP upon elution of the bound enzyme by dispersing the precipitate in NaCl which left the unfolded protein in an unsuitable environment, providing enough time for protein aggregation and leading finally to lower recovered activity compared with application of soluble form of alginate. In addition in the case of alginate capsules, the choice of suitable divalent ion is essential for stability and assistance in refolding.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

The role of detergent in refolding of GdnHCl-denatured arginine kinase from shrimp Fenneropenaeus Chinensis: the solubilization of aggregate and refolding in detergent solutions.

Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.     

A New Artificial Chaperone for Protein Refolding: Sequential Use of Detergent and Alginate

A novel artificial chaperone system using a combination of detergents and alginate was developed to refold three enzymes with totally different structures. Upon dilution of denatured protein in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used alginate, a linear copolymer of d-mannuronic acid and l-guluronic acid, to initiate and complete the refolding process. The results indicated that the extent of refolding assistance for the proteins was different due to detergent structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic and hydrophobic interactions among the capturing and stripping agents used in this investigation. Based on this newly developed method, it is expected that the protein refolding operation can be achieved easily, cheaply and efficiently.     

Artificial chaperone-assisted refolding of chemically denatured alpha-amylase

It is now well established that alpha-cyclodextrin (alpha-CD) is a valuable folding agent in refolding processes of several denatured enzyme solutions. The refolding of Gu-HCl denatured alpha-amylase in the dilution-additive mode revealed that alpha-CD enhanced the refolding yield by 20-30% depending upon alpha-CD concentration. However, the refolding efficiency of the Gu-HCl denatured alpha-amylase through the artificial chaperone-assisted method indicated that alpha-CD enhanced the activity recovery of denatured alpha-amylase by almost 50% and also increased the reactivation rate constant relative to the unassisted control sample. The higher refolding efficiency should be due to different mechanism played by alpha-CD in this technique. In addition, our data indicated that higher refolding yields are obtained when the residual Gu-HCl concentration is low in the refolding environment and when the capture agent is removed not in a stepwise manner from the protein-detergent complexes in the stripping step of the whole process. Collectively, the results of this investigation expand the range of procedural variations used to refold different denatured proteins through artificial chaperone-assisted method.     

Refolding from denatured inclusion bodies,purification to homogeneity and simplified assay of MGDG synthases from land plants

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.     

Fluorimetric study of the artificial chaperone-assisted renaturation of carbonic anhydrase: a kinetic analysis

It is now well accepted that ionic detergents along with alpha- or beta-cyclodextrins can enhance protein refolding yields. In this report, we evaluated the effect of detergent's tail length on the kinetics of denatured carbonic anhydrase refolding along with determining the rate-limiting step of the whole refolding process. A sensitive fluorimetric technique was also developed to follow up the second-by-second fate of the denatured protein while undergoing refolding. In this technique, inclusion complexes are formed between the correctly refolded CA and the fluorescent active site probe, 5-dimethylaminonaphtalene-1-sulfonamide. By this specific technique, it became evident that the rate of detergent stripping from the CA-detergent mixed micelles that also appeared to be the rate-limiting step depends on the beta-CD-detergent association constants which are under the influence of detergent's tail length. Based on these findings, appropriate refolding conditions could be designed to kinetically diminish the rate of off-pathway aggregation.     

A comprehensive structure-function analysis shed a new light on molecular mechanism by which a novel smart copolymer, NY-3-1, assists protein refolding

An in-depth understanding of molecular basis by which smart polymers assist protein refolding can lead us to develop a more effective polymer for protein refolding. In this report, to investigate structure-function relationship of pH-sensitive smart polymers, a series of poly(methylacrylic acid (MAc)-acrylic acid (AA))s with different MAc/AA ratios and molecular weights were synthesized and then their abilities in refolding of denatured lysozyme were compared by measuring the lytic activity of the refolded lysozyme. Based on our analysis, there were optimal MAc/AA ratio (44% MAc), M(w) (1700 Da), and copolymer concentration (0.1%, w/v) at which the highest yield of protein refolding was achieved. Fluorescence, circular dichroism, and RP-HPLC analysis reported in this study demonstrated that the presence of P(MAc-AA)s in the refolding buffer significantly improved the refolding yield of denatured lysozyme without affecting the overall structure of the enzyme. Importantly, our bioseparation analysis, together with the analysis of zeta potential and particle size of the copolymer in refolding buffers with different copolymer concentrations, suggested that the polymer provided a negatively charged surface for an electrostatic interaction with the denatured lysozyme molecules and thereby minimized the hydrophobic-prone aggregation of unfolded proteins during the process of refolding.     

Kinetic aspects of alkaline phosphatase refolding in the presence of alpha-cyclodextrin

To get a better understanding of the molecular aspects of protein folding, the refolding kinetic behavior of guanidine hydrochloride-denatured alkaline phosphatase (ALP) was studied in the presence of alpha-cyclodextrin (alpha-CD) through two different approaches: the dilution additive and the artificial chaperone-assisted methods. It was found that alpha-CD enhanced the recovered activity more than 50% via both approaches while decreased the refolding rate, perhaps due to engaging the hydrophobic patches of the protein in a rigid conformation. In contrast, detergents used in the artificial chaperone method increased the refolding rate significantly. A comparison of the rate constants for the refolding and the activity recovery of denatured ALP in the presence of various concentrations of CD and different kinds of detergents showed that they do not progress in a synchronized pattern. This may be attributed to continuous structural rearrangements in the protein long after the return of enzyme activity. These observations are discussed in terms of kinetic and structural aspects of the refolding pathway.     

Comparative Evaluation of α-Amylase Refolding Through Two Different Artificial Chaperone Systems

Two different artificial chaperone systems were evaluated in this work using either detergents or CDs as the stripping agents. Upon dilution of urea-denatured α-amylase to a non-denaturing urea concentration in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to remove the detergent molecules. Our results by fluorescence, UV, turbidity measurement, circular dichroism, surface tension and activity assay indicated that the extent of refolding assistance was different due to different inter- and intra- molecular interactions in the two different systems. However, the high activity recovery in the presence of detergents, as the stripping agent, suggests that they can constitute suitable replacement for the more expensive and common stripping agent of cyclodextrins.     

Molecularly imprinted polymer-assisted refolding of lysozyme

For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein.