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.     

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.     

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.     

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.     

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.     

Control of aggregation in protein refolding: cooperative effects of artificial chaperone and cold temperature

Refolding of GuHCl-denatured recombinant-human growth hormone (r-hGH) was investigated in both dilution additive and artificial chaperone assisted modes. In both techniques, it was found that CTAB is a better additive (in dilution mode) or a capturing agent (in artificial chaperone method). Neither of the two techniques was capable of complete inhibition of aggregates formed during refolding process. In dilution, using CTAB or alpha-cyclodextrin (alpha-CD) as two different additives, the aggregation was inhibited by almost 55%. However, the extent of inhibition raised to almost 82% in artificial chaperone assisted mode using CTAB as the capturing and alpha-CD as the stripping agents. Maximum inhibition of aggregation (up to 97%) was obtained when the entire process of refolding was done at 4 degrees C. However, under this temperature program, the far-UV CD and intrinsic fluorescence spectra of the refolded samples were not superimposable on their respective native spectra. The spectra superimposibilities were obtained when the refolding process was achieved under a well worked out temperature program: incubation of the sample for 3 min at 4 degrees C after initiation of the stripping step followed by overnight incubation at 22 degrees C. Based on these data, it is expected that higher activity recovery yields of recombinant proteins, particularly at relatively higher protein concentrations, could be achieved by getting a better molecular understanding of major factors responsive for aggregation and refolding pathways.     

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.     

Artificial Chaperone-assisted Refolding of GuHCl-Denatured α-Amylase at Low Temperature: Refolding versus Aggregation

Refolding of GuHCl-denatured α-amylase was investigated using the artificial chaperone-assisted method. Three different cationic detergents (CTAB, TTAB and DTAB) and two nonionic detergents (Tween 80 and Triton X-100) were evaluated as the capturing reagents along with α- and β-CD as the stripping agents. The refolding yields, at a final protein concentration of 0.15 mg/ml, were 82, 71 and 66% in the presence of β-CD and CTAB, TTAB or DTAB, respectively. To improve the refolding yield and to suppress the extent of aggregation, the initial rate of the stripping step was slowed down by maintaining the refolding environment at 4°C for about 3 min followed by raising the temperature to 25°C. Under this thermal procedure, the refolding yield and the extent of aggregation were changed from 82 and 25% at 25°C to 94 and 7% at 4°C, respectively. These findings may assist the activity recovery of recombinant proteins at relatively high concentrations.     

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.     

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.     

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.     

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.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

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.     

Molecularly imprinted poly beta-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 beta-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 beta-CD, a refolding yield of 79% was obtained for denatured alkaline phosphatase using 20 mg/ml of the molecularly imprinted poly (beta-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 beta-cyclodextrin.     

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This "two-step-denaturing and refolding" (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.     

Purification of the Caenorhabditis elegans transposase Tc1A refolded during gel filtration chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12-16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

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.     

Purification of the Caenorhabditis elegans Transposase Tc1A Refolded during Gel Filtration Chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Refolding of chemically denatured alpha-amylase in dilution additive mode

Cyclodextrins (CDs) possess hydrophobic surfaces, which probably shield the hydrophobic surfaces of denatured proteins and prevent the direct interactions between the surfaces which are believed to be responsible for protein aggregation during refolding process. This probability was evaluated by studying the refolding process of denatured alpha-amylase in the presence and absence of alpha-CD, as a dilution additive agent. Our data indicate that in the presence of 100 mM alpha-CD in the refolding buffer, the extent of aggregation reduces by almost 90%. Spectrofluorometric analysis of the refolding intermediate(s) also indicates that the tertiary structure of the refolded alpha-amylase, in the presence of alpha-CD, is very similar to the tertiary structure of the native protein. However, this similarity was distorted upon addition of exogenous hydrophobic (aliphatic or aromatic) amino acids to the refolding buffer, meaning that the hydrophobic interactions between alpha-CD and the denatured protein play significant role in preventing aggregate formation. In addition, by weakening the extent of these hydrophobic interactions by adding polarity-reducing agent (e.g. ethylene glycol) to the refolding buffer, more aggregates were formed. In contrast, strengthening these interactions by enhancing the ionic strength of the refolding buffer made these hydrophobic interactions very strong. Therefore, alpha-CD could not depart from the protein/alpha-CD complex, as it usually does during the process of refolding. As a result, more aggregates were formed in the presence of alpha-CD compared to the corresponding control samples.