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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheimwww.biotechnology-journal.comSatoshi Yamaguchi, Etsushi Yamamoto, Teruhisa Mannenand Teruyuki NagamuneTana Koudelakova, Sarka Bidmanova, Pavel Dvorak,Antonin Pavelka, Radka Chaloupkova, Zbynek Prokop and Jiri DamborskyJohn Blazeck and Hal S. AlperDan Kai, Guorui Jin, Molamma P. Prabhakaran andSeeram RamakrishnaBrandon G. Gerberich and Sujata K. BhatiaTekir and Kutlu Ö. ÜlgenBarbara Thallinger, Endry N. Prasetyo, Gibson S.Nyanhongo and Georg M. GuebitzKungang Li, Wen Zhang and Yongsheng Chen …list of articles published in the January 2013 issue.Methods and Advances approaches. Every year, Special Issue, which covers the latest cutting-edge research and breakthrough technologies. For this issues cover, we chose a design featuring 12 light bulbs, representing the 12 months of the year, each with state-of- the-art research.©Thaut Images, ©Yahia LOUKKAL, all from Fotolia.com. Systems & Synthetic Biology · ISSN 1860-6768 · BJIOAM 8 (1) 1…156 (2013) · Vol.8 · January 2013www.biotechnology-journal.com 1/2013Antimicrobial enzymesProtein engineeringRegenerative medicine © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimBiotechnol. J. 2013, 8, 17…31DOI 10.1002/biot.201200025www.biotechnology-journal.com 1 IntroductionWith the success of the human genome project, many re-searchers are interested in the functional analysis and ap-plication of the gene products: proteins. In this context,the ability to rapidly and inexpensively convert gene se- Protein refolding using chemical refolding additivesSatoshi Yamaguchi1, Etsushi Yamamoto1 Correspondence:Prof. Teruyuki Nagamune, Department of Chemistry &Biotechnology, School of Engineering, The University of Tokyo, E-mail: Received28MAY 2012Revised13JUL2012Accepted26JUL2012 Color online:View the article online to see the figures in color. Biotechnology © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim them in the host cells [2]. The former benefit is an ex-tion. Moreover, many products on the market are pro-, many products on the market are pro-Conversely, inclusion-body-based systems also in-clude a severe bottleneck, which is the requirement of aprotein renaturation step from inclusion bodies [1…3]. Toobtain biologically active protein, inclusion bodies are firstsolubilized by chemical denaturants. Strong chaotropes,such as urea and guanidinium hydrochloride, or strong de-tergents are employed to weaken the noncovalent interac-tions among proteins. In the case of target proteins withtermolecular disulfide bonds (S…S bonds). Such a solubi-lization step is now relatively easy to perform becausewell-established methods are applicable to any protein.However, under these solubilization conditions, the in-tive protein structures rupture and solubilized proteins areoften obtained in a flexible, random-coil state [5]. There-fore, the solubilized polypeptide chains have to be refold-ed into their correct structures to recover their native ac-tivities. The efficient conditions required for refolding gen-(i) the concentration of the denaturants should be re-duced to a level where intramolecular noncovalent inter-actions, such as hydrogen bonding, hydrophobic interac-(ii) the refolding process should be performed under an(iii) the concentration of the denatured protein in therefolding buffer should be maintained at a low level toavoid intermolecular aggregation. To meet these require-approach for optimization of the refolding conditions is often required. In particular, the third requirement is crit-ical to increase the refolding yield because protein refold-ing is a kinetically competitive process between pro numerous technologies were developed to avoid aggre-numerous technologies were developed to avoid aggre-The simple dilution of a solubilized protein solutionfolding. In this method, a solubilized, denatured proteinsolution is diluted by a hundred- to a thousand-fold withan appropriate buffer. Reduction in the concentrations ofboth the denaturant and protein is simultaneouslyachieved by an extremely simple protocol. Protein foldingis a first-order reaction with respect to protein concentra-tion, whereas aggregation is a second- or higher-order re-action [6, 7]. This is because higher concentrations of pro-tein enhance the probability of the collision of proteins, re-sulting in higher aggregation rates. Accordingly, by ade-quately decreasing the protein concentration (typicallysion of protein aggregation was achieved [1, 7]. In addi-tion, when a proper oxidative refolding buffer is employedin the refolding process, the three fundamental require-ments for efficient refolding are simply accomplished byhigh protein dilution. However, considering the cost,time, and loss of protein in the following condensationprocess, protein refolding at higher concentrations is de-sired on both the laboratory and industrial scale. More-over, in industry, high dilution has a cost disadvantagederived from huge reactors, large volumes of refoldingbuffer, and disposal of huge volumes of waste required [3,8]. Most importantly, it is often the case that simple dilu-tion cannot adequately suppress protein aggregation andleads to extremely low protein yields, even at the minimalconcentration required to proceed to the next experimen-Such difficult-to-refold proteins have been reported toform agglutinative refolding intermediates in the foldingprocess [6, 9, 10]. These intermediates are partially foldedwith secondary structures and their tertiary packingthrough the interaction of hydrophobic patches is molten[11]. Based on this knowledge, the competitive model be-tween refolding and aggregation in a refolding process hasbeen proposed as follows (Fig. 1): Initially, partially foldedintermediates are quickly formed through intramolecularhydrogen bonds by decreasing the denaturant concentra-tion. This is followed by the simultaneous refolding and ag-gregation of these intermediates, mainly through intra-and intermolecular hydrophobic interactions, respectively.inates over refolding when the refolding rate is slower.Here, the rate of refolding involving disulfide exchange re-Here, the rate of refolding involving disulfide exchange re-Therefore, in the simple dilution refolding of proteins withmore than two disulfide bonds, aggregation often domi-nates and leads to low refolding yields (Fig. 1). Most se-creted proteins, such as interleukins and growth factors,contain a number of disulfide bonds in the native state tostabilize their tertiary structures [15]. Accordingly, suchsecreted proteins, which are important in the medical andpharmaceutical fields, are difficult targets for refolding dueTo perform high-yield and high-concentration refold-ing, various refolding methods and protocols have beendeveloped, as described in many excellent reviews [1…5].Previous developments can be divided broadly into thefollowing three categories: (i) development of methods toremove denaturants from the solubilized protein solution;(ii) methods involving the management of the physicalconditions used during the refolding process; and (iii)methods in which refolding additives are present for suit-able solution conditions of refolding processes. Herein,progress in the first two categories are only briefly intro-duced because an excellent review clearly introducingsuch progress was recently published [3]. We focus on thefinal category, especially on the idea of developing new re- © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim folding additives or additive-induced conditions orientedfor practical use, rather than for scientific interest. Bysharing such ideas with researchers who are developingnew methodologies for protein refolding, we hope this re-view will contribute to the creation of breakthrough tech-nologies for efficient protein refolding. Additionally, weaim to give researchers in other wide-ranging researchfields incentives to start new studies into the develop-2 Progress in refolding systems 2.1 Methods for removing denaturantsIn dilution-based refolding, at the initial point of dilution,denatured proteins are exposed to the aggregation-pro-around the proteins is rapidly reduced before each dena-tured protein has sufficiently separated from each other(Fig. 2A)[10, 16]. Under such an environment, denaturedproteins immediately aggregate, especially at the highfrom the difference in the speeds of passive diffusion be-tween small-molecule denaturants and relatively largeproteins. Therefore, mixing methods and devices werestudied to rapidly decrease local high concentrations ofdenatured proteins [17]. Furthermore, to increase the sys-tem productivity without increasing the initial proteinconcentrations, fed-batch and continuous dilution wereextensively examined [18, 19]. Recently, laminar flow inmicrofluidic chips was employed to simultaneously con-trol each concentration of denaturants and denatured pro-teins [20]. Although this microfluidic method is not suit-able for industrial preparative refolding owing to its Figure 1. Schematic illustration of oxidative refolding of a difficult-to-refold protein. Partially folded intermediates are quickly formed by removal of denatu-rants, and subsequent refolding and aggregation of these intermediates competitively occurs mainly through the interactions of hydrophobic patches onthe surfaces of proteins. The later step of oxidative refolding includes disulfide bond shuffling, which is catalyzed by reductants and oxidants, and such aslowŽ reaction enhances aggregation in the competitive reaction between aggregation and refolding. Biotechnology © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim minute scale, it may serve as a strong tool for protein re-Dialysis represents a second option for the removal ofdenaturants [1…5, 21, 22]. In dialysis-based refolding, pro-folding. Accordingly, the initial concentration of the de-natured protein can be substantially reduced, comparedwith dilution methods at the same final protein concen-tration. Furthermore, dialysis does not create local highconcentrations of denatured proteins in a refolding solu-tion during the initial stages of refolding. These featuresaddition, stepwise dialysis leads to high refolding yields(Fig. 2B) [5, 22]. As the denaturant concentration de-creases by dialysis, protein molecules become less flexi-ble because of the formation of intra- or intermolecular in-teractions [5]. In this process, it is crucial to control theconcentration of the residual denaturant because unpro-natured to the native state once the concentrations of thedenaturant decrease to a low level where proteins are toorigid to undergo structural rearrangement (Fig. 2B). Witha sufficiently high, but not too high, concentration of de-naturant, the refolding state of proteins can reach equi-librium in the stepwise dialysis approach, whereas pro-teins are transiently exposed to such a midpoint denatu- decreases gradually and uniformly. In a stepwise protocol, at the middle denaturant concentration, where the pathways to produ inhibiting aggregation through isolation of proteins on their surfaces; in standard adsorption methods and a zeolite-based metand N re present the state of protein structures, as described in Fig. 1. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim rant concentration in the simple one-step dialysis ap-proach. Accordingly, stepwise dialysis is more effective inthe following case: an equilibrium that favors correct fold-concentration, and it takes a long time to reach such anly shown to lead to a higher protein yield in the slowŽ re-folding of immunoglobulin-folded proteins or other pro-teins with a number of disulfide bonds [22]. Thus, dialysisunder a suitable solution condition is very effective for theThe third option is to remove the denaturants by chro-matography or the employment of solid phases [23…30].The porous solids were used for trapping denaturants inSEC-based refolding (Fig. 2C) [23, 24]. This chromato-of automation and simultaneous purification capabilities.However, similar to the simple dilution method, the ag-surrounding the protein is rapidly removed, forms at thetop of column. To fix these problems, a denaturant gradi-introduced [24]. Different from the SEC-based methods,solid-assisted methods, in which denatured proteins aretrapped on matrices, have been reported [25…30]. The de-natured proteins with affinity tags such as a hexa-histi-dine tag and a self-splicing protein tag were refolded onligand-modified matrices [26, 27]. In these methods, de-natured proteins are immobilized away from each otheron matrices at high denaturant concentrations and thentriggered to refold by exchange to a refold buffer (Fig. 2C).Accordingly, denatured proteins can be refolded withoutinteractions with other proteins on the matrices. Theseimmobilization methods are elegant and useful on the lab-oratory scale. However, in industrial processes, the costsof expensive proteases for cleaving affinity tags after re-folding may be problematic. On the other hand, nonspe-cific adsorption on various matrices has also been em-ployed in chromatographic refolding methods [28…30].Some proteins were successfully refolded through ion-ex-change chromatography [28] or hydrophobic-interactionchromatography [29]. In these methods, the sometimesproblematic specific tag for adsorption is not required.Proteins are captured in their denatured states on matrixsurfaces (Fig. 2C). This can complicate the refolding of theadsorbed denatured protein on the matrices and mostproteins may actually begin to be refolded after releasefrom the matrices. In addition, in many cases, proteins ad-sorbed on the matrices compete with aggregation whennatured proteins tend to aggregate both before adsorp-tion and after release (Fig. 2C). Therefore, the elaborateoptimization of buffer conditions, such as gradients of de-naturants or eluents, is necessary to obtain high refoldingyields [29]. Recently, zeolites, which are crystalline porousaluminosilica compounds, were reported as novel matri-ces for refolding [30]. Zeolites can strongly capture dena-tured proteins at high denaturant concentrations withoutthe need for any tags (Fig. 2C). Consequently, protein aggregation was efficiently suppressed before adsorp-tion, and then, high refolding yields were reported fortion, and then, high refolding yields were reported forOther unique methods for removing denaturants werepreviously reported. Protein refolding was performed innanoscale aqueous droplets of reversed micelles dis-persed in bulk organic solvents [31]. In this refoldingmethod, a single denatured protein was isolated in suchan aqueous compartment with highly concentrated de-naturants and the denaturant concentration was de-creased successively by mixing with a large amount ofthe reversed micelles without a denaturant. Comparedwith simple dilution methods, the relatively high refoldingyields achieved were probably because the intermolecu-lar interactions between denatured proteins were inhibit-ed due to the isolation effect of the reversed micelles. Sim-ilarly, the liquid…liquid two-phase extraction system wasused to remove denaturants, while inhibiting interactionsbetween denatured proteins [32]. Furthermore, an inter-esting method using an enzyme, urease, to decrease de-naturant concentrations was also reported [33]. This en-zyme can catalyze the hydrolysis of urea to produce NH. This method enables the slow and uniform de-ume refolding buffer. Accordingly, this enzymatic methodhas the same advantages as the dialysis method. Thus, al-though most of these recent works are only proofs of prin-ciple, such unique methods may provide a breakthrough2.2 Physical conditions for high-yield refoldingProtein refolding is usually performed at low temperaturesto reduce protein aggregation [34, 35]. At low tempera-tures, a number of proteins were reported to be unfolded[36] and several oligomeric proteins were dissociated [37].These phenomena can be explained by the exposure of hy-of hydrophobic interactions. Since such hydrophobic interactions are entropy driven due to the release of thewater bound to nonpolar groups, this contribution shouldbeweaker because the entropy contribution to the Gibbsfree energy, , decreases at lower temperatures [37].Accordingly, in the refolding process, lowering the tem-perature leads to the suppression of aggregation and re-folding. Therefore, to increase the refolding yields usingthe dilution method, a temperature-shift procedure wasreported [35]. In this procedure, aggregation of denaturedwhen refolding was initiated and then a temperature jumpwas performed to enhance refolding. When the refoldingprocess included a rapid transition to agglutinative refold- Biotechnology © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ing intermediates, this procedure was effective in avoid-ing intermediates, this procedure was effective in avoid-Similar to low temperatures, high hydrostatic pressurewas employed to effectively suppress unproductive ag-gregation in refolding processes [38, 39]. Intra- or inter-an increase in volume, because of the formation of sol-vent-excluding cavities between the hydrophobic inter-faces and the release of bound water [40, 41]. High pres-sure can compress the volume of systems and lead to thesuppression of hydrophobic interactions. It was reportedthat moderate hydrostatic pressure affected aggregationwithout influencing the folding of a native protein [38, 39].By optimizing conditions such as pressure, temperature,and additives in the buffer, a high-pressure-assisted re-folding method could achieve extremely high refoldingthan the simple dilution method [39]. Furthermore, thismethod has a significant advantage in refolding directlyfrom inclusion bodies by omitting the solubilizing step.Although the equipment for sample pressurization is notubiquitous in biological or bioengineering laboratoriesand not easy to scale to industrial levels, the high-pres-High temperature was also reported to increase re-folding yields in the dilution method [42]. In this method,refolding samples of chemically denatured and reducedproteins were heated for only 5 min at the beginning of refolding and then incubated at a low temperature. As aresult, heating above the melting temperature of proteinscould increase the refolding yield. The increase was explained as follows: intermediates trapped in local energy minima and produc-(ii) the rate of rearrangement of non-native disulfidebonds increased with heating. Currently, high-tempera-ture-assisted refolding was only reported to be an effec-tive approach for one protein. However, this method issimple and appears to be worth testing at least once for3Progress in refolding additives3.1 Stabilizers of native proteins and In most refolding systems, the competitive reaction be-tween productive refolding and unproductive aggrega-tion occurs in aqueous solution. Therefore, the solutionconditions of the refolding buffer are critical to improve re-folding yields. The basal parameters of buffered solutions,such as pH, ionic strength, and buffering agent, affectprotein refolding [12]. Therefore, when a new target pro-tein is first refolded, such parameters must be routinelyoptimized. On this basis, to increase the refolding yields,additives were employed to create an ideal environmentwhere the rate of refolding is increased, the aggregationmediate states. Currently, various additives have been reported as stabilizers of the native state of proteins, enhancers of refolding, and inhibitors of aggregation.These effective additives were found in a broad repertoireof molecular species, such as small synthetic or naturalof co-existing salts changed the solubility of a protein [43].On the basis of salting-in or -out effects, ions in a seriescan be qualitatively ranked and ions that tend to solubi-lize and denature proteins are classified as chaotropes.Conversely, ions categorized as kosmotropes agglutinateproteins and stabilize protein structures [44]. In refoldingmethods, the kosmotropic anion, ammonium sulfate, wasemployed as a stabilizer [45, 46], and such a salt de-creased the rate of protein unfolding from the native state[45]. Similarly, sugars, polyols, betaines, and hydrophilicpolymers were used to stabilize the folded protein andcontribute to an increase in correctly folded protein yields[46…48]. The stabilizing effects of these additives were of-surfaces, which thermodynamically led to the reduction ofprotein surface exposed to the solvent through unfavor-able interactions between protein surfaces and additives[49] (Fig. 3). However, such stabilizers do not only in-crease the refolding yield, but simultaneously enhanceaggregation. Accordingly, stabilizers have always been, stabilizers have always beenThe refolding of difficult-to-refold proteins often in-cludes the formation of more than one disulfide bond. Inthe non-native disulfide bonds, oxidized and reduced glu-tathione (GSSG, GSH, respectively) are commonly usedas disulfide-shufflingŽ reagents [1…4, 12, 50, 51]. In thissystem, GSSG works as an oxidizing agent to promotedisulfide bonds between the thiols of two cysteine sidechains in the protein. Therefore, if only GSSG is added,based purely on statistics, both correct and incorrectcysteine residues. On the other hand, the addition of GSHreduction and oxidation steps, which finally result in theformation of disulfide bonds that give the correct proteinfold. This is because a correct disulfide bond is stabilizedby the free energy derived from the formation of the na-tive conformation [12]. Other thiol…disulfide pairs, such ascysteine and cystine, also exerted similar effects [12, 51].These disulfide-shuffling reagents can drastically increaserefolding yields by increasing the yield of correct disulfidebonds and the rate of refolding when compared with oxidative refolding with only dissolved oxygen [50, 51]. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim To enhance refolding in vitro, foldases that assist thefolding of nascent proteins in vivo were employed. Theemployed foldases were categorized as protein disulfideisomerase (PDI) [13, 52, 53] and peptidyl prolyl isomerase (PPI) [53, 54]. As part of the former enzymes,Dsb families, such as DsbA and DsbC, catalyzed disulfide-shuffling in vivoand in vitro. In detail, these PDIs in theoxidized form can catalyze disulfide formation by reactingwith the thiol group of reduced target proteins, and thosein the reduced form can trigger shuffling by attacking theincorrect disulfide bonds [13]. These foldases increasedthe refolding yield of a protein with multiple disulfidebonds. On the other hand, both subclasses of PPIs, cyclophilin and FK506 binding protein (FKBP), accelerat-ed productive refolding and increased the yield of nativeprotein [54]. They can catalyze the rate-limiting isomer-In biomimetic chemistry, small synthetic reagentswith PDI-like function were developed to improve the re-folding rate [55…61]. At the active site of many PDIs, acommon sequence motif CXXC (C: cysteine, X: anyresidue) is conserved and the one water-exposed thiola value and high nucleophilicity atneutral pH, which leads to rapid nucleophilic attack ondisulfide bonds of folding intermediates. To mimic suchnucleophilicity, reducing reagents with low psuch as aromatic thiols [55], selenoxides [56], and se-lenoglutathione [57], were developed and successfully ledto the rapid formation of multiple disulfide bonds. Moredirectly, peptides consisting of the CXXC motif of disul-fide oxidoreductases were also employed for oxidative re-fide oxidoreductases were also employed for oxidative re-dithiol with the same pKa value and reduction potentialas the CXXC motif of PDI enhanced the rate of disulfideshuffling [59]. Recently, to mimic the hydrophobic regionsaround the active sites of PDI, hydrophobic alkyl chainmodified cyctamines were developed [60]. By these bio-mimetic approaches, the function of foldases could besubstituted with small-molecule mimics. Since the use offoldases has the disadvantages of costly production and Figure 3.Various modes of interaction between a protein and refolding additives and the chemical structures of typical additives in each category. Modified with permission from Elsevier [64]. Biotechnology © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim instability, these mimics are useful for industrial refolding these mimics are useful for industrial refolding3.2 Aggregation inhibitorTo inhibit aggregation, small-molecule additives are fre-quently employed because of their low cost and ease of re-moval after the refolding process [1, 5]. In particular,chaotropes, such as urea and guanidine hydrochloride(GdnHCl), are generally used at relatively low concentra-tions from 0.5 to 2.0 M [7, 62, 63]. Such chaotropes wereoriginally employed as denaturants in refolding process-es. At high urea and GdnHCl concentrations, protein ag-gregates are effectively dissolved and protein unfoldingalso occurs because these chemicals disrupt both intra-and intermolecular interactions of proteins. The mecha-nism underlying chaotropic effects remains the subject ofsome debate [44, 64]. Recently, the most widely acceptedexplanation is the preferential interaction of chaotropeswith protein surfaces (Fig. 3). Such interactions stabilizethe exposed surface of the protein, resulting in a decreasein the free energy of unfolding and an increase in the freeenergy of aggregation [64]. In a standard refoldingmethod, chaotrope concentrations are drastically de-creased to initiate the protein refolding process; however,in some cases, high refolding yields were obtained by us-ing chaotropes at nondenaturing concentrations [7, 62].Under such positive conditions, the aggregation rate wasremarkably decreased compared with the refolding rate.Although the mechanism by which aggregation is selec-tively suppressed remains unclear, the weak interactionof chaotropes with the hydrophobic surface of proteinsmay create a kinetic situation where only intramolecularRudolph and Fischer [65] first reported the function ofacid based chaotropic reagent, on the suppression of ag-gregation and enhancement of protein refolding. Subse-quently, the ability of arginine to increase refolding yieldswas tested with a variety of proteins and was effectiveover the concentration range of 0.4 to 1 M [63, 66, 67]. Cur-rently, -ArgHCl is the most commonly used additive.Compared with GdnHCl, -ArgHCl, which has the sameguanidinium moiety, is a superior aggregation inhibitorfor the refolding process because its protein denaturingeffect is more moderate (Fig. 3). As described above, sucha selective effect of chaotropes is explained by severaltheories and many studies describing the mechanism of-ArgHCl were recently reported [63, 67, 68]. Amongthem, the gap effect theoryŽ suggests that additives larg-er than water, which do not affect the folding of isolatedproteins, can selectively increase the free energy of inter-molecular protein…protein association [67]. This theoryfective aggregation inhibitors for protein refolding (Fig. 3)[69…75]. Amino acid derivatives, such as and glycineamide, increased the refolding yields of someproteins more than proteins more than and polar organic solvents exerted a similar refolding-pro-moting effect on protein refolding [71…73]. Although theirchaotropic effects have not been discussed yet, they pre-sumably operate through the same mechanism. Thus,many small-molecule chaotropes can assist in preparingsuitable solution conditions for protein refolding andsome of them are already commonly used in both labora-tory and industrial settings. However, although their costis relatively low relative to other molecular species, on theindustrial scale, amino acids and their derivatives are verycostly [3]. Therefore, more inexpensive or inexpensive, recyclable chaotropes are required.Additives that bind more strongly to protein surfacesthan chaotropes were also reported as aggregation in-hibitors for refolding. In this category, detergents are mostcommonly used [71, 74…86]. Currently, various kinds of de-tergents, such as cationic, anionic, zwitterionic, and non-range can prevent aggregation and enhance refoldingyields [71, 74…77]. Such suitable refolding conditions wereassumed to require the formation of mixed micelles con-sisting of proteins and detergents, and the relationshipbetween the refolding yields and their critical micellarbetween the refolding yields and their critical micellartein…detergent interactions were extensively studied byindirect and direct methods [64]. However, in many cas-es, detergents inhibit not only aggregation, but also re-folding due to strong interactions between detergentsand refolding intermediates [79, 80]. Therefore, employ-ment of detergents often leads to low refolding yields andextremely long refolding times. In addition, different fromsalts and chaotropic reagents, detergents are difficult toremove from products by dialysis or gel filtration due tothe formation of micelles and their strong interactionswith protein surfaces. Therefore, in subsequent steps, ad-sorbents for detergents, such as reverse-phase chro-Similarly, other compounds, such as cyclodextrin de-rivatives [81, 82], polymers [71, 82…84], and sulfobetaine[85], which can bind to hydrophobic protein surfaces, increased refolding yields. In the refolding processes,these additives must meet two conflicting requirements:(i) they are required to attach to the hydrophobic surfaceto inhibit intermolecular interactions between proteins;and (ii) they are simultaneously required to detach fromthe protein surface, so that inhibition of intramolecular in-teractions does not occur. Therefore, their positive effectsare limited to a narrow optimal concentration where theadditives moderately interact with exposed protein sur-faces. Furthermore, depending on the target protein, such © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim concentration. In most previous studies, commerciallyly employed as aggregation inhibitors by trial-and-errorapproaches. Consequently, optimization of a refolding so-lution needs significant time. Recently, tunableŽ addi-tives, the derivatives of which have systematically alteredhydrophobic moieties that were easily prepared, were re-ported [86…91]. In this approach, the relationships be-tween chemical structures and effectiveness at increas-ing the refolding yield were assessed and then the man-ner in which the chemical substructures confer suitableproperties on protein refolding could be understood. Ion-ic liquids with various hydrophobic tails, cationic heads,and counteranions were investigated and those with botha short alkyl chain and a hydrophilic anion were excellentrefolding additives due to their moderate chaotropic effects (Fig. 4A)[88…91]. Conversely, ionic liquids with along alkyl chain only worked effectively over a limited con-centration range, in a similar manner to detergents. Ionicliquids with an average (mid-length) alkyl chain length ora benzyl group had no positive protein refolding effects(Fig. 4A) [89]. Thus, tuning the properties of additives is asuccessful strategy to obtain tailor-made additives foreach target protein. This strategy can also be expanded toa variety of additives, the mechanisms of which for assisting protein refolding are well known and may potentially result in the rational design of a solution envi-To more easily modulate the properties of aggregationinhibitors, the combined use of an effector and a modula-tor were reported (Fig. 4B) [92]. As described above, refolding additives of detergents often inhibit productiverefolding because such additives strongly bind to the hydrophobic surfaces of refolding intermediates. Accord- Figure 4.Two modes of tunable additives for protein refolding. (A) A small library of chemical additives with systematically altered structures was easilysynthesized as tunable additives. By kinetic analysis of the refolding process, additives with a short hydrophobic tail exerted a positively chaotropic effecton the oxidative refolding of lysozyme, and on the other hand, those compounds with a long tail worked effectively as detergents [89]. Thus, alteration ofthe tail length can control the rates of refolding and aggregation. (B) Schematic illustration of the combined use of a detergent and an organic solvent. Theorganic solvent modulates the interaction between proteins and detergents, resulting in a synergistically positive effect on the refolding yield. The data wasobtained from [92]. U, I, and N represent the state of protein structures, as described in Fig. 1. Biotechnology © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ingly, to modulate protein…detergent interactions, variousorganic cosolvents were added with detergents, and con-sequently, both the refolding and aggregation rates werecontrolled, according to the logtration of the cosolvents. Moreover, the refolding yieldswere synergistically increased by employing polar cosol-vents at a moderate concentration with various deter-gents [92]. Such a combined use of conventional additivesis inexpensive and easy to perform because we do notneed to find or synthesize new, effective additives. Thus,tuning the effect of conventional aggregation inhibitorswith modulator agents may expand the applicationA negatively charged long polymer aided refolding ofcationic proteins by interacting with the protein surfacethrough an electrostatic interaction [93]. It was speculat-ed that the polymer could shield the hydrophobic surfacesof refolding intermediates because of steric hindrance;thus suppressing protein aggregation. Different fromstandard aggregation inhibitors, such polymers do not interact with the hydrophobic patches located on refold-ing intermediates required for packing of the tertiarystructure. Accordingly, these polymers avoid the conven-tional conflicting requirements of aggregation inhibitors,and therefore, unproductive aggregation of refolding intermediates can be specifically suppressed without in-hibition of productive refolding. In addition, such chargedpolymers are removed from the protein surface by chang-ing the pH or employing ion-exchange chromatographyafter refolding, and thus, represent promising tools forThe two conflicting requirements for aggregation inhibitors of protein refolding are overcome by using molecular machines, named molecular chaperones in vivo[94]. Chaperonin GroEL, which is the most well-knownmolecular chaperone, can sequentially capture and release unfolded substrate proteins to assist folding. In detail, the double-ring GroEL tetradecamer encapsulatessubstrate proteins in the central cavity when capped bythe GroES heptamer in an ATP-dependent manner. In thisGroEL/GroES system, the substrate binding site of GroELalters the exposure of its hydrophobic surface through aconformational change in coupling to both ATP hydroly-sis and competitive binding of GroES. This dynamic allosteric alteration of GroEL enables the captured proteinto be released, and additionally, a released, unfolded pro-tein is isolated in each cavity of the GroEL/GroES com-plex to fold without associating with other unfolded pro-teins. Application of such chaperonins toin vitroproteinrefolding has been extensively studied and chaperoninsfrom various bacteria increased the refolding yields bysuppressing unproductive aggregation of target proteins[95, 96]. In addition, only the monomeric polypeptide-binding domains of GroEL increased the refolding yield;however, the chaperone activity of this GroEL was muchlower than the activity of the full GroEL/GroES system [97,98]. Thus, such dynamic allosteric properties and the cav-ity for isolation are not essential for chaperone activity,but the two effects of the GroEL/GroES system are cer-Similar to foldase mimics, the functions of chaperoneswere challenged by substitution with synthetic com-pounds [16, 80, 98…107]. To mimic the allosteric properties mimic the allosteric propertieschaperone-assisted (ACA) refolding, which involves atwo-step dilution procedure (Fig. 5A). In the first step, thedenatured protein solution is diluted with a buffer con-taining detergents to prevent aggregation by forming pro-tein…detergent complexes. In the second step, the pro-tein…detergent complex solution is diluted with a buffercontaining detergent strippers, such as The first step mimics the capture of an unfolded proteinwith GroEL and the second step leads to the release of theprotein, as GroEL allosteric functions. The ACA refoldingapproach is effective in refolding systems for a large vari-ety of proteins [16, 80, 99, 100]. Furthermore, various detergent strippers of oligomeric, polymeric sugars, andcyclodextrin-modified polymers are useful for this method[100…103]. In ACA refolding methods, the detergent strip-pers effectively remove the detergents, so that the dilu-tion ratio in the second refolding step can be substantial-ly lowered compared with conventional simple dilutionmethods (Fig. 5A). Accordingly, similar to the dialysismethod, the protein concentrations at the start of refold-ing can be drastically reduced, resulting in the suppres-ing can be drastically reduced, resulting in the suppres-Smart polymer-assisted refolding was reported as amethod that mimicked the allosteric chaperone function[104]. Temperature-responsive polymers, such as poly(isopropylacrylamide) (PNIPAAm), can alter their hydrophobicity as a function of temperature: their hydrophobicity drastically increases above their low crit-ical solution temperature (LCST). Accordingly, for the ini-tial dilution, the refolding mixture was incubated at ahigher temperature than that of the LCST to inhibit ag-gregation through the formation of strong polymer…pro-tein complexes. Refolding was then started by releasingthe protein from the polymer…protein complexes by low-ering the temperature [104]. Recently, an enzyme-respon-sive surfactant was employed as a changeable aggrega-sive surfactant was employed as a changeable aggrega-surfactant was reported to release captured refolding in-termediates as enzymatically polymerized, more hy-drophilic forms; thus yielding native protein without un-productive aggregation. On the other hand, the function,like the isolation effect observed for chaperonins, was ex-erted by nanogels of self-assembled cholesterol-bearingpullulan [106, 107]. Refolding intermediates were sponta-neously captured into nanogels through hydrophobic in-teractions and the captured proteins were effectively re-leased in their refolded native form upon dissociation of © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim teration of the hydrophobicity of the nanogel by photo-stimulation [107]. This nanogel-assisted refolding suc-cessfully increased the refolding yields of some proteinscompared with the spontaneous refolding system. Thus,in such biomimetic approaches, several artificial chaper-oped and could effectively suppress aggregation of re-folding intermediates by capturing proteins in an aggre-3.3 Application of refolding additivesIn many cases, novel refolding additives are initially eval-uated for their efficacy in the simple dilution methods.However, most of them are compatible with other refold-ing systems. Disulfide-shuffling reagents and chaotropicadditives, for example, have been routinely used in dialy-sis- and SEC-based methods [22, 24]. Molecular chaper-ones were also applied to a reversed micelle system andSEC-based refolding; thus increasing the refolding yield[108, 109]. Recently, the ACA method was actively re-ported to be combined with other techniques [110, 111].The synergistic effects of the combined use with the low-temperature method were studied [110]. In addition, toproach was employed in microfluidic-aided refolding, re-sulting in the effective suppression of aggregation at themixing point [111]. On the other hand, many studies in-volving the combined use of refolding additives, such as following the initial high dilution of the denaturant concentration is suppressed by adding detergents (the first step) and, o protein (the second step). U, M, I, and N represent the state of protein structures, as described in Fig. 1. ( refolding system based on the solid-phase ACA method (left). The protein…detergent complex solution was applied to a cyclodext Biotechnology © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim an aggregation inhibitor and a protein stabilizer, have alsobeen reported for more than 15 years [112]. The combineduse of two foldases and a molecular chaperone representsvery famous work in this research field [12]. In addition,other combinations such as ACA additives andchaotropes [113], and a molecular chaperone and poly-mers [114], have been reported. Thus, the combined useof the reported methods and additives is a promising ap-proach to further increase the refolding yield, especiallywhen the two aspects complement each other. Becausetential for the emergence of a surprising number of valu-Some refolding additives have been introduced to asolid phase for the following reasons: (i) they can be sta-bilized through immobilization on matrices; (ii) insolubleadditives are easy to remove after refolding; (iii) they areic refolding, such as ease of automation, simultaneous pu-rification capabilities, and downsizing of equipment re-quirements, may be available. Foldases and a molecularfolding chromatography [13, 53, 98, 115]. Similarly, small-molecule thiols were immobilized on microspheres andemployed as a solid-phase PDI mimic [116, 117]. Addi-tionally, solid-phase ACA refolding methods were active-ly studied by using various cyclodextrin polymer beads orcyclodextrin-modified microspheres [101, 102, 118, 119].Solid-phase ACA refolding was successfully performed inan expanded-bed column system, which was easy toscale up by using commercially available devices (Fig. 5B)[101]. By increasing the column/loop volume ratio, almostthe same refolding yields as that of a liquid-phase systemwere obtained in such a column system. The loaded cy-clodextrin polymer beads could be reused by simplewashing with water. Thus, the application of refolding ad-ditives to solid-phase systems represents a strong tool forIn the present post-genome era, a rapid and inexpensivemethod for protein production is becoming increasinglyimportant in various research fields. In this context, theinclusion-body-based production system is attractive be-cause of the high protein expression yields and the effi-ciency of upstream processes. However, this productionsystem often has significant drawbacks related to the re-folding step. To date, many methods have been developedto improve the refolding yield. In particular, effective re-folding additives have been explored to create suitableover unproductive protein aggregation in the kineticcompetitive reaction. To increase the refolding rate, ex-cellent reductive/oxidative reagents, which mimic natu-rally occurring foldases, have been developed and their ef-fects are potentially universal in oxidative refolding of anyproteins. On the other hand, many aggregation inhibitorswere also reported, but versatile chemical tools for anyvariability of protein surfaces. Therefore, recently, tunablesynthetic additives and the combined use of an effectorand a modulator were developed for tailor-madeŽ refold-ing. Moreover, understanding the mechanism by whichthese systematically altered additives have an effect onprotein refolding and aggregation enables rational selec-tion and design of refolding additives for a novel target. for the fermentation process and separation-attainable bio reactor sys- controlling biomolecules and living cells, with the aim of creating novel strategies in bioprocess, bioanalysis, drug delivery, and cell engineering. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Therefore, progress in analytical methods to elucidate theeffect of protein-protein and protein-additive interactionson refolding processes is also extremely important [120].In addition, refolding additives that mimicked the func-tion of molecular chaperones were actively studied be-cause chaperones were known to aid folding of a wide va-riety of proteins. Furthermore, some effective combina-tions of refolding additives and conventional techniquesfolding additives to solid-phase techniques successfullyled to suitable methods for industrial refolding. Althoughthe majority of positive results obtained from protein re-folding studies were obtained using several model pro-teins, the present emerging method based on refoldingprocess to a routine one in the laboratory and the manu-5 References[1]Middelberg, A. P. J., Preparative protein refolding. Trends. Biotech-[2]Baneyx, F., Recombinant protein expression in Escherichia coliCurr. Opin. Biotechnol.[3]Eiberle, M. K., Jungbauer, A., Technical refolding of proteins: Do we[4]Baneyx, F., Mujacic, M., Recombinant protein folding and misfold-[5]Tsumoto, K., Ejima, D., Kumagai, I., Arakawa, T., Practical consider-ations in refolding proteins from inclusion bodies.Protein. Expres-[6]Kiefhaber, T., Rudolph, R., Kohler, H. H., Buchner, J. Protein aggre-gation in vitro and in vivoÑa quantitative model of the kinetic com-petition between folding and aggregation. [7]Hevehan, D. L., Clark, E. D. B., Oxidative renaturation of lysozyme at[8]Fahnert, B., Lilie, H., Neubauer, P., Inclusion bodies: Formation andAdv. Biochem. Eng./Biotechnol.[9]Bam, N. B., Cleland, J. L., Randolph, T. W., Molten globule inter -mediate of recombinant human growth hormone: Stabilization with[10]Goldberg, M. E., Rudolph, R., Jaenicke, R., A kinetic study of thecompetition between renaturation and aggregation during the refolding of denatured…reduced egg white lysozyme. [11]Uversky, V. N., Ptitsyn, O. B., Partly foldedŽ state, a new equilibriumstate of protein molecules: Four-state guanidinium chloride inducedunfolding of beta-lactamase at low temperature. [12]Rudolph, R., Lilie, H., In vitro folding of inclusion body proteins.FASEB J.[13]Altamirano, M. M., Garcia, C., Possani, L. D., Fersht, A. R., Oxidativerefolding chromatography: Folding of the scorpion toxin Cn5. [14]Semisotonov, G. V., Uversky, V. N., Sokolovsky, I. V., Gutin, A. M. etal., Two slow stages in refolding of bovine carbonic anhydrase B are[15]Fahey, R. C., Hunt, J. S., Windham, G. C., On the cysteine and cys-tine content of proteins differences between intracellular and extra-[16]Daugherty, D. L., Rozema, D., Hanson, P. E., Gellman, S. H., Artificialchaperone-assisted refolding of citrate synthase. J Biol. Chem.[17]Mannall, G. J., Titchener-Hooker, N. J., Chase, H. A., Dalby, P. A., Acritical assessment of the impact of mixing on dilution refolding.[18]Buswell, A. M., Ebtinger, M., Vertes, A. A., Middelberg, A. P. I., Ef-fect of operating variables on the yield of recombinant trypsinogenfor a plus-fed dilution-refolding reactor. Biotechnol. Bioeng. [19]Katoh, Y., Farshbaf, M., Kurooka, N., Nohara, D. et al., High yield re-folding of lysozyme and carbonic anhydrase at high protein concen-[20]Yamaguchi, H., Miyazaki, M., Briones-Nagata, M. P., Maeda, H., Re-folding of difficult-to-fold proteins by a gradual decrease of denatu-[21]Kelly, S. M., Price, N. C., The unfolding and refolding of pig heart fu-[22]Umetsu, M., Tsumoto, K., Hara, M., Ashish, K. et al., How additivesinfluence the refolding of immunoglobulin-folded proteins in a step-wise dialysis system. Spectroscopic evidence for highly efficient re-folding of a single-chain Fv fragment. J. Biol. Chem.[23]Werner, M. H., Clore, G. M., Gronenborn, A. M., Kondoh, A. et al., Refolding proteins by gel filtration chromatography. FEBS Lett. [24]Gu, Z. Y., Su, Z. G., Janson, J. C., Urea gradient size-exclusion chro-matography enhanced the yield of lysozyme refolding.J. Chro-matogr. A[25]Stempfer, G., Höll-Neugebauer, B., Rudolph, R., Improved refoldingof an immobilized fusion protein. [26]Zahn, R., von Schroetter, C., Wuthrich, K., Human prion proteins ex-Escherichia coliand purified by high-affinity column re-[27]Singh, P. K., Gupta, M. N., Simultaneous refolding and purification ofa recombinant lipase with an intein tag by affinity precipitation with[28]Machold, C., Schlegl, R., Buchinger, W., Jungbauer, A., Matrix as-sisted refolding of proteins by ion exchange chromatography. [29]Li, J. J., Wang, A. Q., Janson, J. C., Ballagi, A. et al., Immobilized Triton-X-100-assisted refolding of green fluorescent protein-Tobac-co Etch virus protease fusion protein using ß-cyclodextrin as the eluent. [30]Nara, T. Y., Togashi, H., Sekikawa, C., Kawakami, M. et al., Use of ze-Colloids Surf. B[31]Hashimoto, Y., Ono, T., Goto, M., Hatton, T. A., Protein refolding byreversed micelles utilizing solid…liquid extraction technique.[32]Shimojo, K., Oshima, T., Naganawa, H., Goto, M., Calixarene-assist-ed protein refolding via liquid…liquid extraction. [33]Okada, J., Maruyama, T., Motomura, K., Kuroki, K. et al., Enzyme-[34]Haase-Pettingell, C. A., King, J., Formation of aggregates from athermolabile in vivo folding intermediate in P22 tailspike matura-[35]Xie, Y., Wetlaufer, D. B., Control of aggregates in protein refolding:[36]Privalov, P. L., Cold denaturation of proteins. Crit. Rev. Biochem. Mol. Biotechnology © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim [37]Weber, G., van t Hoff revisited: Enthalpy of association of protein[38]Gorovits, B. M., Horowitz, P. M., High hydrostatic pressure can re-verse aggregation of protein folding intermediates and facilitate ac-[39]St. John, R. J., Carpenter, J. F., Randolph, T. W., High pressure fostersprotein refolding from aggregates at high concentrations. , 13029…13033.[40]Silva, J. L., Foguel, D., Da Poian, A. T., Prevelige, P. E., The use ofhydro static pressure as a tool to study viruses and other macro -Curr. Opin. Struct. Biol.[41]Weber, G., Thermodynamics of the association and the pressure dis-sociation of oligomeric proteins. J. Phys. Chem.[42]Sakamoto, R., Nishikori, S., Shiraki, K., High temperature increasesthe refolding yield of reduced lysozyme: Implication for the produc-[43]Hofmeister, F., Zur lehre von der wirkung der salze zeite mittheilung.[44]Broering, J. M., Bommarius, A. S., Evaluation of Hofmeister effectson the kinetic stability of proteins.J. Phys. Chem. B2061220619.[45]Goto, Y., Ichimura, N., Hamaguchi, K., Effects of ammonium sulfateon the unfolding and refolding of the variable and constant frag-ments of an immunoglobulin light chain.[46]Kohyama, K., Matsumoto, T., Imoto, T., Refolding of an unstablelysozyme by gradient removal of a solubilizer and gradient additionof a stabilizer.[47]Wang, Z., Landry, S. J., Gierasha, L. M., Srere, P. A., Renaturation ofcitrate synthase: Influence of denaturant and folding assistants. [48]Dong, X. Y., Liu, J. H., Liu, F. F., Sun, Y., Self-interaction of native anddenatured lysozyme in the presence of osmolytes, [49]Bourot, S., Sire, O., Trautwetter, A., Touzé, T. et al., Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coliJ. Biol.[50]Ahmed, A. K., Schaffer, S. W., Watlaufer, D. B., Nonenzymic reacti-vation of reduced bovine pancreatic ribonuclease by air oxidationand by glutathione oxidoreduction buffers. J. Biol. Chem.[51]Raman, B., Ramakrishna, T., Rao, D. M., Refolding of denatured anddenatured/reduced lysozyme at high concentrations. J. Biol. Chem., 1706717072.[52]Lilie, H., Mclaughrin, S., Freedman, R., Buchner, J., Influence of protein disulfide isomerase (PDI) on antibody folding in vitro. J. Biol., 1429014296.[53]Altamirano, M. M., Woolfson, A., Donda, A., Shamshiev, A. et al., Lig-and-independent assembly of recombinant human CD1 by using ox-idative refolding chromatography. Proc. Natl. Acad. Sci. USA[54]Lilie, H., Lang, K., Rudolph, R., Buchner, J., Prolyl isomerases cat-[55]Madar, D. J., Patel, A. S., Lees, W. J., Comparison of the oxidativefolding of lysozyme at high protein concentration using aromatic thiols versus glutathione. [56]Arai, K., Dedachi, K., Iwaoka, M., Rapid and quantitative disulfidebond formation for a polypeptide chain using a cyclic selenoxideChem. Eur. J. [57]Beld, J., Woycechowsky, K. J., Hilvert, D., Diselenides as universaloxidative folding catalysts of diverse proteins.J. Biotechnol.[58]Moroder, L., Besse, D., Musiol, H.-J., Rudolph-Böhner, S. et al., Ox-idative folding of cysteine-rich peptides vs. regioselective cysteine[59]Woycechowsky, K. J., Wittrup, K. D., Raines, R. T., A small-moleculecatalyst of protein folding in vitro and in vivo.[60]Wang, G.-Z., Dong, X.-Y., Sun, Y., Acyl cystamine: Small-molecularfoldase mimic accelerating oxidative refolding of disulfide-contain-[61]Lees, W. J., Small-molecule catalysts of oxidative protein folding.Curr. Opin. Chem. Biol.[62]Orsini, G., Goldberg, M. E., Renaturation of reduced chymo trypsin -ogen-a in guanidine HCl-refolding versus aggregation. J. Biol.[63]Buchner, J., Rudolph, R., Renaturation, purification and characteri-zation of recombinant Fab-fragments produced in Escherichia coli[64]Ohtake, S., Kita, Y., Arakawa, T., Interactions of formulation excipi-ents with proteins in solution and in the dried state. Adv. Drug De-livery Rev.[65]Rudolph, R., Fischer, S., Process for obtaining renatured proteins.[66]Reddy, R. C., Lilie, H., Rudolph, R., Lange, C., the solubility of unfolded species of hen egg white lysozyme. [67]Baynes, B. M., Wang, D. I. C., Trout, B. L., Role of arginine in the sta-bilization of proteins against aggregation.[68]Arakawa, T., Ejima, D., Tsumoto, K., Obeyama, N. et al., Suppressionof protein interactions by arginine: A proposed mechanism of the[69]Hamada, H., Shiraki, K., -Argininamide improves the refolding[70]Ito, L., Shiraki, K., Makino, M., Hasegawa, K. et al., Glycine amideshielding on the aromatic surfaces of lysozyme: Implication for sup-[71]Wetlaufer, D. B., Xie, Y., Control of aggregation in protein refolding:A variety of surfactants promote renaturation of carbonic anhy-[72]Kotik, M., Radford, S. E., Dobson, C. M., Comparison of the refoldinghen lysozyme from dimethyl sulfoxide and guanidinium chloride.[73]Dong, X. Y., Huang, Y., Sun, Y., Refolding kinetics of denatured- reduced lysozyme in the presence of folding aids. J. Biotechnol.[74]Tandon, S., Horowitz, P. M., Detergent-assisted refolding of guanitinium chloride-denatured rhodanese. J. Biol. Chem.[75]Bam, N. B., Cleland, J. L., Randolph, T. W., Molten globule inter -mediate of recombinant human growth hormone: Stabilization with[76]Krause, M., Rudolph, R., Schwarz, E., The non-ionic detergent Brij[77]Mustafi, D., Smith, C. M., Makinen, M. W., Lee, R. C., Multi-blockpoloxamer surfactants suppress aggregation of denatured proteins.[78]Pan, J. C., Wang, J. S., Cheng, Y., Yu, Z. et al., The role of detergentin refolding of GdnHCl-denatured arginine kinase from shrimp : The solubilization of aggregate and refold-[79]Wang, J., Lu, D., Lin, Y., Liu, Z., How CTAB assists the refolding ofBiochem. Eng. J. [80]Rozema, D., Gellman, S. H., Artificial chaperone-assisted refolding ofdenatured-reduced lysozyme: Modulation of the competition be-tween renaturation and aggregation, © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim [81]Karuppiah, N., Sharma, A., Cyclodextrins as protein folding aids.[82]Desai, A., Lee, C., Sharma, L., Sharma, A., Lysozyme refolding withcyclodextrins: Structure…activity relationship. [83]Cleland J. L., Randolph, T. W., Mechanism of polyethylene glycol in-teraction with molten globule folding intermediate of bovine carbonic anhydrase-B. [84]Jiang, Y., Yan, Y. B., Zhou, H. M., Polyvinylpyrrolidone 40 assists therefolding of bovine carbonic anhydrase B by accelerating the re-folding of the first molten globule intermediate. J. Biol. Chem.[85]Vuillard, L., Rabilloud, T., Goldberg, M. E., Interactions of non- detergent sulfobetaines with early folding intermediates facilitateEur. J. Biochem. [86]Singh, R., Flowers, R. A., 2nd., Efficient protein renaturation usingtunable hemifluorinated anionic surfactants as additives. [87]Summers, C. A., Flowers, R. A. 2nd., Protein renaturation by the liquid organic salt ethylammonium nitrate. [88]Lange, C., Patil, G., Rudolph, R., Ionic liquids as refolding additives:-Alkyl and [89]Yamaguchi, S., Yamamoto, E., Tsukiji, S., Nagamune, T., Success-ful control of the aggregation and folding rates during refolding ofdenatured lysozyme by adding [90]Buchfink, R., Tischer, A., Patil, G., Rudolph, R. et al., Ionic liquidsas refolding additives: Variation of the anion. J. Biotechnol. [91]Yamamoto, E., Yamaguchi, S., Nagamune, T., Protein refolding-alkylpyridinium and liquids. [92]Yamamoto, E., Yamaguchi, S., Nagamune, T., Synergistic effects ofdetergents and organic solvents on protein refolding: Control of aggregation and folding rates. [93]Huang, Z., Leong, S. S. J., Molecular-assisted refolding: Study oftwo different ionic forms of recombinant human fibroblast growth[94]Taguchi, H., Chaperonin GroEL meet the substarate protein as a[95]Buchner, J., Schemit, M., Fuchs, M., Jaenicke, R. et al., GroE facil-itates refolding of citrate synthase by suppressing aggregation[96]Teshima, T., Kohda, J., Kondo, A., Yohda, M. et al., Affinity purifica-tion of fusion chaperonin Cpn60-(His)strain MS and its use in facilitating protein refolding and[97]Zahn, R., Buckle, A. M., Perrett, S., Johnson, C. M. et al., Chaper-one activity and structure of monomeric polypeptide binding do-mains of GroEL. Proc. Natl. Acad. Sci. USA, 1502415029.[98]Altamirano, M. M., Golbik, R., Zahn, R., Buckle, A. M. et al., Re-folding chromatography with immobilized min-chaperones. [99]Nath, D., Rao, M., Artificial chaperone mediated refolding of xylanase from an alkalophilic thermophilic tions for in vitro protein renaturation via a folding intermediate.Eur. J. Biochem.[100]Machida, S., Ogawa, S., Shi, X. H., Takaha, T. et al., Cycloamyloseas an efficient artificial chaperone for protein refolding. FEBS Lett.[101]Mannen, T., Yamaguchi, S., Honda, J., Sugimoto, S. et al., Expand-ed-bed protein refolding using a solid-phase artificial chaperone.[102]Yamaguchi, S., Hong, C., Mannen, T., Tsukiji, S. et al., Solid-phaseartificial chaperone-assisted refolding using insoluble beta- cyclodextrin-acrylamide copolymer beads. Biotechnol. Lett.[103]Khodagholi, F., Eftekharzadeh, B., Yazdanparast, R., A new artificialchaperone for protein refolding: Sequential use of detergent and al-[104]Lu, D., Liu, Z. X., Zhang, M. L., Liu, Z. et al., The mechanism of PNIPAAm-assisted refolding of lysozyme denatured by urea.[105]Morimoto, N., Ogino, N., Narita, T., Akiyoshi, K., Enzyme-respon-sive artificial chaperone system with amphiphilic amylose primer.[106]Nomura, Y., Ikeda, M., Yamaguchi, N., Aoyama, Y. et al., Protein refolding assisted by self-assembled nanogels as novel artificial[107]Hirakura, T., Nomura, Y., Aoyama, Y., Akiyoshi, K., Photorespon-sive nanogels formed by the self-assembly of spiropyrane-bearingpullulan that act as artificial molecular chaperones. [108]Sakono, M., Kawashima, Y., Ichinose, H., Maruyama, T. et al., Direct refolding of inclusion bodies using reversed micelles.[109]Luo, M., Guan, Y.-X., Yao, S.-J., On-column refolding of denaturedlysozyme by the conjoint chromatography composed of SEC andJ. Chromatogr. B.[110]Yazdanparast, R., Esmaeili, M. A., Khodagholi, F., Control of aggregation in protein refolding: Cooperative effects of artificialInt. J. Biol. Macromol. [111]Yamamoto, E., Yamaguchi, S., Sasaki, N., Kim, H.-B. et al., Artificialchaperone-assisted refolding in a microchannel. [112]Nohara, D., Matsubara, M., Tano, K., Sakai, T., Design of optimumrefolding solution by combination of reagents classified by specif-[113]Dong, X. Y., Shi, J. H., Sun, Y., Cooperative effect of artificial chap-erones and guanidinium chloride on lysozyme renaturation at high[114]Nian, R., Kim, D. S., Tan, L., Kim, C.-W. et al., Synergistic coordina-tion of polyethylene glycol with ClpB/DnaKJE bichaperone for re-folding of heat-denatured malate dehydrogenase.Biotechnol. Prog.[115]Guan, Y.-X., Fei, Z.-Z., Luo, M., Jin, T. et al., Chromatographic re-folding of recombinant human interferon gamma by an immobi-lized sht GroEL191-345 column. J. Chromatogr. A [116]Shimizu, H., Fujimoto, K., Kawaguchi, H., Improved refolding of denatured/reduced lysozyme using disulfide-carrying polymeric[117]Woycechowsky, K. J., Wittrup, K. D., Raines, R. T., A small-moleculecatalyst of protein folding in vitro and in vivo.[118]Esnaeili, M. A., Yazdanparast, R., Beta-cyclodextrin-bonded silicaassists alkaline phosphatase and carbonic anhydrase refolding in asolid phase assisted refolding approach. [119]Badruddoza, A. Z. M., Hidajat, K., Uddin, M. S., Synthesis and char-acterization of ß-cyclodextrin-conjugated magnetic nanoparticlesand their uses as solid-phase artificial chaperones in refolding ofcarbonic anhydrase bovine. J. Colloid Interface Sci. [120]Basu, A., Li, X., Leong, S. S. J., Refolding of proteins form inclusionbodies: Rational design and recipes. Appl. Microbiol. Biotechnol.