A.Kumar et al.Prog.Polym.Sci.32 (2007)1205-1237 1215 [104 n that of t separation and thus r in o the olymer from renatured proteins can be easily achieved under the o 4).This is due to the following refoldin non-denaturing temperature,upon protein refold- protein being refolded via hydrophobic interaction. ing.The mechanism of PNiPAAm-assisted protein As a result,an improved refolding yield is obtained. folding on bovine carbonic anhydrase B as model particularly at a high temperature [108).In another work,dextran-grafted-PNiPAAm (DGP)was pre um pared and artif assis prot refolding of me intermediates via hydro In this eas the func on of tu 11051.Another a pplication of pNiPaam for the phobic gment helps to form the comple renaturation of B-lactamase from inclusion bodies protein being refolded and the presence of hydro has shown that PNiPAAm was more effective than philic segment is to accommodate and disperse the PEG in enhancing protein renaturation.At a folded or partially folded protein. The result has concentration of 0.1%(w/v),PNiPAAm improved shown that DGP favors high refolding yield as by41% olding assisted by surfactan hydr ws.the which h total lysozyme activity reco bout 70%6 ir such a way to match the tein during its refolding process and also the demands of batch dilution.The mechanism revealed that when different protein refolding PNiPAAm gels were added into the refolding Eudragit S-100.a pH-responsive polymer is buffer,the hydrophobic interactions between dena supp to increase the rate of refolding and tured proteins and gels could prevent the refolding percentage of den ature prote and this aggrega to as 1g0 (10 protei nind red 7.Recently. Liu et e polyme ature-responsive PNiPAAm grafted with uld be regained within 10 min during the refolding B-cyclodextrin (a weakly hydrophobic stripper) study.It has been proposed that Eudragit S-100 for protein refolding.where cetyltrimethylammo- could help in reversing protein aggregation in nium bromide (CTAB)was taken as surfactant amyloid based diseases [109].Eudragit S-100 has Lysozyme was used as model protein and the also been exploited for simultaneous refolding and CTAB r the result show that not only purification of xylanase.It has been found that B-denatured lysozyme microwave-treated Eudragit S-100 also gave better B-CD-g-PNIPAAM →O+ Unfolded compierte B-CD-g-PNIPAAM ctan Fig.4.Schematic view of the stimuli-artificial cha assisted protein refolding in
molecular assemblies of SP [104]. Since the LCST of PNiPAAm is only slightly higher than that of the ambient conditions for protein processing, the separation and thus recycling of the polymer from renatured proteins can be easily achieved under non-denaturing temperature, upon protein refolding. The mechanism of PNiPAAm-assisted protein folding on bovine carbonic anhydrase B as model protein was investigated and the results of fluorescence analysis and equilibrium studies indicate that PNiPAAm enhances protein refolding by the formation of complexes with aggregation-prone folding intermediates via hydrophobic interactions [105]. Another application of PNiPAAm for the renaturation of b-lactamase from inclusion bodies has shown that PNiPAAm was more effective than PEG in enhancing protein renaturation. At a concentration of 0.1% (w/v), PNiPAAm improved the yield of b-lactamase activity by 41% compared to 26% with PEG [106]. PNiPAAm gels had also been used in renaturation of lysozyme. With the addition of fast responsive PNiPAAm gel beads, the total lysozyme activity recovery was about 70% in 3 h, as compared to about 40% achieved by simple batch dilution. The mechanism revealed that when PNiPAAm gels were added into the refolding buffer, the hydrophobic interactions between denatured proteins and polymer gels could prevent the aggregation of refolding intermediates, thus enhancing protein renaturation [107]. Recently, Liu et al [108], prepared an artificial chaperon, composed of temperature-responsive PNiPAAm grafted with b-cyclodextrin (a weakly hydrophobic stripper) for protein refolding, where cetyltrimethylammonium bromide (CTAB) was taken as surfactant. Lysozyme was used as model protein and the result showed that b-CD-g-PNiPAAm not only strips CTAB from the CTAB-denatured lysozyme complex with the b-CD segment, which was proved by fluorescence emission spectroscopy, but also inhibits the formation of protein aggregates during the following refolding step (Fig. 4). This is due to the PNiPAAm segment that interacts with the protein being refolded via hydrophobic interaction. As a result, an improved refolding yield is obtained, particularly at a high temperature [108]. In another work, dextran-grafted-PNiPAAm (DGP) was prepared and characterized for its use as artificial chaperon to assist protein refolding of model proteins like lysozyme and bovine carbonic anhydrase. In this case, the function of tunable hydrophobic segment helps to form the complex with protein being refolded and the presence of hydrophilic segment is to accommodate and disperse the folded or partially folded protein. The result has shown that DGP favors high refolding yield as compared to refolding assisted by surfactant. Here the hydrophobicity can be tuned by varying the temperature ranges from above the LCST of DGP to lower temperature, which can be programmed in such a way to match the protein hydrophobicity during its refolding process and also the demands of different protein refolding. Eudragit S-100, a pH-responsive polymer is supposed to increase the rate of refolding and refolding percentage of denatured protein and this was found to assist refolding of a-chymotrypsin, which is known to bind to the polymer rather nonspecifically. Complete activity of a-chymotrypsin could be regained within 10 min during the refolding study. It has been proposed that Eudragit S-100 could help in reversing protein aggregation in amyloid based diseases [109]. Eudragit S-100 has also been exploited for simultaneous refolding and purification of xylanase. It has been found that microwave-treated Eudragit S-100 also gave better ARTICLE IN PRESS Fig. 4. Schematic view of the mechanism of ‘‘temperature-stimuli-artificial chaperone’’ assisted protein refolding in vitro. A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1215
1216 A.Kumar et al.Prog.Polym.Sel.32 (2007)1205-1237 results as compared to untreated Eudragit S-100 in formed.Thus.an attractive system for a surface terms of refolding/purification after denaturation grafting is formed with large interconnected pores with 8M urea.It is believed that polymers bind to ensuring high surface area available for grafting and hydrophobic sites on the proteins and prevent efficient mass transport of monomers.Currently aggregation [110]. most of the work has focused on hydrogels tha harply to sm change s in temperature o n ar he endo ie nd her have gate of bioche strength the biomolecular 11-31.Som gels also have been red to in basic life sciences.The application of SPand thei respond to specific biomolecule or chemical triggers bioconjugates in solution have thus shown potential such as glucose [115.116].This stimulus response of and cost-effective applications in bioseparation of gels makes it possible to exploit them as smart proteins and othe bioparticles for basic life science and numerous applications of these res and een p in m have reversib to nev e the netu with hette of th otic fo high vield.Furthermore.ongoing research is car. the Flory equation [117]: expressed ving the new applications of SPs in solution form and investigating different biological and non- P=RT[In(1-0)++zo2 biological systems. +V.(./oo3-/2 3.Covalently cross-linked,reversible and physical Here,V,is the volum T the The most extensive ations and es-lin heen ca rried out on hyd prepared gel.In Flor quation,the first three term solution These smart gels are represent a"swelling force"of the network due to conventional procedure wherein synthesis takes the energetically favorable mixing of polymer chains place at room temperature and provides hydrogels with the solvent molecules,while the last term is an of small pore sizes. Smart macroporous hydrogels "elastic retractive force" which tries to bring the network how are re papers e ck to its unstrained state. he equil the lts from a anc 10 Thus fo of th froe cnditions and providing ther with inte ndent on its c slink der ing properties have emerged which are called Volume transitions are discontinuous for networks cryogels [114].Cryogels are obtained at tempera which have charged polymer chains and/or stifl tures below the melting temperature of the solvent chains Whereas phase transitions in chemically At subzero temperatures most of the solvent is ross-linked networks are well understood. the frozen while the dissolved substances are concer phase transitions in physically cross-linked net trate in sh nqui WOrK hyar ogen-be net Ork hav ase t of th only recently e physica hase the local monomer cor is muc on.The higher than the monomer concentration in the equilibrium initial reaction mixture.The gel formation occurs swelling capacity and the phase transitions in such in this liquid microphase and the crystals of frozen gels[118]. solvents perform like porogen.After melting the ice The thermoresponsive. PNiPAAm gels have crystals.a system of large interconnected pores is attracted great attention for their scientific interest
results as compared to untreated Eudragit S-100 in terms of refolding/purification after denaturation with 8 M urea. It is believed that polymers bind to hydrophobic sites on the proteins and prevent aggregation [110]. Hence, the properties of linear free chains of SPs in solution are well studied and endowed for the above-discussed applications. These studies advance the understanding of biochemical processes and biomolecular interactions of various biomolecules in basic life sciences. The application of SP and their bioconjugates in solution have thus shown potential and cost-effective applications in bioseparation of proteins and other bioparticles for basic life sciences research and other industrial applications. The application of SP in solution form have shown promise to new advancement of protein folding procedure as well, as it enhanced and rectified the protein refolding process with better outcome and high yield. Furthermore, ongoing research is carving the new applications of SPs in solution form and investigating different biological and nonbiological systems. 3. Covalently cross-linked, reversible and physical gels The most extensive investigations on SPs have been carried out on hydrogels that swell in aqueous solutions. These smart gels are synthesized through conventional procedure wherein synthesis takes place at room temperature and provides hydrogels of small pore sizes. Smart macroporous hydrogels have also been synthesized by various approaches, which show different applications which are reviewed in recent papers [111–113]. Recently hydrogel of large pore size synthesized in moderately frozen conditions and providing them with interesting properties have emerged which are called cryogels [114]. Cryogels are obtained at temperatures below the melting temperature of the solvent. At subzero temperatures most of the solvent is frozen, while the dissolved substances are concentrated in small non-frozen regions, so called ‘‘liquid microphase’’. As the volume of the non-frozen liquid microphase is much less than that of the solid phase, the local monomer concentration is much higher than the monomer concentration in the initial reaction mixture. The gel formation occurs in this liquid microphase and the crystals of frozen solvents perform like porogen. After melting the ice crystals, a system of large interconnected pores is formed. Thus, an attractive system for a surface grafting is formed with large interconnected pores ensuring high surface area available for grafting and efficient mass transport of monomers. Currently most of the work has focused on hydrogels that respond sharply to small changes in temperature or pH [5]. But other gels have also been investigated that respond to changes in ionic strength, solvent, light intensity, and electric or magnetic fields [11–13]. Some gels also have been engineered to respond to specific biomolecule or chemical triggers, such as glucose [115,116]. This stimulus response of gels makes it possible to exploit them as smart materials and numerous applications of these materials have been established. The reversible volume phase transition in gels occurs because of the ‘‘osmotic forces’’ which swell or collapse the network structure. The basic features of the osmotic forces are expressed qualitatively by the Flory equation [117]: p ¼ RTflnð1 ^Þ þ ^ þ w^2 þ Vsðne=V0Þð^1=3 ^=2Þg. Here, Vs is the molar volume of the solvent, ø the volume fraction of the network, R the gas constant, T the absolute temperature, w the interaction parameter and (ne/V0) is cross-linked density in prepared gel. In Flory equation, the first three terms represent a ‘‘swelling force’’ of the network due to the energetically favorable mixing of polymer chains with the solvent molecules, while the last term is an ‘‘elastic retractive force’’ which tries to bring the network back to its unstrained state. The equilibrium swelling capacity of the gel results from a balance of these two forces. Thus, for a given gel-solvent system, the swelling capacity of the gel is strongly dependent on its cross-link density. Volume transitions are discontinuous for networks which have charged polymer chains and/or stiff chains. Whereas phase transitions in chemically cross-linked networks are well understood, the phase transitions in physically cross-linked networks (e.g. hydrogen-bonded networks) have gained attention only recently. The physical crosslinks are weak and temporary and can be disrupted reversibly by imposing a deformation. Therefore, deformation is likely to affect the equilibrium swelling capacity and the phase transitions in such gels [118]. The thermoresponsive, PNiPAAm gels have attracted great attention for their scientific interest ARTICLE IN PRESS 1216 A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237