1210 A.Kumar et al.Prog.Polym.Sci.32 (2007)1205-1237 (EHEC),ethylene oxide-propylene oxide (EOPO) stages and enables a 10-fold enzyme concentration random co-polymer and EOPO block co-polymer while maintaining more than 95%of the initial [55,56].There are also other examples wherein enzyme activity.Such system shows cost viability as thermoresponsive polymers such as EOPO co- compared to many polymer/polymer and polymer/ polymers [57,58]or poly(N-vinylcaprolactam-co- salt aqueous two-phase extraction systems [60]. vinyl imidazole)[54]form two-phase systems with Partitioning of pure a-amylase and amyloglucosi- dextran and have been used to purify proteins. dase as well as cell-free extract of a hyperthermo- Aqueous two-phase systems have even been formed stable a-amylase in different ATPSs has demon- with polymers where both polymers are thermo- strated the potential for partitioning of enzymes responsive and it is possible to recycle both used in extractive bioconversion of starch.The polymers by temperature-induced phase separation partition behavior of pure a-amylase and amylo- [59].This is a modified and improved form of the glucosidase in four ATPSs,namely,PEO-PPO/ ATPS system than the generally used system where (NH)2SO4,PEO-PPO/MgSO4,polyethylene glycol one of the polymers is thermoresponsive and the (PEG)/(NH4)2SO4,and PEG/MgSO4 has also been other polymer is dextran or a starch derivative.The evaluated [61].The partitioning behavior of three polymers mostly used in these works are proteins (lysozyme,BSA,and apolipoprotein A-1) EO50PO50,a random co-polymer of 50%ethylene in water/HM-EOPO two-phase systems has been oxide (EO)and 50%propylene oxide (PO),and a studied and the effect of various ions,pH,and hydrophobically modified random co-polymer of temperature on protein partitioning was monitored. EO and PO with aliphatic C14H29-groups coupled This approach has useful potential as it involves to each end of the polymer (HM-EOPO).In only one polymer for phase formation [62].BSA aqueous solution both polymers will phase separate and lysozyme were partitioned in the thermosepa- above a critical temperature (cloud point for rated water/HM-EO two-phase system of the EO50%PO50%50C,HM-EOPO,14C)and this cationic polymer at different pH,salt and SDS will for both polymers lead to formation of an concentrations [63].The use of both a low-cost upper water phase and a lower polymer enriched starch derivative (maltodextrin)as replacement for phase.When EO50PO50 and HM-EOPO are mixed dextran and a co-polymer of thermoreactive EOPO in water,the solution will separate in two phases was investigated.The partitioning behavior of three above a certain concentration i.e.an aqueous two- model proteins:BSA,lysozyme and trypsin was phase system is formed analogous to PEG/dextran analyzed in order to evaluate the capability of this system.The partitioning of three proteins,bovine novel ATPS for protein separation and it was found serum albumin(BSA),lysozyme and apolipoprotein that the protein recovery was in the range of A-1,has been studied in the EO50PO50/HM- 60-98%[64].A new type of ATPS has recently EOPO system.It was shown that the yield of 78% been established which uses modified starch deriva- and purification factor 5.5 of apolipoprotein A-1 tive and thermoresponsive polymer of VCL as phase can be achieved [59].Aqueous two-phase partition- forming polymers [52].It is also reported that ing of endo-polygalacturonase(endo-PG)produced thermoseparating ATPS for extraction of recombi- by Kluyveromyces marxianus strains was carried out nant cutinase fusion protein from E.coli homo- on systems containing the thermoseparating poly- genate can be scaled up to pilot scale [65].The mer Ucon 50-HB-5100 (a random co-polymer of application of pH-responsive polymers like poly- 50%EO and 50%PO)as one of the phase-forming ethyleneoxide-maleic acid co-polymer [66]as phase- component.On testing the partitioning efficiency forming polymers in ATPS has also been reported. of the enzyme on different ATPSs comprised of The polymers,just like proteins,contain two pH- Ucon 50-HB-5100 (Ucon)/polyvinyl alcohol (PVA triggerable functionalities (NH-and COO-)that 10,000),Ucon 50-HB-5100/hydroxypropyl starch make them exhibit pH-responsive behavior.Poly- (Reppal PES100)and Ucon 50-HB-5100/ diallylaminoethanoate-dimethyl sulfoxide(PAEDS) (NH4)2SO4 it was found that Ucon 50-HB-5100/ co-polymer is a polyelectrolyte that is almost (NH4)2SO4 was the most efficient for enzyme completely water-insoluble in acidic conditions. partitioning,in comparison with total protein which This behavior makes it a potential candidate for strongly partitioned to the salt-rich phase at 22C. industrial applications since it can be effectively The proposed separation scheme for endo-PG recovered from solution by pH-controlled precipita- purification consists of three in series extraction tion.Furthermore,in applications such as protein
(EHEC), ethylene oxide–propylene oxide (EOPO) random co-polymer and EOPO block co-polymer [55,56]. There are also other examples wherein thermoresponsive polymers such as EOPO copolymers [57,58] or poly(N-vinylcaprolactam-covinyl imidazole) [54] form two-phase systems with dextran and have been used to purify proteins. Aqueous two-phase systems have even been formed with polymers where both polymers are thermoresponsive and it is possible to recycle both polymers by temperature-induced phase separation [59]. This is a modified and improved form of the ATPS system than the generally used system where one of the polymers is thermoresponsive and the other polymer is dextran or a starch derivative. The polymers mostly used in these works are EO50PO50, a random co-polymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO), and a hydrophobically modified random co-polymer of EO and PO with aliphatic C14H29–groups coupled to each end of the polymer (HM–EOPO). In aqueous solution both polymers will phase separate above a critical temperature (cloud point for EO50%PO50% 50 1C, HM–EOPO, 14 1C) and this will for both polymers lead to formation of an upper water phase and a lower polymer enriched phase. When EO50PO50 and HM–EOPO are mixed in water, the solution will separate in two phases above a certain concentration i.e. an aqueous twophase system is formed analogous to PEG/dextran system. The partitioning of three proteins, bovine serum albumin (BSA), lysozyme and apolipoprotein A-1, has been studied in the EO50PO50/HM– EOPO system. It was shown that the yield of 78% and purification factor 5.5 of apolipoprotein A-1 can be achieved [59]. Aqueous two-phase partitioning of endo-polygalacturonase (endo-PG) produced by Kluyveromyces marxianus strains was carried out on systems containing the thermoseparating polymer Ucon 50-HB-5100 (a random co-polymer of 50% EO and 50% PO) as one of the phase-forming component. On testing the partitioning efficiency of the enzyme on different ATPSs comprised of Ucon 50-HB-5100 (Ucon)/polyvinyl alcohol (PVA 10,000), Ucon 50-HB-5100/hydroxypropyl starch (Reppal PES100) and Ucon 50-HB-5100/ (NH4)2SO4 it was found that Ucon 50-HB-5100/ (NH4)2SO4 was the most efficient for enzyme partitioning, in comparison with total protein which strongly partitioned to the salt-rich phase at 22 1C. The proposed separation scheme for endo-PG purification consists of three in series extraction stages and enables a 10-fold enzyme concentration while maintaining more than 95% of the initial enzyme activity. Such system shows cost viability as compared to many polymer/polymer and polymer/ salt aqueous two-phase extraction systems [60]. Partitioning of pure a-amylase and amyloglucosidase as well as cell-free extract of a hyperthermostable a-amylase in different ATPSs has demonstrated the potential for partitioning of enzymes used in extractive bioconversion of starch. The partition behavior of pure a-amylase and amyloglucosidase in four ATPSs, namely, PEO–PPO/ (NH4)2SO4, PEO–PPO/MgSO4, polyethylene glycol (PEG)/(NH4)2SO4, and PEG/MgSO4 has also been evaluated [61]. The partitioning behavior of three proteins (lysozyme, BSA, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied and the effect of various ions, pH, and temperature on protein partitioning was monitored. This approach has useful potential as it involves only one polymer for phase formation [62]. BSA and lysozyme were partitioned in the thermoseparated water/HM-EO two-phase system of the cationic polymer at different pH, salt and SDS concentrations [63]. The use of both a low-cost starch derivative (maltodextrin) as replacement for dextran and a co-polymer of thermoreactive EOPO was investigated. The partitioning behavior of three model proteins: BSA, lysozyme and trypsin was analyzed in order to evaluate the capability of this novel ATPS for protein separation and it was found that the protein recovery was in the range of 60–98% [64]. A new type of ATPS has recently been established which uses modified starch derivative and thermoresponsive polymer of VCL as phase forming polymers [52]. It is also reported that thermoseparating ATPS for extraction of recombinant cutinase fusion protein from E. coli homogenate can be scaled up to pilot scale [65]. The application of pH-responsive polymers like polyethyleneoxide–maleic acid co-polymer [66] as phaseforming polymers in ATPS has also been reported. The polymers, just like proteins, contain two pHtriggerable functionalities (NH3 +– and COO– –) that make them exhibit pH-responsive behavior. Polydiallylaminoethanoate-dimethyl sulfoxide (PAEDS) co-polymer is a polyelectrolyte that is almost completely water-insoluble in acidic conditions. This behavior makes it a potential candidate for industrial applications since it can be effectively recovered from solution by pH-controlled precipitation. Furthermore, in applications such as protein ARTICLE IN PRESS 1210 A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237
A.Kumar et al.Prog.Polym.Sci.32 (2007)1205-1237 1211 partitioning,the protein-like structure of the poly- bifunctional mode of affinity precipitation does not mer is expected to enhance protein-polymer inter- utilize SP,it thus falls beyond the scope of this actions [67]. review.Hetero-bifunctional format of affinity pre- More interesting application has been shown for cipitation is a more general approach,wherein the separation of animal cells by coupling an affinity ligands are covalently coupled to soluble- antibody (against a cell surface protein)to a tempe- insoluble polymers to form an affinity macroligand rature-sensitive SP such as PNiPAAm(Fig.2).The (AML).The macroligands could be synthesized ATPS composed of PEG and dextran was devel- either by covalent linking of the ligands (directly or oped where PNiPAAm was used as a ligand carrier through a short spacer)or by co-polymerization of for specific separation of animal cells.Monoclonal ligands to a water-soluble SP.An ideal polymer for antibodies were conjugated with the carrier and affinity precipitation must contain reactive groups added to the polyethylene glycol 8000-dextran T500 for ligand coupling,show moderate interaction with aqueous two-phase system.About 80%of the the ligand or impurities to prevent non-specific co- animal cells which specifically bind to the antibody- precipitation of impurities,give complete phase polymer conjugate partitioned to the top phase of separation of the polymer upon a change of medium ATPS.As a model system,CD34-positive human property,form polymer precipitates that are com- acute myeloid leukemia cells (KG-1)were specifi- pact,to allow easy separation and to exclude cally separated from human T lymphoma cells trapping of impurities into a gel structure,be easily (Jurkat)by applying anti-CD34 conjugated with solubilized after the precipitate is formed,the PNiPAAm in the ATPS [53]. precipitation-solubilization cycle must be repeata- ble many times with good recovery,be available and 2.1.2.Affinity precipitation cost effective. The selective precipitation of a target molecule The polymer-ligand conjugate firstly forms a from a mixture is a very attractive approach in complex with the target protein and phase separa- bioseparations.Precipitation can be highly selective tion of the complex is triggered by small changes in technique for protein purification or enrichment. environment,resulting in transition of backbone Traditionally precipitation of the target protein is into an insoluble state.The target protein is then achieved by the addition of large amounts of salts, either eluted from insoluble macroligand-protein like ammonium sulfate,organic solvents miscible complex or the precipitate is dissolved,the protein with water,like acetone or ethanol or by the gets dissociated from the macroligand and the addition of polymers,like PEG [68].It is not ligand-polymer conjugate is reprecipitated without expected to have high selectivity to be achieved by the protein which remains in the supernatant in a traditional precipitation techniques,as the selectiv- purified form (Fig.3).Various ligands,such as ity of precipitation is limited to the differences in triazine dyes,sugars,protease inhibitors,antibodies, integral surface properties of protein molecules. and nucleotides have been successfully used for Thus,the introduction of high selectivity to the affinity precipitation. precipitation techniques is of great importance. There is a range of different proteins/enzymes Affinity precipitation of proteins using SP emerged which have been purified successfully by affinity in the early 1980s.Since then it has evolved as a precipitation using pH-responsive polymers [72].In technique capable of simple,fast,and efficient general,a specific ligand is chemically coupled to the purification of a variety of proteins [69-71].As a polymer backbone which latter binds to the target general rule,there are five basic steps in affinity protein in solution and the protein-polymer com- precipitation:(i)carrying out affinity interactions in plex is precipitated by change of pH as it renders the free solution,(ii)precipitation of the affinity polymer backbone insoluble.But in some cases the reagent-target protein complex from the solution, polymer itself has the affinity for the target protein (iii)recovery of the precipitate,(iv)dissociation and and the polymer acts as a macroligand.Chitosan recovery of the target molecule from the complex, was used to precipitate lysozyme or lectins such and finally,(v)recovery of the affinity reagent. as wheat germ agglutinin and similarly Eudragit Affinity precipitation methods have two main S-100 was used as a macroligand for the binding approaches which have been described in the and precipitating xylanase or lactate dehydrogenase literature [70],as precipitation with homo-and or endopoly-galacturonase [73,74].The pH-respon- hetero-bifunctional ligands.However,as the homo- sive SP have been used successfully in affinity
partitioning, the protein-like structure of the polymer is expected to enhance protein–polymer interactions [67]. More interesting application has been shown for the separation of animal cells by coupling an antibody (against a cell surface protein) to a temperature-sensitive SP such as PNiPAAm (Fig. 2). The ATPS composed of PEG and dextran was developed where PNiPAAm was used as a ligand carrier for specific separation of animal cells. Monoclonal antibodies were conjugated with the carrier and added to the polyethylene glycol 8000-dextran T500 aqueous two-phase system. About 80% of the animal cells which specifically bind to the antibody– polymer conjugate partitioned to the top phase of ATPS. As a model system, CD34+-positive human acute myeloid leukemia cells (KG-1) were specifi- cally separated from human T lymphoma cells (Jurkat) by applying anti-CD34 conjugated with PNiPAAm in the ATPS [53]. 2.1.2. Affinity precipitation The selective precipitation of a target molecule from a mixture is a very attractive approach in bioseparations. Precipitation can be highly selective technique for protein purification or enrichment. Traditionally precipitation of the target protein is achieved by the addition of large amounts of salts, like ammonium sulfate, organic solvents miscible with water, like acetone or ethanol or by the addition of polymers, like PEG [68]. It is not expected to have high selectivity to be achieved by traditional precipitation techniques, as the selectivity of precipitation is limited to the differences in integral surface properties of protein molecules. Thus, the introduction of high selectivity to the precipitation techniques is of great importance. Affinity precipitation of proteins using SP emerged in the early 1980s. Since then it has evolved as a technique capable of simple, fast, and efficient purification of a variety of proteins [69–71]. As a general rule, there are five basic steps in affinity precipitation: (i) carrying out affinity interactions in free solution, (ii) precipitation of the affinity reagent–target protein complex from the solution, (iii) recovery of the precipitate, (iv) dissociation and recovery of the target molecule from the complex, and finally, (v) recovery of the affinity reagent. Affinity precipitation methods have two main approaches which have been described in the literature [70], as precipitation with homo- and hetero-bifunctional ligands. However, as the homobifunctional mode of affinity precipitation does not utilize SP, it thus falls beyond the scope of this review. Hetero-bifunctional format of affinity precipitation is a more general approach, wherein affinity ligands are covalently coupled to soluble– insoluble polymers to form an affinity macroligand (AML). The macroligands could be synthesized either by covalent linking of the ligands (directly or through a short spacer) or by co-polymerization of ligands to a water-soluble SP. An ideal polymer for affinity precipitation must contain reactive groups for ligand coupling, show moderate interaction with the ligand or impurities to prevent non-specific coprecipitation of impurities, give complete phase separation of the polymer upon a change of medium property, form polymer precipitates that are compact, to allow easy separation and to exclude trapping of impurities into a gel structure, be easily solubilized after the precipitate is formed, the precipitation–solubilization cycle must be repeatable many times with good recovery, be available and cost effective. The polymer–ligand conjugate firstly forms a complex with the target protein and phase separation of the complex is triggered by small changes in environment, resulting in transition of backbone into an insoluble state. The target protein is then either eluted from insoluble macroligand–protein complex or the precipitate is dissolved, the protein gets dissociated from the macroligand and the ligand–polymer conjugate is reprecipitated without the protein which remains in the supernatant in a purified form (Fig. 3). Various ligands, such as triazine dyes, sugars, protease inhibitors, antibodies, and nucleotides have been successfully used for affinity precipitation. There is a range of different proteins/enzymes which have been purified successfully by affinity precipitation using pH-responsive polymers [72]. In general, a specific ligand is chemically coupled to the polymer backbone which latter binds to the target protein in solution and the protein–polymer complex is precipitated by change of pH as it renders the polymer backbone insoluble. But in some cases the polymer itself has the affinity for the target protein and the polymer acts as a macroligand. Chitosan was used to precipitate lysozyme or lectins such as wheat germ agglutinin and similarly Eudragit S-100 was used as a macroligand for the binding and precipitating xylanase or lactate dehydrogenase or endopoly-galacturonase [73,74]. The pH-responsive SP have been used successfully in affinity ARTICLE IN PRESS A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1211
1212 A.Kumar et al.Prog.Polym.ScL.32(2007)1205-1237 copper ading with dissociation of the complex by imidazole addition Keys:re polymer 在 ·copper io \②impurities imidazole Fig.3.Scheme of metal chelate affinity precipitation of proteins.Reproduced from 179]with permission e of the metal ions and the target tein binds the metal polymer which shows some non-specific interactions with other proteins between the histidine on the protein and the metal a more general use has not taken place for these ion.Many proteins both containing natural metal- polymers. on binding residues and recombinant proteins On the other hand.a thermoresponsive polymer containing His-tag residues have been purified using late atnnity precipitation [/8].I herelo sprea agge cles (with gaine h the co-ordi groups chromatographic mat like n al chelating affinity metal loaded r m the mixt precipitation 75-781.By combining the versatile The precipitated compex is solubilized by reversin properties of metal affinity with affinity precipita- the precipitation conditions and the target molecule tion,the technique presents enormous potential as a is dissociated from the precipitated polymer by selective separation strategy and makes this metho using imidazole or EDTA as eluting agent.The rge proc at elevate esta VC In ad y,pur 1791 as in netal-chelate ingle His. D ecipitation.In metal chelating affinity precipit ments)from recombinant escherichia coli cell tion,metal ligands like imidazole are covalently culture broth was performed.Quantitative precipi- coupled to the reversible soluble-insoluble SP by tation of the Hiso-scFv fragments was tested at radical co-polymerization 80.The co-polymers different loads of the cell supernatant using Cu(II) carrying metal-chelating ligands are charged with and Ni(Il)loaded co-polymers of vinylimidazole
precipitation of many proteins, but because of the charged character of the polymer which shows some non-specific interactions with other proteins a more general use has not taken place for these polymers. On the other hand, a thermoresponsive polymer is expected to deliver better performance, because of its uncharged nature. Their wide spread application by using metal as affinity ligand has gained usefulness by adopting the technique in a nonchromatographic format like metal chelating affinity precipitation [75–78]. By combining the versatile properties of metal affinity with affinity precipitation, the technique presents enormous potential as a selective separation strategy and makes this method more simple and cost effective when the intended applications are for large-scale processes. Extensive efforts are being made in this direction for establishing thermosensitive polymers, PNiPAAm or PVCL [79] as effective SPs in metal-chelate affinity precipitation. In metal chelating affinity precipitation, metal ligands like imidazole are covalently coupled to the reversible soluble–insoluble SP by radical co-polymerization [80]. The co-polymers carrying metal-chelating ligands are charged with metal ions and the target protein binds the metalloaded co-polymer in solution via the interaction between the histidine on the protein and the metal ion. Many proteins both containing natural metalion binding residues and recombinant proteins containing His-tag residues have been purified using metal chelate affinity precipitation [78]. Therefore, His-tagged protein or cells or bioparticles (with surface accessible co-ordinating groups) can be purified through the precipitation of target molecule–metal loaded polymer complex from the mixture. The precipitated complex is solubilized by reversing the precipitation conditions and the target molecule is dissociated from the precipitated polymer by using imidazole or EDTA as eluting agent. The biomolecule is recovered from the co-polymer by precipitating the latter at elevated temperature in the presence of NaCl. In a recent study, purification of extracellularly expressed six histidine-tagged single chain Fv-antibody fragments (His6-scFv fragments), from recombinant Escherichia coli cell culture broth was performed. Quantitative precipitation of the His6-scFv fragments was tested at different loads of the cell supernatant using Cu(II) and Ni(II) loaded co-polymers of vinylimidazole ARTICLE IN PRESS Fig. 3. Scheme of metal chelate affinity precipitation of proteins. Reproduced from [79] with permission. 1212 A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237
A.Kumar et al.Prog.Polym.Sci.32 (2007)1205-1237 1213 and NiPAAn (-poly( am than for Cu(-p M(VI-NiPAAm).but selectivity etal-binding was better in the former case.The bound His fragments were recovered almost completely charged with Ni(ll)were able to interact with a (>95%)by elution with 50mM EDTA buffer,pH His-tag on the target proteins.Purifications of two 8.077八. His-tagged enzymes.B-D-galactosidase and chlor- Besides protein metal-ion amphenicol a etyltransferase,were used to demon- charged )can singl strate th application nded using this al affinity prec h and plasmid DNA by affinity pitation [811.The separation method utilizes the interaction of meta idazole and over 85 was observed in both cases.The recovered ELPs ions to the aromatic nitrogens in exposed purines in were reused with no observable decrease in the single-stranded nucleic acids [82].Alternatively purification performance.This has been the first plasmid DNA can also be selectively captured at report exploiting the features of ELPs for protein all scales with an appropriate amount AM on affinity condition he capabilit purin ons by mp ggers and whose ran 人 eful hod in future.not only for purific ation but also fo affinity interaction)by increasing the temperature diverse applications in bioseparation such as dna the Aml-plasmid DNa complex is precipitated purification and environmental remediation [86] After filtration and washing.the precipitate is re- Another interesting example has been a one-po dissolved and the specifically bound plasmid DNA affinity precipitation purification of carbohydrate binding pro tein reported A (Co 4 an by Sun et al ly respons whea ctin poly hich used in the t inant Pblock ELP. or polysaccharide-containing cor nounds such as rate-binding n glucan [84).The thermally reversible soluble-inso- was demonstrated luble PNiPAAm-dextran derivative(DD)conjugate Other types of metal-chelating polymers for affinity has been synthesized by conjugating amino-termi precipitation of proteins were reported b nated PNiPAAm to DD via ethyl-3-(3-dimethy highly branched co-polymers o NiPAAm and 1.2 oe wa (UM mon es fr pol clo es I AAm-c MA ously immu nized with the derivatized dextran 241 transfer ows the imidazole functionality in the of the reneating tide.VPGVG which The LCST of the co-polymers can be controlled by behave very similar to PNiPAAm polymers have the amount of hydrophobic and GMA co-monomers been shown to undergo reversible phase transi tions within a wide range of conditions [85,86] The co-polymers These have been us is terminal to purly a His- fic syste 87.88 BRCA- ent (a prote on affinity on via t o LP rpeaune' ed ly m Itose for affi- nce of mity pre cipitation of thermolabile a-glucosidase was
(VI) and NiPAAm [Cu(II)-poly(VI-NiPAAm) and Ni(II)-poly(VI-NiPAAm)]. The precipitation effi- ciency with Ni(II)-poly(VI-NiPAAm) was lower than for Cu(II)-poly(VI-NiPAAm), but selectivity was better in the former case. The bound His6-scFv fragments were recovered almost completely (495%) by elution with 50 mM EDTA buffer, pH 8.0 [77]. Besides protein purification, the metal-ion charged co-polymer of poly(VI-NiPAAm) can also be applied for the separation of single stranded nucleic acids like RNA from double stranded linear and plasmid DNA by affinity precipitation [81]. The separation method utilizes the interaction of metal ions to the aromatic nitrogens in exposed purines in single-stranded nucleic acids [82]. Alternatively plasmid DNA can also be selectively captured at all scales with an appropriate amount of AML under appropriate affinity conditions. The AML in this case is a conjugate between oligomeric AML precursor and a single-stranded oligonucleotide, whose sequence is complementary to a specific affinity motif in the plasmid DNA (triple helix affinity interaction). By increasing the temperature the AML-plasmid DNA complex is precipitated. After filtration and washing, the precipitate is redissolved and the specifically bound plasmid DNA is released [83]. Similarly lectins, concanavalin A (ConA) and wheat germ lectin (WGL) when conjugated to PNiPAAm, these lectin–polymer conjugates were used in the purification of various polysaccharides or polysaccharide-containing compounds such as glucan [84]. The thermally reversible soluble–insoluble PNiPAAm–dextran derivative (DD) conjugate has been synthesized by conjugating amino-terminated PNiPAAm to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide and the conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran [24]. Recently elastin like polymers (ELPs) consisting of the repeating penta-peptide, VPGVG which behave very similar to PNiPAAm polymers have been shown to undergo reversible phase transitions within a wide range of conditions [85,86]. These, ELPs have been used as terminal tags in recombinant systems to facilitate recombinant protein purification [87,88] and have recently been used for conjugating to metal binding ligands for affinity purification via temperature-triggered precipitation [89]. ELPs with repeating sequences of [(VPGVG)2(VPGKG) (VPGVG)2]21 were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole- 2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP biopolymers. Biopolymers charged with Ni(II) were able to interact with a His-tag on the target proteins. Purifications of two His-tagged enzymes, b-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the application of metal affinity precipitation using this new type of affinity reagent. The bound enzymes were easily released by the addition of either EDTA or imidazole and over 85% recovery was observed in both cases. The recovered ELPs were reused with no observable decrease in the purification performance. This has been the first report exploiting the features of ELPs for protein purification based on metal-affinity purification. The capability of modulating purification conditions by simple temperature triggers and their low cost of preparation will probably make the ELPbased metal-affinity precipitation a useful method in future, not only for protein purification but also for diverse applications in bioseparation such as DNA purification and environmental remediation [86]. Another interesting example has been a one-pot affinity precipitation purification of carbohydratebinding protein reported by Sun et al [90]. By designing thermally responsive glyco-polypeptide polymers, which were synthesized by selective coupling of pendant carbohydrate groups to a recombinant triblock ELP, glyco-affinity precipitation purification of carbohydrate-binding protein was demonstrated. Other types of metal-chelating polymers for affinity precipitation of proteins were reported by synthesizing highly branched co-polymers of NiPAAm and 1,2- propandiol-3-methacrylate (GMA: glycerol monomethacrylate), poly(NiPAAm-co-GMA) using the technique of RAFT polymerization using a chain transfer agent that allows the incorporation of imidazole functionality in the polymer chain-ends. The LCST of the co-polymers can be controlled by the amount of hydrophobic and GMA co-monomers incorporated during co-polymerization procedures. The co-polymers demonstrated LCST below 18 1C and were successfully used to purify a His-tagged BRCA-1 protein fragment (a protein implicated in breast cancer) by affinity precipitation [91,92]. An interesting example of the use of poly(N-acryloylpiperidine) terminally modified with maltose for affi- nity precipitation of thermolabile a-glucosidase was ARTICLE IN PRESS A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237 1213
A.Kumar et al.Prog.Polym.ScL.32(2007)1205-1237 demonstrated 1931 The use of the polymer with 2.2.Protein folding extremely low LCST (soluble below at 4C and completely insoluble above 8C)made it possible Protein refolding is an important step in the to use the technique for purification of thermolabile production of many functional recombinant pro a-glucosidase from cell-free extract of Saccharomyces teins.Modest changes in the protein's environment cerevisiae achieving 206-fold purification with 68% t str anges that can a reoYl dern D precipitation is readily combined with ur cloning of latio in ATPSs Partitionng ems the Gram-ne ive bacterium E coli has with ligand-polymer coniugate is usually directed to been the most commonly used syst tem for the the upper hydrophobic phase of ATPSs formed by production of heterologous proteins due to ease of PEG and dextran/hydroxypropyl starch,wherea large-scale and high-density cultivation.However. the use of E.coli for large-scale protein production partiti frequently plagued by th formation of insolubl phase. the precipi pr po protein aggregate ,th mer complex is prom oted by char s,in cyobl tain dy e protein acetatelactate dehydrogenase,and protein e using a conjugate of Eudragit S-100 with the surfactant is a common practice which inhibits triazine dye Cibacron Blue [96]and immunoglobu- protein aggregation in protein refolding procedure 1inG[91,92, respectively approach was The hydr ophobicity of the surfactant is the im demonstrate by the purification portant factor facilit inders Pu rmation pro mylas weet po mylase [97,98]and ud he the Sp ate animal cells by polymers have found p ntially int crafting the smart AMLs by coupling an antibody (against a cell surface protein)to a SP [99] advantage over conventional surfactant as their Combination of partition ing with afhnity precipita hydrophobicity can be tion improves yield and purification factor and fgircasmrioatioaofproteimfompariculale rom re olded pro can achieve whic stream a a bl the has AMI facilita ently been ed th introd and develops three-phase i PEG bound to functior nal ligand.PEC was bound to a thermoreactive hydrophobic head ration of proteins using smart affinity ligands (poly (propylene oxide)-phenyl group (PPO-Ph). [100.1011.In this method.a water-soluble polv- Refolding of bovine carbonic anhydrase was exam- mer is floated as an interfacial precipitate by ined in the presence of PPO-Ph-PEG at various adding ammonium sulfat 【ertiary-butanol. emp ratures The refolding yield carbonic The polymer (appropriatel sen)in the anhydra se was strongly enna an PPO-Ph sence polymer protein comple this artificial cha which A wheat germ agglutinin activity recover and 40-fold purification)and wheat germ lipase of denatured protein in a fashion similar to that of a (94%activity recovery and 27-fold purification) natural chaperone was developed by using SP have been purified using chitosan as a macroaffinity These artificial chaperons have been used to assist ligand [102]. the refolding of bovine carbonic anhydrase using
demonstrated [93]. The use of the polymer with extremely low LCST (soluble below at 4 1C and completely insoluble above 8 1C) made it possible to use the technique for purification of thermolabile a-glucosidase from cell-free extract of Saccharomyces cerevisiae achieving 206-fold purification with 68% recovery. Affinity precipitation is readily combined with other protein isolation techniques, e.g. partitioning in ATPSs [94]. Partitioning of protein complexed with ligand–polymer conjugate is usually directed to the upper hydrophobic phase of ATPSs formed by PEG and dextran/hydroxypropyl starch, whereas most of the proteins present in crude extracts or cell homogenates partition into lower hydrophilic phase. Then the precipitation of the protein–polymer complex is promoted by changing pH. Trypsin was purified using conjugate of soybean trypsin inhibitor with hydroxypropylcellulose succinate acetate [95], lactate dehydrogenase, and protein A using a conjugate of Eudragit S-100 with the triazine dye Cibacron Blue [96] and immunoglobulin G [91,92], respectively. The approach was also successfully demonstrated by the purification of microbial xylanases, pullulanases, wheat germ a-amylase, and sweet potato a-amylase [97,98] and purification of lectins from wheat germ, potato and tomato. Other attractive extension of this approach has been to separate animal cells by crafting the smart AMLs by coupling an antibody (against a cell surface protein) to a SP [99]. Combination of partitioning with affinity precipitation improves yield and purification factor and allows easier isolation of protein from particulate feed streams. A new concept has recently been introduced where AML facilitated three-phase partitioning and develops three-phase partitioning into a more selective and predictable technique for bioseparation of proteins using smart affinity ligands [100,101]. In this method, a water-soluble polymer is floated as an interfacial precipitate by adding ammonium sulfate and tertiary-butanol. The polymer (appropriately chosen) in the presence of a protein for which it shows affinity, selectively binds to the protein and floats as a polymer–protein complex. By using this approach wheat germ agglutinin (99% activity recovery and 40-fold purification) and wheat germ lipase (94% activity recovery and 27-fold purification) have been purified using chitosan as a macroaffinity ligand [102]. 2.2. Protein folding Protein refolding is an important step in the production of many functional recombinant proteins. Modest changes in the protein’s environment can bring about structural changes that can affect its function. Modern DNA cloning techniques have made possible the over-expression of recombinant proteins in various host systems. Among the many systems, the Gram-negative bacterium E. coli has been the most commonly used system for the production of heterologous proteins due to ease of large-scale and high-density cultivation. However, the use of E. coli for large-scale protein production is frequently plagued by the formation of insoluble protein aggregates, in cytoplasm or periplasm, thus reducing the yield of soluble, active proteins. To attain the native structure and function of proteins, the refolding process is a major challenge in currently ongoing biochemical research. Using surfactant is a common practice which inhibits protein aggregation in protein refolding procedure. The hydrophobicity of the surfactant is the important factor which facilitates or hinders the conformational transition of unfolded protein, depending on the magnitude of the intramolecular hydrophobic force of the protein. With the appreciation of varying hydrophobicity of the SP, these polymers have found potentially interesting applications in the field of protein folding. SP have distinct advantage over conventional surfactant as their hydrophobicity can be manipulated simply by temperature or pH and simple separation of SP from refolded protein can easily be achieved which makes them available for the refolding of different proteins. The utility of SP was studied for protein refolding in ATPS. The system consisted of modified PEG bound to functional ligand. PEG was bound to a thermoreactive hydrophobic head (poly (propylene oxide)–phenyl group (PPO–Ph). Refolding of bovine carbonic anhydrase was examined in the presence of PPO–Ph–PEG at various temperatures. The refolding yield of carbonic anhydrase was strongly enhanced and aggregate formation was suppressed by addition of PPO–Ph– PEG at a specific temperature of 50–55 1C [103]. An artificial chaperone, which can decrease protein aggregation and increase reactivation yield of denatured protein in a fashion similar to that of a natural chaperone was developed by using SP. These artificial chaperons have been used to assist the refolding of bovine carbonic anhydrase using ARTICLE IN PRESS 1214 A. Kumar et al. / Prog. Polym. Sci. 32 (2007) 1205–1237