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–poly￾mer 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 immunoglobu￾lin 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 precipita￾tion 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 biosepa￾ration of proteins using smart affinity ligands [100,101]. In this method, a water-soluble poly￾mer is floated as an interfacial precipitate by adding ammonium sulfate and tertiary-butanol. The polymer (appropriately chosen) in the pre￾sence 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 pro￾teins. 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 im￾portant 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 appre￾ciation of varying hydrophobicity of the SP, these polymers have found potentially interesting applica￾tions 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 exam￾ined 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