Artificial Chaperone-Assisted Refolding of Bovine Carbonic

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Artificial Chaperone-Assisted Refolding of Bovine Carbonic Anhydrase Using Molecular Assemblies of Stimuli-Responsive Polymers Noriko Yoshimoto, Takuya Hashimoto, Matundu Menayame Felix, Hiroshi Umakoshi, and Ryoichi Kuboi* Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan Received December 17, 2001; Revised Manuscript Received September 27, 2002

An artificial chaperone, which can decrease the protein aggregation and increase the reactivation yield of denatured protein in a fashion similar to natural chaperone, was newly developed using stimuli-responsive polymers. It has previously been reported that the addition of poly(propylene oxide)-phenyl-poly(ethylene glycol) (PPOn-Ph-PEG) with the unit number of PPO (n) 33 could enhance the refolding of bovine carbonic anhydrase (Kuboi et al. J. Chromatogr. B 2000, 243, 213). PPO-Ph-PEG with a large PPO chain (n ) 50) was synthesized and the surface properties were characterized by both the relative fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) and the fluidity determined by diphenylhexatriene (DPH). The variation of ANS intensity and DPH fluidity is shown in a diagram as functions of temperature and polymer concentration. The high values of ANS intensity and fluidity of PPO50-Ph-PEG were obtained in a relatively wide conditional range (more than 0.08 mM and more than 15 °C) although the conditions showing the high values of PPO33-Ph-PEG were restricted (more than 0.1 mM and more than 40 °C). It was also found that molecular assemblies of PPOn-Ph-PEG with diameters of 7-18 nm were formed in the above conditions. On the basis of the surface properties of their polymer self-assemblies, the possibility of using them as an artificial chaperone was investigated. The effect of the addition of PPOn-Ph-PEG on the reactivation yield of a model protein, carbonic anhydrase from bovine (CAB), and the optical density of the solution was examined at various temperatures and concentrations. The reactivation yield of CAB was strongly enhanced and the aggregate formation (the optical density) was suppressed by adding PPOn-Ph-PEG in the above conditions, which show high ANS intensity and DPH fluidity. Especially in the presence of 0.1 mM PPO50-Ph-PEG, the reactivation yield of CAB reached approximately 100% at 40-55 °C. It was thus found that self-assemblies of the present polymer could be utilized as an artificial chaperone by selecting suitable stimuli conditions. 1. Introduction Nowadays, the development of a protocol for the efficient refolding of the target recombinant protein has become an important issue because a variety of genetically engineered proteins for use in the medical and bioindustrial area have been produced as inclusion bodies in the host cells. In 1995, Rozema and Gellman pioneered the artificial chaperoning method for efficient protein refolding. This method included the following two steps:1 (i) the capture step, in which denatured protein was refolded in a buffer containing ionic detergent to prevent protein aggregation, and (ii) the stripping step, in which the detergents were removed by the addition of cyclodextrin. Since their research was published, many researchers have applied their method to the refolding of various proteins.2-6 Although their method is attractive for its practicality in bioprocess design including the efficient refolding of proteins, it is even more important for designing a stimuli-responsive biomaterial to mimic chaperone ma* To whom correspondence may be addressed. TEL and FAX: +81(0)6-6850-6285. E-mail: [email protected].

chinery (i.e., GroEL and GroES) for the design of the artificial biomaterials. Of the variety of other states along with the actual refolding pathways of proteins, extensive studies have been carried out in recent years on the intermediate state of proteins because of its intriguing features and practical importance in the refolding process. The protein in this state is known to have a secondary structure like the native state but does not have a closely packed tertiary structure.7,8 Recently, a systematic approach on the protein folding process has been reported in a series of works related to the evaluated values of proteins in various conformations.9 The variation of the surface properties of proteins during their denaturation and refolding processes can be quantitatively characterized using the aqueous two-phase partitioning method.10,11 The evaluated local hydrophobicity of a protein was found to play an important role not only in the protein denaturation or aggregate-formation processes10,11 but also in interaction with liposomes,12-14 heat shock proteins,15 and hydrophobic ligands.16 A biomimetic material, which can improve the reactivation yield of denatured protein and is

10.1021/bm015662a CCC: $25.00 © 2003 American Chemical Society Published on Web 10/15/2003

Refolding with Polymer Assemblies

known as an “artificial chaperone”,17 can be designed and developed based on the quantitative surface properties of the target protein and chaperones (i.e., liposomes and GroEL). In the early 1990s, Cleland and co-workers first investigated the chaperone-like function of the amphiphilic polymer (poly(ethylene glycol)).18,19 Some researchers in polymer engineering have recently tried to design an artificial chaperone by using ionic/nonionic detergent,20 polyols,21 and hydrophobically modified pulluran.22 Chimerical materials composed of polyelectrolytes and antibodies were also used for the reactivation of polymeric protein.23 Recently, Kawaguchi and co-workers have reported that the microsphere prepared by vinyl-polymers modified with the functional groups (methacryl acid and thiol) has a chaperone-like action.6 However, a method for designing an artificial chaperone has not been researched. Considerable research attention has recently been focused on polymers that can spontaneously and reversibly change their structure and properties in response to external chemical and/or physical stimuli such as pH and temperature. These polymers, known as smart polymers24 or stimuli-responsive polymers, sense a stimulus as a signal, judge the magnitude of this signal, and then alter their function in direct response. The stimuli responsive polymer has similar functions to those of natural macromolecules (such as protein, enzyme, and chaperone), which can react and adopt themselves to environmental stimuli. It is considered possible to design stimuli-responsive polymers to assist and enhance protein refolding, which can be used as artificial chaperones in the place of natural chaperones. The final purpose of this study is to establish a method for designing an artificial chaperone that can decrease the aggregate formation and increase the reactivation yield of denatured proteins. In our previous paper, the refolding of model protein (bovine carbonic anhydrase, CAB) was reported to be enhanced by the addition of poly(propylene oxide)-phenyl-poly(ethylene glycol) (PPOn-Ph-PEG; n ) unit number of PO).25 The behaviors of self-assemblies of PPOn-Ph-PEG and their surface properties were first characterized at various temperatures and at various polymer concentrations by using hydrophobic fluorescence probes, such as 1-anilino-8-naphtlene sulfonate (ANS) and diphenylhexatriene (DPH). The refolding of CAB was performed in the presence of the polymer assemblies, whose characteristics were appropriately controlled. 2. Experimental Section 2.1. Materials. Bovine carbonic anhydrase (CAB, EC 4.2.1.1, Mw ) 28.8 kDa) was purchased from Sigma Chemical Co. Ltd. (St. Louis, MO). Guanidine hydrochloride (GuHCl) used as a denaturant of CAB was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The phase-forming polymers in aqueous two-phase systems such as dextran 100k-200k (Dex) (Mw ) 100k-200k) and poly(ethylene glycol) (PEG) 1540, 4K, 8K (Mw ) 1.5k, 3k, 8k) were purchased from Wako. Triton X-405 was purchased from Sigma. All other reagents used in this study were of analytical grade. 2.2. Synthetic Polymers. The structural formulas of the ligand used in this study, poly(propylene oxide)-phenyl-

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Figure 1. Structure of PPOn-Ph-PEG.

poly(ethylene glycol) (PPOn-Ph-PEG), is shown in Figure 1. The ligands used here have hydrophobic heads (such as stimuli responsive polymer; poly(propylene oxide) (PPOn)) with various chain lengths (n ) 33, 50) and hydrophilic polymer (ethylene oxide, PEG) chains with the same molecular weights (average of molecular weight about 8K). PPOn-Ph-PEG was synthesized as previously reported.25 In brief, Ph-PEG was obtained by transalkylation of Triton X-405.26 Poly(propylene oxide) bromide, prepared through the bromination of PPO, was made to react with Ph-PEG. The formation process of the final product was confirmed by measuring IR spectra and chemical shift by NMR analysis. All other reagents used in this study were of analytical grade. 2.3. Refolding of CAB in the Presence of PPOn-PhPEG. The refolding experiment was performed using the CAB as a model protein. For protein refolding, 60 µL of denatured protein solution (5 M GuHCl) was diluted with the refolding buffer (0.1 M Tris-hydrochloride buffer, pH 8.0) to a total volume of 3 mL.19 Before the initiation of CAB refolding, PPOn-Ph-PEG was added to the refolding buffer in a concentration of 0.5-0.8 mM for PPO33-PhPEG and 0.02-0.3 mM for PPO50-Ph-PEG. The refolding buffer was also preheated to a temperature of 25-60 °C for PPO33-Ph-PEG and 10-55 °C for PPO50-Ph-PEG. The dilution ratio was fixed at 50:1 in all experiments we conducted. After the above refolding experiments, the activity of CAB and the optical density of the solution were evaluated at a temperature of 25 °C. The activity of CAB was determined from the hydrolysis rate of substrates (p-nitrophenyl acetate, p-NPA, 1 mM) at the wavelength of 348 nm in the 50 mM Tris-HCl buffer with 5 mM ethylenediaminetetraacetic acid (EDTA).27 The refolding of CAB can be stopped by the addition of EDTA. The aggregate formation of the CAB during the refolding process was monitored using an optical density of the solution as a measure.19 The reactivation yield was determined as a percentage by selecting the activity of native CAB at the same concentration at 25 °C as a standard. 2.4. Characterization of Polymer Surface. The local hydrophobicity (LH) of proteins was determined by the aqueous two-phase partitioning method.11 The surface properties of PPOn-Ph-PEG (n ) 33, 50) were characterized using hydrophobic fluorescence probes, 1-anilino-8-naphthalene sulfonate (ANS) and diphenylhexatriene (DPH). The local hydrophobicity of the microenvironment of PPOn-PhPEG and its self-assemblies was estimated from the relative fluorescence intensity of ANS (λex ) 400 nm, λem ) 470 nm). ANS dissolved in dioxane was added to the polymer solution to give a final concentration of 200 µM. In the preexperiments, the changes in fluorescence intensity of the proteins and functional ligands were determined as a function

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Figure 2. Dependence of polymer concentration on the surface tension: open circle, PPO33-Ph-PEG; open square, PPO50Ph-PEG; closed circle, Ph-PEG.

of the ANS concentration, and the value at 200 µM shows the plateau of the curve against the ANS concentration. Membrane fluidity of the self-assemblies of the stimuliresponsive polymers was determined by the following method using fluorescent probes, DPH.28 The DPH was oriented into the self-assemblies in the following way. A solution of DPH (in ethanol) was added to the polymer solution to maintain the polymer/probe molar ratio at 250 ([DPH]final ) 2 mM). The mixture was then incubated at least for 1 h at room temperature with gentle stirring. Probes in the aqueous solution of the self-assemblies of polymers were negligible since they have little fluorescence in water. The fluorescence polarization of samples was measured with a spectrofluorometer to which excitation and analyzing polarizers (FP 2010, JASCO) were fitted. The sample was excited with vertically polarized light (360 nm) and then the emission intensity at 430 nm both parallel (I|) and perpendicular (I⊥) to the excited light was recorded. Then, polarization of DPH was calculated from the following equation P ) (I| - I⊥)/(I| + I⊥) The membrane fluidity of liposomes was determined from the inverse values (1/P) by using the P values. All experiments for the measurements of fluorescent intensity were measured by excitation at 400 nm on a FP-777 (JASCO Co. Ltd., Japan) with a thermoregulated cell compartment. 2.5. Measurements. The average diameter and size distribution of the self-assemblies of PPO-Ph-PEG were analyzed with a DLS-700 Ar system (Otsuka Electric, Osaka, Japan) equipped with an argon laser (scattering angle was 90°). Measurements were made at 25 and 55 °C. 3. Results and Discussion 3.1. Characteristics of Molecular Assemblies of PPOnPh-PEG. 3.1.1. Formation of Self-Assemblies of PPOnPh-PEG. It has been reported that block copolymers composed of propylene oxide and ethylene oxide formed micelle-like self-assemblies, which could be controlled by polymer concentration and temperature.29-32 The possibility of forming self-assemblies of PPOn-Ph-PEG (n ) 33, 50) was first investigated. Figure 2 shows the relationship between the polymer concentration and the surface tension

Figure 3. Dependence of polymer concentration on the average size of self-assemblies of PPO33-Ph-PEG and PPO50-Ph-PEG: open circle, PPO33-Ph-PEG, 55 °C; closed circle, PPO33-Ph-PEG, 25 °C; open square, PPO50-Ph-PEG, 25 °C; close triangle, Ph-PEG, 25 °C.

of the polymer solution at a temperature of 25 °C. In all the polymer solutions tested here, the surface tension was decreased by increasing the polymer concentration and reached constant values above the specific concentration of polymer, that is, at the critical aggregation concentration (CAC). The CAC value of both PPOn-Ph-PEG was lower than that of Ph-PEG (CAC, 0.18 mM) and the CAC values for PPO33-Ph-PEG and PPO50-Ph-PEG were 0.12 and 0.05mM, respectively. It was found that self-assemblies of PPOn-Ph-PEG were formed above CAC, and the PPO50Ph-PEG could form self-assemblies at a lower polymer concentration. As shown in Figure 3, the average size of the selfassemblies of PPOn-Ph-PEG was analyzed using the dynamic light scattering (DLS) method. The size did not significantly change (2-2.5 nm) in the case of the control polymer (Ph-PEG) prepared at a concentration of 0.1-1.0 mM at 25 °C. The average size of the molecular assemblies of the PPO33-Ph-PEG was 4.7 nm at the high polymer concentration (0.8 mM) although it was 2.3-2.4 nm at the low polymer concentration (less than 0.2 mM). However, the average size of the molecular assemblies of the PPO33Ph-PEG became 6-8 nm and the aggregation number estimated from the average diameter was 5-8 with a polymer concentration of more than 0.2 mM at a temperature of 55 °C. On the other hand, the average size of self-assemblies of PPO50-Ph-PEG was linearly increased at 25 °C by increasing the polymer concentration and became 15-17 nm at a concentration of more than 0.5 mM. The aggregation number of the PPO50-Ph-PEG was estimated as 55-75. It has previously been reported that the block copolymer of EO and PO (Pluronic series) forms core-corona type selfassemblies.33 It is considered that similar self-assemblies of core-corona type could be formed as in the case of Pluronic based on the structural similarity. The diameter, which was determined by SANS, and the aggregation number of the self-assemblies has been reported to be 7-18 nm and 50-70, respectively. In both cases the size was varied by altering the chain length of PO and EO.34 The above results on the average size and the aggregation number of PPOnPh-PEG corresponded well with the previous findings of


Figure 4. Dependence of (a) temperature and (b) PPO33-Ph-PEG concentration on relative fluorescence intensity of ANS at various conditions.

Pluronic. In addition, the increase of the mole fraction of PO, which forms the hydrophobic core, was also shown to be greater, and it is suggested this is due to the increase of the hydrophobic attractive force. The difference in the size and aggregation number of PPOn-Ph-PEG with PO unit numbers of 33 and 50 was thought to be due to the difference in the hydrophobic attractive force of the PPO chain. It was found that the PPOn-Ph-PEG formed selfassemblies with a diameter of 5-17 nm, and the behaviors of self-assemblies can be controlled by the polymer concentration and temperature. 3.1.2. Surface Characteristics of Self-Assemblies of PPOn-Ph-PEG at Various Concentrations and Temperatures. In our previous reports, it has been reported that the local hydrophobicity and fluidity were important factors in the design of an artificial chaperone.12,25 The surface properties of self-assemblies of PPOn-Ph-PEG were characterized, especially in relation to hydrophobicity, using the hydrophobic fluorescent probe (i.e., ANS and DPH). Figures 4 and 5 show the dependence of the relative fluorescence intensity of ANS (IANS) of PPO33-Ph-PEG and PPO50-Ph-PEG on the (a) temperature and (b) polymer concentration. In the case of self-assemblies of PPO33-PhPEG at CAC (0.1 mM), the IANS values did not change below 40 °C and increased transiently up to the top values above 45 °C (Figure 4a). As shown in Figure 4b, the IANS values were also plotted against the polymer concentration. The IANS values were significantly increased by increasing the polymer concentration and became 8-10 times greater than those at 0.1 mM in the every temperature range (25-55 °C). A similar tendency was also obtained in the case of PPO50Ph-PEG, and the transition region of the IANS variation was


Figure 5. Dependence of (a) temperature and (b) PPO50-Ph-PEG concentration on relative fluorescence intensity of ANS at various conditions.

observed at a temperature of 20 °C in all polymer concentrations tested here (Figure 5a). The IANS values were also found to increase with the increase of the polymer concentration and reached saturated values above 25 °C. It has previously been reported that the intensity of the hydrophobic fluorescent probe, DPH, was used for confirmation of the formation of self-assemblies of Pluronic.29,30 The above results imply that self-assemblies of PPO33-Ph-PEG formed the hydrophobic core, where hydrophobic ANS can be partitioned, and the hydrophobicity of the core can be controlled by temperature and concentration. Fluidity, which can be determined by DPH, was also analyzed in order to investigate the dynamic properties of the hydrophobic core of the self-assemblies of PPOn-PhPEG. The dependence of the fluidity of PPO33-Ph-PEG and PPO50-Ph-PEG on (a) temperature and (b) polymer concentration is shown in Figures 6 and 7. In the case of PPO33-Ph-PEG, the fluidity value did not significantly change and was 4.5-4.8 near the CAC (0.05-0.1 mM) at all temperatures tested here, although the values varied at a concentration of more than 0.2 mM (Figure 6a). As shown in Figure 6b, the values were decreased by increasing the polymer concentration at 25 °C and were increased at 40 °C. In addition, the fluidity value at 55 °C indicated maximal value at a concentration of 0.2 mM. The variation of fluidity of PPO50-Ph-PEG is also shown in Figure 7. In contrast to the case of PPO33-Ph-PEG, the fluidity values indicated more than 5.0 at all temperatures and concentrations. As shown in Figure 7a, the values were about 5.3 near CAC at all temperatures. The values increased significantly to 7.0-8.0 above CAC and reduced to 5.5-6.5 at the high temperature. Fluidity was also plotted in Figure 7b. The values increased from 5 to 8 at a concentration of 0.1-0.2

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Figure 6. Dependence of (a) temperature and (b) PPO33-Ph-PEG concentration on fluidity of DPH at various conditions.

Figure 7. Dependence of (a) temperature and (b) PPO50-Ph-PEG concentration on fluidity of DPH at various conditions.

mM at 15-45 °C and increased from 5 to 6.5 at 0.3 mM at 55 °C. The fluidity determined from the anisotropy of DPH has previously been reported to be due to dynamic properties of micelle-like structures, where the fluidity was 4-5 in the previous results.29,30 3.1.3. A Diagram for Surface Characteristics of SelfAssemblies of PPOn-Ph-PEG. The results for the ANS

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intensity and fluidity are summarized in a diagram of the polymer concentration and temperatures as shown in Figure 8. The diagrams for the ANS intensity of PPO33-Ph-PEG and PPO50-Ph-PEG are shown in parts a and b of Figure 8 and those for fluidity are shown in parts c and d of Figure 8. In the case of PPO33-Ph-PEG (Figure 8a,c), the characteristics of polymers can be classified as the following conditional region on the basis of the ANS intensity and fluidity. In the case of the PPO50-Ph-PEG, the characteristics of the self-assemblies were similar to those of the classification region ii, except for the region colored blue in Figure 8b. In addition, fluidity was extremely high above 0.12 mM at all temperatures. Among the above conditional regions, it is considered that the self-assemblies prepared in region ii had a strong hydrophobic core with high fluidity of the microenvironment, judging from the results on ANS intensity and fluidity. In addition, region ii of the PPO50Ph-PEG was wider than that of PPO33-Ph-PEG because of the strong attractive force of the longer PPO chain. In our previous study, the chaperone-like function of the PPO33-Ph-PEG was reported to be induced at 0.1 mM at a temperature of 55 °C,25 where both the local hydrophobicity and fluidity were shown to indicate high values. In the previous report on the liposome-assisted refolding of proteins, the local hydrophobicity and membrane fluidity were key factors in controlling the refolding of bovine carbonic anhydrase and lysozyme.12,14 In addition, the local hydrophobicity of target protein was also reported to be an important factor.15 It has been reported that the local hydrophobicity of the surface of various proteins was maximized during the process of their conformational change.10 In our simple model for protein refolding assisted by chaperone-like materials (PPOn-Ph-PEG and liposomes), the protein at the intermediate state formed a complex with the chaperone-like materials, and the refolding was induced on their surface because of the induction of the conformational change due to the fluidized surface.12,14,25 The self-assemblies of PPO33-Ph-PEG and PPO50-PhPEG in region ii were thought to be suitable for use as an artificial chaperone based on the previous model for the design of an artificial chaperone and the above characteristics of the polymers. 3.2. Application of the Self-Assemblies of PPOn-PhPEG as an Artificial Chaperone. The possibility of applying the self-assemblies of PPOn-Ph-PEG as an artificial chaperone was investigated through refolding experiments on a model protein (CAB). First, the effect of the addition of self-assemblies of PPOnPh-PEG on the CAB refolding was investigated. The refolding of CAB was initiated by a 50:1 dilution of CAB denatured with 5 M GuHCl. Before initiation of CAB refolding, the PPOn-Ph-PEG was added to the refolding buffer (0.1 mM for both polymers) and preheated to a temperature of 55 °C to form self-assemblies of PPOn-PhPEG in region ii described in the previous section. Figure 9 shows the time course of the reactivation yield (Figure 9a) of CAB and optical density (OD340) of the solution (Figure 9b) in the presence and absence of molecular assemblies of PPOn-Ph-PEG. As shown in Figure 9a, the reactivation



Figure 8. Map of relative ANS intensity ((a) and (b)) and fluidity ((c) and (d)) of PPOn-Ph-PEG as a function of temperature and polymer concentration. Maps for PPO33-Ph-PEG are shown in (a) and (c) and those for PPO50-Ph-PEG in (b) and (d). Colors in (a) and (b) indicate the range of the values of ln IANS: red, 1.2-1.6; yellow, 0.8-1.2; light green, 0.4-0.8; dark green, 0-0.4, blue, -0.4 to 0; purple, -0.8 to -0.4. Colors in (c) and (d) indicate the range of the values of fluidity: red, 8-9; yellow, 7-8; light green, 6-7; dark green, 5-6, blue, 4-5; purple, 3-4. Table 1. Summary of Conditional Regions Classified by the Characteristics of Polymers

Cpoly (mM)

T (°C)

iii

0.1 0.1-0.3 0.1 0.1-0.3

40 >45 40

v

0.3-0.8

10 nm)

yield was increased by increasing the time in the absence of PPOn-Ph-PEG and the values reached a plateau (45%) in 10 min. In the presence of PPOn-Ph-PEG, the reactivation yield of CAB increased to 78% in 10 min. The value at the plateau was 1.7 times greater than that in the absence of PPO33-Ph-PEG. In the presence of PPO50-Ph-PEG, the reactivation yield increased to 83% in 10 min and reached 100% in 40 min, showing that the denatured CAB was fully refolded. The reactivation yield of CAB in the presence of POPC liposome is also shown in Figure 9a, where the value

at the plateau was only 71%. The aggregate formation of CAB was also studied by measuring the optical density of the solution as shown in Figure 9b. The increase in the turbidity of the solution can be interpreted as the aggregate formation in the solution. After the initiation of CAB refolding, the optical density in the absence of PPO-PhPEG was increased by increasing the time and the values reached 0.5 in the values of OD340. A similar curve was obtained in the presence of PO-Ph-PEG, which does not form self-assemblies. On the other hand, the OD340 values at the plateau in the presence of both PPO33-Ph-PEG and PPO50-Ph-PEG indicated lower values than those in the absence of PPOn-Ph-PEG, showing that the formation of CAB aggregation was prevented in the presence of PPOnPh-PEG. A poly(propylene oxide) group (PPO), as well as poly(ethylene oxide) group (PEO), is known as a stimuli responsive polymer, which reacts and adapts its structure through recognition of temperature change. The effect of temperature on the CAB refolding process in the presence of the self-assemblies of PPOn-Ph-PEG was investigated. CAB refolding was performed at various temperatures and the CAB activity and the OD340 were measured in the above

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Figure 9. Time course of reactivation yield of CAB in the presence of PPOn-Ph-PEG (n ) 33, 50) and liposome. The refolding of CAB denatured with 5 M GuHCl was initiated by a 50:1 dilution of the denatured sample at 50 °C. The final concentration of CAB and PPOn-Ph-PEG was 0.1 mg/mL and 0.1 mM, respectively. Symbols: closed circle, control; open triangle, Ph-PEG; double circle, liposome prepared by 1-palmytoyl-2oleoyl-sn-glycero-3-phosphocholine;13 open circle, PPO33-Ph-PEG; open square, PPO50-Ph-PEG.

Figure 10. Temperature dependence of maximal reactivation yield of CAB in the presence of PPOn-Ph-PEG (n ) 33, 50): closed circle, control; triangle, Ph-PEG; open circle, PPO33-Ph-PEG; open square, PPO50-Ph-PEG. The refolding of CAB denatured with 5 M GuHCl was initiated by a 50:1 dilution of the denatured sample. The final concentrations of CAB and PPOn-Ph-PEG were 0.1 mg/mL and 0.1 mM, respectively.

conditions (30-55 °C). Figure 10 shows the effect of temperatures on (a) the reactivation yield and (b) aggregate formation (OD340) after 40 min of CAB refolding. In the absence of PPOn-Ph-PEG, the reactivation yield did not change at a temperature of 25-45 °C and decreased at a higher temperature range (Figure 10a). On the other hand, the reactivation yield of CAB was increased by increasing the temperature and the values were maximized in the specific temperature range (48-52 °C) in the presence of PPO33-Ph-PEG (Figure 10a). The maximum value of CAB activity was 78%. No CAB activity was observed at the high temperature. In the presence of PPO50-Ph-PEG, the reactivation yield of the CAB was significantly improved to 95-100% at almost all temperatures below 55 °C. As shown in Figure 10b, the OD340 values as a measure of aggregate formation of CAB increased at a temperature greater than 48 °C, where the aggregation of CAB was found to be formed in the absence of PPOn-Ph-PEG. On the other hand, aggregate formation was prevented in the specific temperature range of 48-55 °C in the presence of PPOn-Ph-PEG. CAB is a monomeric protein and is often used for the study of protein refolding due to its slow refolding rate compared with other proteins. Cleland and Wang18,19 have investigated the conformational change and the formation of microsized inactive aggregates by using quasi-elastic light scattering. It has also been demonstrated that the reactivation

yield of CAB is enhanced by the addition of a molecular chaperone (such as GroELp), liposomes, and hydrophobic ligands (such as PEG and Triton X-series).11-13,15,16 It has also been reported that the local hydrophobicity of the chaperones and the other chaperone-like materials could play an important role in the refolding of denatured proteins.12,14 As shown in Figure 1, PPOn-Ph-PEG, which has been used in this study, has a hydrophobic headgroup (PPOn-Ph group) and a hydrophilic tail (PEG) along the molecules. The effect of the type of headgroup bound to the PEG molecule has already been investigated,16 and the hydrophobicity of the headgroup was shown to be an important factor in interaction with the CAB in the intermediate state. Similarly in this case, the local hydrophobicity of PPO-Ph-PEG at various temperatures was considered to be an effective factor governing the refolding process of CAB. In our series of studies, it has been shown that CAB refolding was dependent on the local hydrophobicity of CAB and additives (such as GroEL and liposomes).11-13,15,16 The surface properties of both CAB and PPO-Ph-PEG were characterized, especially, in relation to their local hydrophobicity. The temperature regions, where the local hydrophobicity of (i) CAB and (ii) PPOn-Ph-PEG was increased, are shown as follows: (i) 60 °C and (ii) 42-53 °C (n ) 33) and less than 55 °C (n ) 50). In specific conditions, the complex of CAB at an intermediate state and PPOn-Ph-


PEG was thought to be formed because of hydrophobic interaction. CAB aggregation is considered to be suppressed because the intermolecular interaction of CAB is reduced by forming a complex of CAB and PPOn-Ph-PEG (Figure 10b). After this complex is formed, the CAB at an intermediate state is considered to be refolded to a native state and native CAB is released from the polymer surface (Figure 10a) because of the sharp change in surface properties of the polymer, which may be caused by the membrane fluidity of their self-assemblies at temperature, although further investigation on the mechanism is needed. 4. Conclusion The possibility of designing an artificial chaperone, which decreases aggregate formation and increases the reactivation yield of CAB, was investigated by using the stimuliresponsive polymer (PPOn-Ph-PEG). First, the possibility of forming self-assemblies of PPOn-Ph-PEG was investigated. The surface properties of the self-assemblies of PPOnPh-PEG, such as relative fluorescent intensity of ANS and fluidity of DPH, were found to be controlled by the polymer concentration, temperature, and chain length of the PPO group. The surface characteristics of PPOn-Ph-PEG were summarized in a diagram in relation to polymer concentration and temperature. The effective conditions for protein refolding were suggested based on the diagram. The chaperonelike activity of the self-assemblies of PPOn-Ph-PEG at optimal conditions was also investigated by selecting CAB as a target protein. In the presence of PPO33-Ph-PEG, the refolding yield of CAB was increased 1.7 times and the aggregate formation was repressed when a suitable condition for heating was selected, where the local hydrophobicity of both PPO33-Ph-PEG and CAB increased. It is confirmed that almost all CAB was refolded over a relatively wide temperature range (less than 55 °C), especially when PPO50Ph-PEG was added to the refolding buffer. It is considered that the present stimuli-responsive polymer can be used as an artificial chaperone and can be applied for protein refolding. Nomenclature LH ) local hydrophobicity ()∆ ln Kpr) OD340 ) optical density of the solution at 340 nm as a measure of the formed aggregate in the solution Ry ) reactivation yield of CAB

Acknowledgment. This work was partly supported the Grant-in-Aid for Scientific Research (No. 12450333, No. 13750736, and No. 146553106) from the Ministry of Education, Science, Sports and Culture of Japan and the Sumitomo Foundation (No.010400). References and Notes (1) Rozema, D.; Gellman, S. H. Artificial ChaperonessProtein Refolding via Sequential Use of Detergent and Cyclodextrin. J. Am. Chem. Soc. 1995, 117, 2373-2374. (2) Rozema, D.; Gellman, S. H. Artificial Chaperone-Assisted Refolding of Carbonic Anhydrase B J Biol. Chem. 1996, 271 (7), 3478-3487. (3) Rozema, D.; Gellman, S. H. Artificial Chaperone-Assisted Refolding of Denatured-Reduced Lysozyme: Modulation of the Competition between Renaturation and Aggregation. Biochemistry 1996, 35 (49), 15760-15771.

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Artificial Chaperone-Assisted Refolding of Bovine Carbonic

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