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-hydroxyalkyl) N-methylimidazolium chlorides
Institut für Biotechnologie, Martin-Luther-Universität Halle/Wittenberg, 06120 Halle (Saale), Germany
Reprint requests to: Christian Lange, Institut für Biotechnologie, Martin-Luther-Universität Halle/Wittenberg, Kurt-Mothes-Str.3, 06120 Halle (Saale), Germany; e-mail: christian.lange{at}biochemtech.uni-halle.de; fax: +49-345-55-27013.
(RECEIVED May 19, 2005; FINAL REVISION July 19, 2005; ACCEPTED July 19, 2005)
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Abstract |
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-hydroxy-alkyl)-N-methylimidazolium chlorides on the renaturation of two model proteins, namely hen egg white lysozyme and the single-chain antibody fragment ScFvOx. All tested ionic liquids acted as refolding enhancers, with varying efficacies and efficiencies. The results of the refolding screening could be interpreted by taking into account the effect of the studied ionic liquids on protein aggregation, together with the systematic variations of their influence on the stability of native proteins in solution. More hydrophobic imidazolium cations carrying longer alkyl chains were increasingly destabilizing, while terminal hydroxylation of the alkyl chain made the salts more compatible with protein stability. The studied ionic liquids can be classified as preferentially bound, slightly to moderately chaotropic cosolvents for proteins. Keywords: refolding; aggregation; stability; calorimetry; ionic liquids
Abbreviations: BSA, bovine serum albumin • DTT, dithiothreitol • GuHCl, guanidinium chloride • L-ArgHCl, L-arginine monohydro-chloride. Names of ionic liquids are abbreviated as indicated in Table 1
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Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051596605.
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Introduction |
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Renaturation processes are determined by the competition between productive refolding and unproductive side reactions, i.e., the formation of misfolded species and the aggregation of denatured protein (Goldberg et al. 1991; Kiefhaber et al. 1991). Additives may act on any step of these reactions. The refolding of a given protein may be promoted by stabilizing its native state, by accelerating the kinetics of the correct folding reaction, and by suppressing unspecific aggregation of the unfolded polypeptide and/or intermediates on the folding pathway. It has been suggested that amino acids (arginine, proline, lysine) (Rudolph and Fischer 1990; Arakawa and Tsumoto 2003; Reddy K. et al. 2005), polyamines (putrescine, spermidine, spermine) (Rudolph et al. 1995; Kudou et al. 2003), and mild detergents (Tandon and Horowitz 1988; Wetlaufer and Xie 1995; Krause et al. 2002), but also the denaturing agents GuHCl and urea themselves (Orsini and Goldberg 1978) act as suppressors of aggregation, while other substances such as sugars, polyalcohols, and ammonium sulfate improve refolding yields by stabilizing the native conformation of proteins (Sawano et al. 1992; Michaelis et al. 1995).
Recently, ionic liquids, i.e., organic salts with melting points below 100°C, have received increasing attention as reaction media for chemical and biocatalytic reactions (for reviews, see, e.g., Kragl et al. 2002; Park and Kaszlaukas 2003; van Rantwijk et al. 2003). These salts generally consist of combinations of organic cations, namely derivatives of N,N'-substituted imidazolium, N-substituted pyridinium, tetraalkylated ammonium, and tetraalkylated phosphonium, and either organic or inorganic anions. Their solution properties are in many ways fascinating and are amenable to be engineered by changes in substitution patterns, although up to now no absolutely reliable set of rules exists to predict the properties of any novel ionic liquid.
Summers and Flowers (2000) were the first to explore ionic liquids, namely tetraethyl and tetrabutyl ammonium nitrate, as additives for protein refolding. When present during the oxidative refolding of hen egg white lysozyme in concentrations up to 0.5 M, these salts effectively suppressed aggregation and led to a significant increase in refolding yields. The pure liquid tetra-alkyl ammonium nitrates were found to denature the protein. This was not entirely expected, as many enzymes had been shown to retain their activities in reaction mixtures containing ionic liquids up to very high concentrations. Preliminary experiments in our laboratory, e.g., had shown that recombinant tissue-type plasminogen activator could be refolded with increasing yields in solutions containing up to 94% (w/v) of the ionic liquid 1-butyl-4-methyl-pyridinium tetrafluoroborate (Rudolph et al. 2005). Apparently, the efficacy of ionic liquids as refolding enhancers or, more generally speaking, their differential interaction with various protein states, is as variable as any other solvent property within this class of compounds, and one might anticipate that it is also a function of the particular properties of the protein in question.
In order to study systematic trends in the properties of imidazolium-based ionic liquids, we investigated the renaturation of two model proteins, namely hen egg white lysozyme and the anti-oxazolone single-chain antibody fragment ScFvOx (Fiedler and Conrad 1995), in the presence of a series of N'-alkyl and N'-(-hydroxy-alkyl)-N-methylimidazolium chlorides, with alkyl chain lengths varying between two and six carbon atoms. The results of the refolding screening could be interpreted very well by on the one hand taking into account the effect of the studied ionic liquids on protein aggregation, and on the other hand their effect on the stability of native proteins in solution.
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-hydroxylated alkyl methylimidazolium salts than for their non-hydroxylated analogs.
ScFvOx
In order to assess the general validity of the results obtained with lysozyme, a renaturation screening for the recombinant single-chain antibody fragment ScFvOx was included in the study. ScFvOx was refolded from a crude inclusion body solubilizate. The refolding time for this experiment was expanded to 4 d, due to an apparently slow progress of the refolding reaction, in agreement with earlier reports of very slow refolding kinetics for antibody fragments and antibody fragment-based immunotoxins (e.g., Buchner and Rudolph 1991; Brinkmann et al. 1992). The oxidative refolding of ScFvOx was first performed at concentrations of ionic liquids ranging from 0.5 M to 4 M. The results of a typical series of experiments are represented in Figure 1, C and D
. As solubilized inclusion bodies were used as starting material, the exact concentration of ScFvOx polypeptide in the refolding mixture could not be determined. Refolding yields are therefore reported in terms of absolute concentrations of active ScFvOx.
Significant renaturation yields could only be obtained in the presence of the ionic liquids EMIM Cl, OH-EMIM Cl, and OH-PMIM Cl, and in the presence of L-ArgHCl, in concentrations between 0.5 M and 1.0 M, while the salts carrying longer alkyl side chains at the substituted methylimidazolium cations did not act as effective refolding enhancers for ScFvOx. For the ionic liquids that did promote renaturation, optimum refolding conditions appeared to be shifted to lower concentrations compared to the results obtained with lysozyme. Therefore, for EMIM Cl, OH-EMIM Cl, and OH-PMIM Cl, the renaturation screening was extended to a lower concentration range and performed at more closely spaced concentration intervals (data not shown). Maximum refolding yields were observed in the presence of 0.5 M EMIM Cl, 0.8 M OH-EMIM Cl, and 0.3 M OH PMIM Cl, respectively.
Aggregation
It became apparent during the renaturation screening experiments that the tested ionic liquids exerted a significant effect on the formation of aggregates from reduced-denatured protein under refolding conditions. At concentrations of the organic salts above 0.5 M, no aggregation of misfolded polypeptide was observable by visual inspection, in contrast to the refolding reactions performed in the absence of organic salts, where significant amounts of precipitate were formed. In order to corroborate this observation, aggregate formation during refolding was followed by light scattering. The experiments were performed in the presence of 0.5 M and 2 M of the studied ionic liquids. In the absence of added organic salts, an instantaneous formation of microscopic aggregates was observed, as indicated by the jump in the light scattering signal upon addition of denatured-reduced lysozyme, which was followed by an increasingly noisy signal of decreasing amplitude, representing the growth of aggregates up to the final precipitation of macroscopic clots formed from misfolded protein material (Fig. 2A, inset). In the presence of 2 M of all of the studied ionic liquids, aggregate formation was completely suppressed under our experimental conditions, while at 0.5 M the strength of this effect was apparently correlated with the hydrophobicity of the substituted methylimidazolium cation (Fig. 2A,B). Aggregation was most effectively suppressed by HMIM Cl, while in the presence of the N-methylimidazolium chlorides with shorter N'-alkyl substitutions, as well as of their terminally hydroxylated analogs, some formation of aggregates could still be observed to set in after a lag phase.
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In order to investigate the denaturing effect of the N'-substituted N-methylimdiazolium chlorides in more detail, we performed a series of differential scanning microcalorimetry (DSC) experiments with lysozyme in the presence of these salts. Under the employed buffer conditions, at pH 4.0, 5.0, and 6.0, lysozyme showed a reversible thermal unfolding transition (Fig. 3A). In the presence of 1 M of all imidazolium-based ionic liquids, a significant reduction in the stability of lysozyme was observed. In Figure 3B the DSC traces for the thermal unfolding in the presence of 1 M OH-EMIM Cl, BMIM Cl, and HMIM Cl are shown as examples. Again, a clear systematic effect of the alkyl chain length on the denaturing effect of the salts was observed for both the N'-alkylated and the N'--hydroxyalkylated N-methylimidazolium chlorides (Fig. 4A–C). These tendencies were matched by the results of guanidinium chloride-induced unfolding experiments comparing the effects of BMIM Cl and OH-EMIM Cl (Fig. 5). While more hydrophobic cations carrying longer alkyl chains were more destabilizing, a terminal hydroxylation of the alkyl chain made the salts more compatible with protein stability.
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It is interesting to compare the results for the studied ionic liquids to the properties of the amino acid salt L-ArgHCl, one of the most widely used additives for protein renaturation. L-ArgHCl has also been shown to suppress the aggregation of unfolded protein, without destabilizing the native state of the model proteins employed in the corresponding studies (Arakawa and Tsumoto 2003; Reddy K. et al. 2005). However, in some instances a slight reduction in the thermodynamic stability of proteins in the presence of L-ArgHCl was observed (Taneja and Ahmad 1994; Lin and Timasheff 1996).
Occasionally, we observed stabilization of native protein activity in solution in the presence of nondenaturing concentrations of the studied ionic liquids (data not shown), which we ascribe to the prevention of irreversible decay processes, namely aggregation. This observation suggests that it may be worthwhile to explore the possible application of ionic liquids in the formulation of protein preparations. Stabilization of protein activity and structure by other ionic liquids has been reported (Lozano et al. 2001; Baker et al. 2004; de Diego et al. 2004).
The efficacy of the studied N'-substituted N-methylimidazolium chlorides as refolding enhancers for the tested model proteins was found to be comparable to, and in some instances even better than, that of L-ArgHCl. As the substituted imidazolium cations in these salts may be easily modified by variation of the substitution pattern, their salts can potentially be optimized as cosolvents for any particular refolding problem. Variation of the anion provides an additional dimension for the tailoring of the solvent properties of imidazolium-derived ionic liquids that should be explored.
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280 of 2.63 mL mg –1 cm–1 for native and 2.37 mL mg–1 cm–1 for denatured lysozyme (Saxena and Wetlaufer 1970).
ScFvOx
The coding DNA sequence of recombinant anti-oxazolone single-chain antibody fragment (ScFvOx), fused to sequences coding for an N-terminal hexahistidine tag and a C-terminal myc tag, was obtained from the expression construct ScFvOx/pHEN-1 (Fiedler and Conrad 1995), kindly provided by Dr. U. Fiedler (IPK, Gatersleben, Germany). The coding sequence was inserted between the unique NdeI and BamHI restriction sites of the bacterial expression vector pET15b(+). ScFvOx protein was expressed in E. coli BL21 (DE3) cells that had been cotransformed with the helper plasmid pUBS520 (Brinkmann et al. 1989). The expressed protein formed inclusion bodies, which contained more than 50% (w/w) ScFvOx polypeptide, as estimated by the analysis of Coomassie-stained SDS polyacryl-amide gels. Inclusion body solubilizate was prepared according to Rudolph et al. (1997). Briefly, harvested E. coli cells were homogenized at 4°C in 5 mL per 1 g wet weight of resuspension buffer (0.1 M Tris/HCl, 1 mM EDTA [pH 7.0]) containing 0.3 mg mL–1 lysozyme, and incubated for 30 min. The cells were disrupted in a French press, and cellular DNA was digested for 30 min after adding MgCl2 and Benzonase to final concentrations of 3 mM and 10 µg mL–1, respectively. The solution was mixed with 0.5 volumes of 60 mM EDTA, 6% (v/v) Triton X-100, 1.5 M NaCl (pH 7.0), and incubated for a further 30 min at 4°C. The inclusion bodies were pelleted by centrifugation at 31,000g for 10 min at 4°C, washed with 40 mL of resuspension buffer per 1 g of E. coli wet cell weight, and harvested by a final centrifugation step.
For refolding, inclusion bodies were solubilized in 1 mL of solubilization buffer (6 M GuHCl, 0.1 M Tris/HCl [pH 8.0], 100 mM DTT, 1 mM EDTA) per 10 mg wet weight for 2 h at 25°C. The solubilization mixture was acidified to pH 4.0 by the addition of 1 M HCl and cleared by centrifugation, followed by two dialysis steps against 100 volumes of 4 M GuHCl, 10 mM HCl at room temperature and one dialysis step against 200 volumes of 4 M GuHCl at 4°C. This preparation served as a starting material for refolding experiments. Protein concentrations of the ScFvOx inclusion body solubilizates were determined by the Bradford method (Bradford 1976), using bovine serum albumin in the presence of a constant final concentration of GuHCl as standard. The extinction coefficient 280 for denatured ScFvOx (49,740 M–1 cm–1, corresponding to 1.731 mL mg–1 cm–1) was calculated from the amino acid composition (Gill and von Hippel 1989). The concentration of a sample of purified refolded ScFvOx was determined using the calculated extinction coefficient, and this sample served as a standard for the calibration of the antigen-binding assay (see below).
Protein renaturation
Lysozyme
The reductive denaturation and the oxidative refolding of lysozyme were carried out as described (Reddy K. et al. 2005). Briefly, the renaturation of the protein was initiated by rapid mixing of reduced denatured lysozyme with 60 volumes of degassed renaturation buffer (3 mM GSH, 0.3 mM GSSG, 1 mM EDTA, 0.1 M Tris/HCl [pH 8.2]), containing organic salts as indicated. The final concentration of lysozyme was 280 µg mL–1. For the screening experiments, the samples were incubated overnight at room temperature and analyzed for lysozyme activity after 18–24 h. Native lysozyme, incubated under the same conditions, without added organic salts, served as control.
ScFvOx
For the refolding screening, ScFvOx inclusion body solubilizate was diluted to a final concentration of 80 µg mL–1 into refolding buffer (2.5 mM GSH, 2.5 mM GSSG, 1 mM EDTA, 80 mM Tris/HCl [pH 8.7]), containing organic salts as indicated. Renaturation was allowed to proceed for 96 h at 15°C.
Activity assays
Lysozyme activity
Lysozyme activity was assayed by measuring the lysis of Micrococcus lysodeikticus (75 µg mL–1 lyophilized cells in 66 mM sodium phosphate buffer (pH 6.2), 25°C). Lysozyme samples were added to the stirred assay mixture, and activities were determined by measuring the decrease in turbidity at 450 nm over the initial 30 sec.
Antigen binding of ScFvOx
Concentrations of active ScFvOx were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well ELISA microtiter plates (NUNC) were coated overnight with 120 µL per well of ~25 µg mL–1 BSA-oxazolone conjugate in 0.1 M sodium carbonate buffer (pH 9.6). The conjugate was prepared according to Alfthan et al. (1995) and contained ~25 mol coupled 4-ethoxy-methylen-2-phenyl-2-oxazoline-5-one per mol of BSA. After coating, the plates were washed three times with ELISA blocking reagent (Roche Diagnostics), followed by blocking for 90 min at room temperature. Samples and standards were diluted into blocking reagent to a final volume of 100 µL per well, loaded onto the plates, incubated for 90 min, and unbound material washed off with three changes of blocking reagent. For the detection of bound ScFvOx, the plates were incubated for 1 h with 100 µL per well of a dilution (1:5000) of mouse anti-C-myc antibody (Roche Diagnostics) in blocking reagent. After one washing step with blocking reagent, the plates were incubated with horseradish peroxidase-coupled chicken anti-mouse-IgG (Chemicon) (1:3000). The plates were washed with substrate buffer (3.25 mM sodium perborate, 40 mM sodium citrate, 60 mM sodium phosphate [pH 4.5]) before applying 1 mg mL–1 2,2'-azino-di-(3-ethylbenzthi-azoline-6-sulfonic acid) (ABTS) in substrate buffer. Color development was followed at 405 nm in a Sunrise microplate reader (Tecan). Bound ScFvOx was determined from the increase in the absorption at 405 nm over 30 min.
Aggregation measurements
The formation of aggregates during the oxidative refolding of lysozyme was monitored by measuring the intensity of scattered light at 360 nm in a Hitachi F-4500 fluorescence spectrophotometer. In these experiments, the oxidative refolding of lysozyme was carried out as described above at 25°C under stirring, in the presence of a constant final concentration of 80 µM GuHCl carried over from the denatured protein solution.
Differential scanning calorimetry
Differential scanning calorimetry (DSC) experiments were carried out with a VP-DSC instrument (Microcal). For the measurements at pH 6.0, native lysozyme at a concentration of 2 mg mL–1 was dialyzed against buffers containing 11 mM citrate/NaOH, 1 mM EDTA, and organic salts in the indicated concentrations. For the measurements at pH 5.0 and 4.0, the solutions contained 20 mM citrate/NaOH and 290 mM acetate/NaOH, respectively. Degassed dialysis buffer was used to fill the instrument and to record the baseline. Prior to loading into the sample cell, the protein solutions were diluted with the respective dialysis buffer to a concentration of ~0.5 mg mL–1 and degassed. Temperature scans were performed from 10°C to 100°C with a scan rate of 90 K h–1. All samples were found to show a reversible thermal unfolding transition. After transformation of the heat capacity data from time-based to temperature- based and subtracting the buffer/buffer baselines, the corrected DSC traces were fitted using SigmaPlot 8.0 software (Jandel Scientific) to a two-state model, taking into account a linear temperature dependency of the heat capacity of the native state and a second-order polynomial for that of the denatured state.
Guanidinium chloride-induced folding/unfolding
The GuHCl-dependent unfolding of lysozyme was monitored by tryptophan fluorescence. For the equilibrium stability experiments, [GuHCl] series were prepared by mixing 50 µg mL–1 native lysozyme in buffer containing 11 mM citrate/NaOH (pH 6.0), and ionic liquids as indicated, with an equal concentration of denatured lysozyme in the same buffer, additionally containing 6 M GuHCl. The samples were equilibrated for 18 h at 30°C before fluorescence measurements were carried out. The excitation wavelength was set to 280 nm, and the fraction of unfolded protein was calculated from the change in fluorescence intensity at 360 nm.
Ionic liquids
The ionic liquids employed in this study were obtained from various sources, as indicated in Table 1, and used without further purification. In all cases they were more than 98% pure, as indicated by the respective manufacturers. N-Butyl- N'-methylimidazolium chloride (BMIM Cl), N-(2-hydroxy-ethyl)-N'-methylimidazolium chloride (OH-EMIM Cl), and N-(3-hydroxypropyl)-N'-methylimidazolium chloride (OH-PMIM Cl) were kind gifts from Dr. M. Dunkel (DEGUSSA AG, Trostberg, Germany) and Dr. B. Schulz (NIGU Chemie, Waldkraiburg, Germany). All salts studied were found to be miscible with aqueous buffers in any ratio, except for N-hexyl-N'-methylimidazolium chloride (HMIM Cl), which was occasionally found to form two-phase systems with aqueous buffers at mixing ratios below 10% (w/w). Densities were determined gravimetrically at room temperature to an accuracy of ±10 mg mL–1.
Other reagents
Ultra-pure guanidinium hydrochloride (GuHCl) was obtained from ICN Biomedicals. L-Arginine hydrochloride (L-ArgHCl) was from Ajinomoto. Lyophilized cells of Micrococcus lysodeikticus were purchased from Sigma-Aldrich. Benzonase was from Merck. All other reagents were of analytical grade or of higher purity.
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Acknowledgments |
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References |
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) the N'-(
) and BMIM Cl (•).