Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India
1 To whom correspondence should be addressed. e-mail: mn_gupta{at}hotmail.com
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: -chymotrypsin/chemical denaturation/inclusion bodies/pH-responsive polymers/protein folding
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The recovery of biological activity in the case of inclusion bodies is generally carried out by solubilizing these dense intracellular aggregates by unfolding protein molecules using denaturants such as 8 M urea or 6 M guanidine hydrochloride. It is for this reason that many attempts have been made to optimize the folding of denatured proteins. Such efforts include the use of water-soluble polymers (Middelberg, 2002). The pioneering work of Wang and coworkers (Cleland et al., 1992
) indicated that polyethylene glycol, a water-soluble polymer, facilitates correct folding via hydrophobic interaction with initial folding intermediates that are prone to aggregation. In this respect, such polymers act similar to molecular chaperonins (Jaenicke and Seckler, 1999
).
We have been working with a commercially available enteric polymer Eudragit S-100 for its application in bioseparation and biocatalysis (Sardar et al., 2000; Sharma and Gupta, 2002
). This non-toxic methylmethacrylate polymer is a reversibly soluble polymer, which precipitates at slightly acidic pH (
4.8) and redissolves around pH 6.0. (The exact transition point for solubility depends upon medium properties such as ionic strength.) This pH-responsive behavior is the result of the presence of both free carboxyl groups and hydrophobic moieties. At lower pH, carboxyl groups are protonated and hydrophobic interactions dominate; this leads to precipitation of the polymer. In view of the presence of hydrophobic moieties, this polymer was thought to be a promising candidate for facilitating protein folding. In this work, this polymer is evaluated for facilitating correct folding of urea-denatured
-chymotrypsin. It is shown that this may be one of the best additives for assisting correct folding of proteins in vitro.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-Chymotrypsin was assayed using benzoyl tyrosine ethyl ester as the substrate (Walsh and Wilcox, 1970
). The enzyme unit is defined as the amount of enzyme which hydrolyzes 1 µmol of the ester per minute at pH 7.8 and at 25°C, under specified conditions.
Unfolding and refolding of -chymotrypsin
-Chymotrypsin (from bovine pancreas, 3x crystallized from 4x crystallized chymotrypsinogen, Cat. No. C-4129; Sigma Chemical Co., St Louis, MO, USA) was used without further purification. The stock solution of Eudragit S-100 (Rohm Pharma GmbH, Weiterstadt, Germany) was prepared as described earlier (Sardar et al., 2000
). Eudragit S-100 is a copolymer of methacrylic acid and methyl methacrylate (in a molar ratio of 1:2), with molecular weight in the range of 200 000250 000 (Product Sheet; Rohm Pharma GmbH).
-Chymotrypsin (10 mg) was dissolved in 0.05 M TrisHCl buffer pH 8.2, containing 8 M urea (molecular biology grade; Sisco Research Laboratories, Mumbai, India) and 100 mM dithiothreitol (DTT) and incubated at 25°C for 4 h. A 0.1 ml aliquot of this unfolded enzyme was diluted with 0.1 ml of Eudragit S-100 (stock solution of 5%) and the final volume was made up to 1 ml with 0.05 M TrisHCl buffer, pH 8.2. The diluted sample was then assayed for enzyme activity.
Native gel electrophoresis of -chymotrypsin (Betts et al., 1999)
Gels were cast at room temperature. The stacking gel was 4% acrylamide/bisacrylamide, whereas the resolving gel was 10% acrylamide/bisacrylamide. The concern that stacking of aggregation intermediates will promote further aggregation in the gel was taken care of by comparing the distribution of multimers in the same sample separated by electrophoresis with and without a stacking gel. Gels and running buffer were equilibrated at 4°C before loading chilled samples. Sample loading and electrophoresis were performed in a cold room (45°C). The samples, collected at different time intervals, were stored on ice. The efficiency of cold quenching was determined by assaying the samples at various time intervals after chilling. The gel was stained with Coomassie Blue G-250 at room temperature for 45 min and then destained.
Inactivation of -chymotrypsin by organic solvent
-Chymotrypsin (10 mg) was suspended in 1 ml of 90% dioxane (in 0.05 M TrisHCl, pH 7.8) and shaken at 25°C. After 1 h, the residual enzyme activity was measured in the following ways (Soler et al., 1997
). (i) Aliquots were withdrawn at different time intervals and directly assayed in the presence of the organic co-solvent. (ii) Aliquots of the suspended enzyme were directly assayed using the standard protocol, in the absence of organic co-solvent. (iii) Aliquots were withdrawn and washed thoroughly with the assay buffer and incubated in the same for 24 h; they were then assayed as usual. (iv) Aliquots were withdrawn and washed thoroughly with the assay buffer. The samples were then incubated in 0.05 M TrisHCl buffer, pH 8.2, containing 8 M urea and 100 mM DTT for 2 h at 25°C. The denatured samples were then renatured with 0.5% Eudragit S-100, as described earlier.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Figure 1 shows that the presence of Eudragit S-100 reduces the unfolding of -chymotrypsin in the presence of various concentrations of urea. Unfolding of
-chymotrypsin leads to increase in emission intensity of intrinsic fluorescence (Desie et al., 1986
), hence fluorescence emission is considered a sensitive tool to monitor the extent of unfolding. Figure 2 shows the effect of the presence of polymer during the refolding of urea-denatured enzyme. The presence of polymer not only led to a higher regain of activity but the refolding rate was also much faster. In the absence of the polymer, only 31% of the original activity could be regained after 15 h. No further regain of activity was observed beyond this time. On the other hand, the presence of the polymer led to a swift regain of 100% activity within 10 min.
|
|
|
|
|
|
The work of Betts et al. (Betts et al., 1999) has established that native gel electrophoresis constitutes one of the simplest and perhaps the best method for detection of aggregation intermediates during unfolding and refolding of proteins. Figure 5 shows that urea-denatured
-chymotrypsin, 5 min after being placed in the refolding buffer, consisted of dimers and multimers (lane A). Even after 1 h, higher molecular weight multimers, with a considerable extent of aggregation, could still be observed (lane B). However, if the refolding buffer contained Eudragit S-100, within 5 min, a considerable number of higher molecular weight multimers disappeared (lane C). After 1 h (in the presence of Eudragit S-100; lane D), only monomeric
-chymotrypsin could be observed at a position similar to the position of native
-chymotrypsin; lane E). Comparison of lanes C and D indicate that with time, proteinprotein interactions are replaced by polymerprotein interactions. Thus, the latter are favored thermodynamically and lead to correct refolding in the presence of the polymer.
|
Soler et al. (Soler et al., 1997) have reported that
-chymotrypsin gets inactivated in the presence of 90% dioxane. Table III shows that placing
-chymotrypsin in 90% dioxane for 1 h at 25°C led to complete inactivation of the enzyme. Dilution with the aqueous assay buffer led to slow and only
10% regain of enzyme activity. The organic solvent induced denaturation could be reversed after unfolding the inactivated enzyme by 8 M urea (Table III). After urea denaturation, whereas dilution with simple assay buffer led to only 5.5% regain of the activity after 1 h, dilution with Eudragit S-100 led to a fairly rapid regain of the activity. Approximately 78% activity could be regained within 5 min and after 1 h, 100% activity regain was observed.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Solubilization of inclusion bodies is usually carried out in 8 M urea or 6 M guanidine hydrochloride at alkaline pH and in the presence of a reducing agent like ß-mercaptoethanol or dithiothreitol (Jaenicke and Rudolph, 1989). In the case of
-chymotrypsin, Hibbard and Tulinsky (Hibbard and Tulinsky, 1978
) have shown the massive penetration of the interior of the enzyme by urea, in contrast to guanidine hydrochloride which showed no significant changes in the protein interior. In fact, urea bound within protein interiors is presumed to stabilize the partly folded protein chains. This double-edged denaturation/activation of enzymes has been usefully employed for enhancing enzyme activity in organic solvents (Guo and Clark, 2001
). Thus, for the purpose of unfolding/refolding proteins in inclusion bodies, urea appears to be a more suitable choice as it is expected to stabilize the unfolded intermediates and simultaneously help unfolding the insoluble protein bodies sufficiently to correct any misfolded structures. As the aim of the present work was to use Eudragit S-100 in refolding denatured
-chymotrypsin, as a model system for possible future application to recovery of biological activity from inclusion bodies, 8 M urea was chosen as the denaturant.
Refolding of denatured -chymotrypsin
In the case of recovery of active proteins from inclusion bodies, the unfolding step (which solubilizes the insoluble and misfolded protein moieties) is followed by the refolding step. The protein refolding is initiated by reduction in the concentration of denaturant. Two competing processes at this stage are the first order refolding and higher order aggregation. Thus, carrying out refolding at dilute protein concentration (<1050 µg ml1) reduces aggregation and leads to a varying extent of regain of protein activity. However, such low concentrations are impractical for obtaining refolded proteins in an economical way. This has been the motivation for the search for additives which facilitate correct refolding, even at a practical range of protein concentrations. As Middelberg (Middelberg, 2002) has pointed out, the search for conditions for efficient refolding of denatured proteins continues to be an ongoing effort. Recently, a temperature-sensitive polymer, poly(N-isopropylacrylamide), has been used for refolding of carbonic anhydrase B (Chen et al., 2003
). Even with an initial protein concentration of 5 mg ml1 (diluted 100 times with the refolding buffer), not more than 75% of activity could be regained. The time period was not clearly mentioned but appeared to be more than 1 h. The refolding yield decreased rapidly with increase in protein concentration; with an initial protein concentration of 15 mg ml1, the reported refolding yield was 39%. It is also considered advisable to use denaturants at non-denaturing conditions at the refolding stage. The presence of these denaturants at much lower concentrations is presumed to prevent aggregation and hence promote correct folding (Clark et al., 1999
). Thus, all refolding steps in this work were carried out in the presence of 0.8 M urea. In the present work, the use of the pH-responsive polymer as an additive in the refolding buffer led to a 100% regain of activity within 10 min, even when the initial protein concentration was 12.5 mg ml1 (diluted 10 times with the refolding buffer). In fact, we have not come across a more efficient refolding additive in the literature. Our approach is fast, consists of a single step and leads to complete recovery of the enzyme activity.
Renaturation of -chymotrypsin inactivated by dioxane
The use of enzymes and other proteins in a predominantly non-aqueous environment has emerged as an important approach in organic synthesis and the resolution of enantiomers (Gupta, 2000). Thus, understanding their instability/inactivation in organic solvents is vital to enlarging the scope of non-aqueous enzymology. Soler et al. (Soler et al., 1997
) showed that
-chymotrypsin, inactivated in 90% dioxane, did not show significant renaturation even after 24 h. However, unfoldingrefolding gave complete recovery in the case of the immobilized preparation. The polymer-assisted refolding worked equally well in the current work even when the enzyme was free and not anchored to a stabilizing matrix. The activity regain was much faster and complete recovery within 1 h was observed.
Possible role of the polymer in folding
Eudragit S-100 is a stimulus-responsive or smart polymer (Roy and Gupta, 2003). It is a pH-responsive polymer, which is soluble at pH 8.2, the pH used during the refolding experiments. At this pH, it is an anionic polymer consisting of carboxylate moieties. It also has hydrophobic structural components in the form of esterified carboxylate groups. Cleland et al. (Cleland et al., 1992
) concluded in their work that both polyethylene glycol and the chaperonin GroEL, bind to hydrophobic structures in the partially unfolded protein molecules and thus inhibit their aggregation without altering their rate of folding. The mode of increasing percent refolding (to correct structure) by Eudragit S-100 is likely to be similar. Chen et al. (Chen et al., 2003
), while working with a temperature-responsive smart polymer, have also explained the role of such a polymer in assisting protein refolding in a similar way. However, unlike the results of Cleland et al. (Cleland et al., 1992
) with polyethylene glycol, Eudragit S-100 in fact did alter the rate of refolding. Such an acceleration of refolding by anionic polymers has been reported by Rentzeperis et al. (Rentzeperis et al., 1999
), who implicated electrostatic interactions in the rate acceleration of the polyanion-mediated refolding. It is likely that Eudragit S-100 achieves the acceleration of refolding in a similar way.
Potential in vitro and in vivo applications
As mentioned in the Introduction, water-soluble polymers (and chaperonins in vivo) are assumed to assist correct refolding by preventing aggregation by attachment to exposed hydrophobic portions of the polypeptide. We have not come across any study where the refolding additives are shown to dissociate first the preformed aggregates, and then push the folding of the monomeric protein along the right pathway. The native gel electrophoresis data (Figure 5) shown in this work indicate this new role of refolding additives. It is possible that other workers have not observed it because most of the refolding studies have been done with fairly dilute solutions. Thus, it is possible that upon extensive dilution, refolding additives merely had to assist refolding of monomers since, in these cases, dilution prevented aggregate formation. It may be intriguing to determine whether molecular chaperonins also play a similar role in vivo.
One of the key approaches in understanding protein folding has been to study refolding of unfolded proteins. Besides, recovery of activity from over-expressed proteins (in the form of inclusion bodies) and involvement of protein aggregation in the case of several human diseases (Sadana, 2000; Carrio and Villaverde, 2001
) are two important areas wherein studying protein unfoldingrefolding is relevant. The use of a non-toxic, pH-responsive polymer in the present work has the additional advantage that the polymer can be separated and reused merely by precipitation around pH 5.0. Most of the proteins, including
-chymotrypsin, are stable around this pH region. The possibility of refolding at fairly high protein concentration provides one with a practical method for renaturation.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carrell,R.W. and Lomas,D.A. (1997) Lancet, 350, 134138.[CrossRef][ISI][Medline]
Carrio,M.M. and Villaverde,A. (2001) FEBS Lett., 489, 2933.[CrossRef][ISI][Medline]
Chen,Y.-J., Huang,L.-W., Chiu,H.-C. and Lin,S.-C. (2003) Enzyme Microb. Technol., 32, 120130.[CrossRef][ISI]
Clark,E.D.B., Schwarz,E. and Rudolph,R. (1999) Methods in Enzymol., 309, 217236.[ISI][Medline]
Cleland,J.L. and Wang,D.I.C. (1990) Biochemistry, 29, 1107211078.[ISI][Medline]
Cleland,J.L., Hedgepeth,C. and Wang,D.I.C. (1992) J. Biol. Chem., 267, 1332713334.
Desie,G., Boens,N. and De Schryver,F.C. (1986) Biochemistry, 25, 83018308.[ISI][Medline]
Guo,Y. and Clark,D.S. (2001) Biochim. Biophys. Acta, 1546, 406411.[ISI][Medline]
Gupta,M.N. (2000) Methods in Non-Aqueous Enzymology. Birkhauser Verlag, Basel.
Hibbard,L.S. and Tulinsky,A. (1978) Biochemistry, 17, 54605468.[ISI][Medline]
Jaenicke,R. and Rudolph,R. (1989) In Creighton,T.E. (ed.), Protein Structure. A Practical Approach. IRL Press, Oxford, pp. 191223.
Jaenicke,R. and Seckler,R. (1999) In Bukau,B. (ed.), Molecular Chaperones and Folding Catalysts. Regulation, Cellular Function and Mechanisms. Harwood Academic Publishers, Amsterdam, pp. 407436.
Middelberg,A.P.J. (2002) Trends Biotechnol., 20, 437443.[CrossRef][ISI][Medline]
Rentzeperis,D., Jonsson,T. and Sauer,R.T. (1999) Nat. Struct. Biol., 6, 569573.[CrossRef][ISI][Medline]
Roy,I. and Gupta,M.N. (2003) Chem. Biol., 10, 111.[CrossRef][ISI][Medline]
Sadana,A. (2000) In Gupta,M.N. (ed.), Methods in Non-Aqueous Enzymology. Birkhauser Verlag, Basel, pp. 195211.
Sardar,M., Roy,I. and Gupta,M.N. (2000) Enzyme Microb. Technol., 27, 672679.[CrossRef][ISI][Medline]
Sharma,A. and Gupta,M.N. (2002) Biotechnol. Bioeng., 80, 228232.[CrossRef][ISI][Medline]
Soler,G., Bastida,A., Blanco,R.M., Fernandez-Lafuente,R. and Guisan,J.M. (1997) Biochim. Biophys. Acta, 1339, 167175.[ISI][Medline]
Walsh,K.A. and Wilcox,P.E. (1970) In Perlmann,G.E. and Lorand,L. (eds), Methods in Enzymology. Academic Press, New York, Vol. 19, pp. 3141.
Received June 17, 2003; revised September 26, 2003; accepted October 21, 2003