An approach for protein to be completely reversible to thermal denaturation even at autoclave temperatures

Masahiro Iwakura1,, Dai Nakamura, Tatsuyuki Takenawa and Yasushi Mitsuishi

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Reversibility of protein denaturation is a prerequisite for all applications that depend on reliable enzyme catalysis, particularly, for using steam to sterilize enzyme reactors or enzyme sensor tips, and for developing protein-based devices that perform on–off switching of the protein function such as enzymatic activity, ligand binding and so on. In this study, we have successfully constructed an immobilized protein that retains full enzymatic activity even after thermal treatments as high as 120°C. The key for the complete reversibility was the development of a new reaction that allowed a protein to be covalently attached to a surface through its C-terminus and the protein engineering approach that was used to make the protein compatible with the new attachment chemistry.

Keywords: complete reversibility/cyanocysteine-mediated reaction/dihydrofolate reductase/immobilized enzyme/thermal denaturation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Denaturation of proteins can occur under a variety of physical conditions, including high temperature, very low or high pH, and very high pressure (Tanford, 1968Go; Klibanov, 1983Go). In many cases, the denaturation is only partially reversible (Klibanov, 1983Go; Volkin and Klibanov, 1987; Stempfer et al., 1996Go); thermal denaturation in particular often results in irreversible inactivation (Klibanov, 1983Go). Because of this irreversibility, it is not possible to use steam to sterilize enzyme reactors or enzyme sensor tips, greatly limiting their applications. Complete reversibility is also a limiting factor in the development of protein-based devices that perform on–off switching of the protein function such as enzymatic activity, ligand binding and so on by physical means. If complete reversibility of thermal denaturation could be achieved, the availability of protein catalysts would greatly increase and thermal sterilization, as well as on–off control, of enzyme reactors would become possible.

In contrast to the efforts made to improve thermal stability through protein engineering (Liao et al., 1986Go; Shortle, 1992Go; Malakauskas and Mayo, 1998Go), few attempts have been made to improve thermal reversibility (Volkin and Klibanov, 1987; Iwakura et al., 1995Go; Iwakura and Honda, 1996Go), nor has a general strategy been developed. We have been able to improve the reversibility of thermal denaturation of an enzyme by circularization (Iwakura and Honda, 1996Go), namely, by cross-linking the N- and C-termini. Even then, however, the denaturation was not completely reversible. Because the primary cause of irreversible inactivation is now recognized to be aggregation of proteins during unfolding and refolding (Speed et al., 1996Go; Stempfer et al., 1996Go; Altamirano et al., 1997Go), preventing intermolecular interactions would be a simple way to achieve reversible protein denaturation (Klibanov, 1983Go; Volkin and Klibanov, 1987; Stempfer et al., 1996Go) and immobilization of proteins to a solid surface is a straightforward way to separate protein molecules from each other. Stempfer et al. report that the refolding yield of a protein after denaturation was greatly improved when the protein was immobilized on a solid surface (Stempfer et al., 1996Go). They also discovered, however, that solvent conditions that caused the denatured protein to be adsorbed to the surface at multiple points resulted in a drastic decrease in recovery from denaturation. Therefore, it appears that in order to achieve reversible denaturation, the protein must be immobilized to the surface at a single site. It is also necessary to have a thermostable covalent linkage connecting the protein to the solid support in order to prevent loss of immobilized protein.

Many reactions, including disulfide bond formation, have been used to immobilize proteins to surfaces through covalent bonds (Mosbach, 1976Go; Srere and Uyeda, 1976Go). Almost all such reactions, however, rely on the chemical reactivity of amino acid side chains (Srere and Uyeda, 1976Go), making it difficult to immobilize a protein at a single site. Therefore, if one could use either the N- or C-terminus as the site of immobilization, then attachment at a single position becomes possible. Recently, we found an interesting reaction route involving chemical cleavage at a cysteine residue (Figure 1Go) (Takenawa et al., 1998Go; Ishihama et al., 1999Go). The cleavage reaction, studied extensively by Catsimpoolas et al. (1966) and Jacobson et al. (1973), occurs at the peptide bond of a cyanocysteine residue formed by cyanylation of an SH group using the reagent 2-nitro-5-thiocyanobenzoic acid (NTCB) (Degani and Patchornik, 1971GoDegani and Patchornik, 1974Go). The reaction occurs under mild conditions and is highly efficient. Cyanylation of the SH group of the cysteine residue causes the carbonyl carbon at an X-cyanocysteinyl linkage to become susceptible to attack by an OH acting as a nucleophile (hydrolysis: route 1). Alternatively, the cyanocysteine residue can be converted to a dehydroalanine residue by a ß-elimination reaction (route 2). The use of a primary amine as a nucleophile results in the formation of an amide bond (aminolysis) both intra- (route 3) and inter-molecularly (route 4) (Ishihama et al., 1999Go). The route 4 reaction is quite appealing, not only because it takes place under moderate conditions without any catalysts, but also because it allows the formation of an amide bond between the C-terminus of the protein and an NH2-group on the solid support. It is noteworthy that the reaction produces homogeneously immobilized protein because only the route 4 reaction produces the immobilized protein, allowing the other reaction products and unreacted protein to be removed by washing the resin such as with guanidine–HCl. To examine the effectiveness of the route 4 reaction, dihydrofolate reductase (DHFR) was used as a model protein.



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Fig. 1. Reaction routes involved in the cyanocysteine-mediated immobilization reaction (route 4) of a protein having a C-terminal extension of (Gly)n-cyanocysteine-Xaa [A] and a solid support containing a primary amine (NH2-) as a reacting group [B]. Reaction route 1 is a hydrolysis reaction that cleaves the Gly-cyanocysteine (X-cyanocysteine) linkage to form a protein with a (Gly)n C-terminal extension [C]. Reaction route 2 is a ß-elimination reaction that converts Gly-cyanocysteine-Xaa to Gly-dehydroalanine-Xaa [D]. Reaction route 3 is an intramolecular aminolysis reaction to form a protein with an intramolecular cross-link [E]. Reaction route 4 is an intermolecular aminolysis reaction to produces a protein immobilized at its C-terminal end through the (Gly)n-linker [F]. Xaa indicates an amino acid.

 
DHFR (EC 1.5.1.3) is a monomeric, two domain protein that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using the reducing cofactor NADPH (Blakley, 1984Go). DHFR is a clinically important enzyme not only as the target of a number of antifolate drugs, such as trimethoprim (TMP) and methotrexate (MTX), but also because it can be used to produce l-leucoverin, an anti-cancer drug, in a stereospecific manner (Uwajima et al., 1990Go). DHFR from Escherichia coli is denatured at temperatures higher than 40°C. This denaturation is only partially reversible and is accompanied by aggregation and precipitation (Iwakura et al., 1995Go). An engineered disulfide bond inside DHFR, designed to reduce local flexibility, did not improve the thermal stability or the reversibility, although overall conformational stability as measured by reversible guanidine–HCl denaturation is apparently improved (Villafranca et al., 1986Go). A Cys-free DHFR (Iwakura et al., 1995Go) and a circularized form (Iwakura and Honda, 1996Go) showed improved, but not complete, reversibility. Thus, DHFR is a good model for examining our new approach to obtaining an enzyme with complete reversibility against thermal denaturation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Materials

Amino-cellulofine that was a thermally resistant resin containing primary amines (~20 µmol NH2/ml gel) was purchased from Seikagaku Kogyo Co. (Tokyo, Japan). DEAE-Toyopearl 650M was from Tosoh Co. (Tokyo, Japan). All primer DNAs for mutagenesis were synthesized by JbioS Ltd (Saitama, Japan).

Plasmid construction and protein purification

The construction of a Cys-free double mutant DHFR (Cys85Ala + Cys152Ser), AS-DHFR, has been previously described (Iwakura et al., 1995Go). Genes for AS-DHFRs tethered with Gly-Gly-Gly-Gly-Cys, Gly-Gly-Gly-Gly-Cys-Ala, Gly-Gly-Gly-Gly-Cys-Gly and Gly-Gly-Gly-Gly-Cys-Val peptides at the C-terminus were constructed by PCR using the primers listed in Table IGo and the plasmid pDFHR20 (Iwakura et al., 1995Go) as a template. The amplified DNA contained BamHI sites (GGATCC) at both termini, the DHFR gene with tethered sequence, stop codon (TAA), and a ribosome binding site (GGAGG) and overexpression promoter that were derived from pDHFR20. The resulting genes were inserted into the BamHI site of a high copy vector, pUC19, which was used to transform E.coli JM109 cells to TMP-resistance, which is a good indication for the overproduction of DHFR in E.coli cells (Iwakura et al., 1983Go). Recombinant plasmids were isolated from several colonies on an agar plate containing 200 µg/ml ampicillin and 100 µg/ml TMP. DNA sequence of the BamHI insert of the isolated plasmids was determined and the plasmid with designed sequence was selected. The E.coli strain with the selected plasmid was used for protein purification of the designed tethered DHFR.


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Table I. DNA primers used for the construction of DHFR mutants
 
Purification of the DHFR variants was carried out primarily by MTX-affinity chromatography, taking advantage of adequate pre-purification steps from cell-free extracts (Iwakura et al., 1992Go). Purified protein was stored as precipitated form in 10 mM phosphate buffer pH 7.0 containing 1 mM EDTA, 14 mM 2-mercaptoethanol and saturated ammonium sulfate. Protein concentration of the DHFR variants was determined by the absorbance at 280 nm using the extinction coefficient ({varepsilon}280 = 31 100 M–1 cm–1; Touchette et al., 1986Go).

Enzyme assay

The activity of free DHFR was determined spectrophotometrically at 15°C by following the disappearance of NADPH and DHF at 340 nm ({varepsilon}340 = 11 800 M–1 cm–1; Hillcoat et al., 1967Go). The standard assay mixture contained 50 µM DHF, 100 µM NADPH, 14 mM 2-mercaptoethanol, 1x MTEN buffer [50 mM 2-morpholinoethanesulfonic acid, 25 mM tris(hydroxymethyl)aminomethane, 25 mM ethanolamine and 100 mM NaCl pH 7.0; Morrison and Stone, 1988] and the enzyme in a final volume of 1.0 ml. The reaction was started by the addition of DHF. The unit of enzyme activity was defined as µmol tetrahydrofolate formed per minute.

Conversion of Cys residue to cyanocysteine residue by chemical modification with NTCB

Precipitated (stored) protein was collected by centrifugation at 20 000 g for 20 min. The collected protein was dissolved in a small volume of 10 mM phosphate buffer pH 8.0 containing 2.5 mM EDTA and 1 mM dithiothreitol to make a protein concentration of ~0.1 mM, and then, incubated at 37°C for 30 min to completely reduce the SH group. Excess thiol compounds were removed by gel filtration through a column of Sephadex G-15 (1.0x12 cm). The SH group of the protein (~50 µM) was cyanylated by chemical modification with 5 mM NTCB in 10 mM phosphate buffer pH 7.0 containing 2.5 mM EDTA for 4 h at room temperature. In preparation for the reaction with octylamine, the protein was dialyzed three times against 1 mM phosphate buffer pH 6.5 at 4°C, and then lyophilized. The lyophilized protein was dissolved in an adequate volume of buffer and used for the reaction with octylamine. For the immobilization reaction, modified protein was applied to a column of Sephadex G-15 (1.0x12 cm), which had been equilibrated with 10 mM borate buffer pH 9.5 to remove the chemical reagents and to exchange the buffer solution. The modified protein thus obtained was immediately used for immobilization reaction with amino resin.

LC–MS measurements

Liquid chromatography–electrospray mass spectrometric (LC–MS) measurements were performed as previously described (Takenawa et al., 1998Go). Separation of protein was carried out with an acetonitrile gradient from 35 to 55% containing 0.1% TFA on an L-column ODS (2.1x150 mm; Kagaku-hin Kensa Kyoukai, Tokyo, Japan) on a Shimadzu HPLC system lC-10A.

Others

DNA sequencing was performed on an ABI PRIZM 310 Genetic analyzer. N-terminal amino acid sequences were determined by Edman degradation on a Beckman System Gold LF3000 protein sequencer equipped with an on-line PTH amino acid analyzer.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Design of a mutant DHFR protein to be immobilized

A Cys-free DHFR (AS-DHFR) was used in this study. PCR mutagenesis was used to extend the C-terminus of AS-DHFR with the Gly-Gly-Gly-Gly-Cys, Gly-Gly-Gly-Gly-Cys-Ala, Gly-Gly-Gly-Gly-Cys-Gly and Gly-Gly-Gly-Gly-Cys-Val peptides, which allow the protein to covalently bind to a solid support. A schematic drawing of the tethered DHFRs is shown in Figure 2Go. The sole Cys-SH residue in the tethered DHFRs was converted to S-cyanocysteine by a treatment with NTCB.



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Fig. 2. Schematic representation of AS-DHFR with a C-terminal extension of (Gly)4-cyanocysteine-Xaa. Ala-85 and Ser-152 are both Cys residues in wild-type DHFR. The positions of the six Lys residues are indicated. The carbonyl carbon at the Gly-cyanocysteine (X-cyanocysteine) linkage is the target of nucleophilic attack of octylamine [1] or the amino resin [2].

 
Reaction with octylamine

The efficiency of the route 4 reaction with 10 mM octylamine was studied (reaction with [1] in Figure 2Go). The route 4 product could be separated from the other products by HPLC with L-column ODS and made it possible to determine the efficiency of the reaction by LC–MS measurement, probably because of the increased hydrophobicity of the attached octylamine. Figure 3AGo shows the time course of the reaction, indicating that the reaction yield was dependent on the identity of the Xaa residue in the cyanocysteine-Xaa sequence, and that Ala appears to the best choice as Xaa. Figure 2BGo shows the pH dependence of the reaction. At the optimum pH of 9.5, more than 50% of the protein had reacted with octylamine. The molecular mass of the product was determined to be 18 291 Da, in good agreement with the calculated value of 18 290 Da for AS-DHFR-(Gly)4-CONH-(CH2)7-CH3.



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Fig. 3. (A) Effect of Xaa on the route 4 reaction with octylamine as a nucleophile. Closed circles, Xaa=Ala; open diamonds, Xaa=Gly; open squares, Xaa=Val; open circles, no Xaa. Reaction was carried out in 10 µM AS-DHFR-(Gly)4-cyanocysteine-Xaa, 10 mM octylamine and 50 mM borate buffer pH 9.0 at room temperature. (B) pH dependence of the route 4 reaction with octylamine. Reaction was carried out in 10 µM AS-DHFR-(Gly)4-cyanocysteine-Ala, 10 mM octylamine and 50 mM borate buffer (indicated pH) at room temperature.

 
Immobilization of the tethered DHFR on amino-cellulofine resin

To apply the route 4 reaction for the site-specific immobilization, 5 ml of amino-cellulofine were mixed with 10 ml of AS-DHFR-(Gly)4-cyanocysteine-Ala (25 µM, total 250 nmol protein) at room temperature in 10 mM borate buffer pH 9.5. After mixing for 5 min, about 50% of protein remained in solution, while the rest of the protein was probably adsorbed to the amino-resin by ionic interaction. In fact, when the reaction was carried out in the same buffer containing 0.5 M KCl, it was observed that almost all the protein was found in solution. After 24 h incubation at room temperature, solid KCl was added to bring the KCl concentration to 0.5 M and the amount of protein in solution was measured by absorbance at 280 nm. The total amount of protein in solution was 154 nmol, indicating that about 40% of the protein (96 nmol of DHFR/5 ml gel) was immobilized to the resin through the route 4 reaction. The immobilized gel was washed with 10 mM phosphate buffer pH 7.0 containing 5 M guanidine–HCl, 1 mM EDTA and 14 mM 2-mercaptoethanol to remove non-covalently bound protein, then equilibrated with the same buffer without guanidine–HCl. The amount of immobilized DHFR was also measured using MTX. The washed immobilized gel (5 ml gel) was incubated with 0.1 mM MTX in the phosphate buffer pH 7.0, then washed with the same buffer containing 1 M KCl to remove MTX bound to the amino-resin by electrostatic interaction. The MTX bound to immobilized DHFR was eluted with 5 M guanidine–HCl and the concentration of the eluted MTX was determined spectrophotometrically at 302 nm at pH 12 ({varepsilon}302 = 22 100 M–1 cm–1). The amount of the eluted MTX was 101 nmol, which was in agreement with the value obtained by direct spectrophotometric measurement of the protein (96 nmol).

Activity measurement

Figure 4Go shows a schematic drawing of a flow reactor for measuring the activity of the immobilized DHFR. Since the degree of immobilization was high (~20 nmol enzyme/ml gel), it was difficult to obtain the relatively small amounts of DHFR needed for the standard assay conditions (~200 pmol/ml of free DHFR). Therefore, in order to keep the enzyme reaction below saturation levels, reaction conditions using as little as 100 µl of gel (~2 nmol enzyme) with the flow rate as high as 2 ml/min were necessary even at a temperature as low as 15°C. Under these conditions, the reaction went to ~80% completion as the mixture flowed over the column. In this study, we use the ‘percentage conversion value’ defined as follows: using the flow reactor as shown in Figure 4Go, first, the absorbance at 340 nm was measured with tubing in place of the reaction column, A(1); second, the absorbance was measured with the reaction column in place, A(2); third, the absorbance of the reaction mixture containing an excess of free DHFR was measured, A(3). The percentage conversion was defined as 100x[A(1)–A(2)]/[A(1)–A(3)].



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Fig. 4. Schematic drawing of the flow reactor for measuring the activity of immobilized DHFR. The reaction mixture and reaction column were located in the same water bath. The temperature of the flow cell housing in the spectrophotometer was kept at 15°C.

 
At flow rates of 2 and 5 ml/min, the percentage conversion values were 80 and 43%, respectively. Based on the obtained percentage conversion values and calculated reaction times, i.e. the length of time that the reaction mixture is in contact with the gel, the activities of the immobilized DHFR at flow rates of 2 and 5 ml/min were estimated to be 0.95 and 1.27 units (µmol product/min), respectively. Enzyme assays are usually carried out under homogeneous conditions and the initial reaction rate, which is measured at the region corresponding to a very low percentage conversion, is used to determine the activity. In order to achieve this low percentage conversion in the flow reactor, it was necessary to use the higher flow rate (5 ml/min). The specific activity obtained at the higher flow rate (6.4x108 units/mol of DHFR) is in good agreement with the specific activity obtained for free DHFR (3x108 units/mol DHFR at 15°C) suggesting that the immobilized DHFR is fully active.

Thermal treatment of immobilized DHFR

Temperature dependence of the activity of the immobilized enzyme is shown in Figure 5AGo. The percentage conversion value increased with increasing temperature between 15 and 25°C. At temperatures between 25 and 55°C, the percentage conversion value was as high as 95%, which probably represents saturation level using this measurement technique due to diffusion or mixing problem with using the small amount of gel and high flow rate as 2 ml/min. 100% conversion could be attained at a flow rate as low as 0.5 ml/min even at a lower temperature than 10°C. At temperatures higher than 60°C, the percentage conversion value decreased with increasing temperature, indicating that the immobilized enzyme is thermally denatured with an apparent Tm of ~70°C. To test the reversibility of the immobilized enzyme, the reaction column shown in Figure 3Go was held at various temperatures between 30 and 90°C for 30 min; the temperature was then lowered to 15°C and the percentage conversion value was measured. Thermal treatment between 100 and 120°C was accomplished by removing the reaction column from the flow reactor and placing it in an autoclave with the timer set for 5 min at various temperatures. After autoclaving, the treated column was put back into the flow reactor and the percentage conversion value was measured at 15°C. In the case of the autoclaved samples, it usually required a total of more than 30 min to raise the temperature to the set values and to lower the temperature below 100°C for each 5 min treatment. Despite this long period of time spent at high temperatures, the immobilized enzyme showed complete reversibility against the thermal treatment up to 120°C (Figure 5BGo). Free enzyme with a similar concentration (20 nmol/ml) showed irreversible inactivation with an apparent Tm of ~52°C (in 10 mM phosphate buffer pH 7.0) and ~59°C (in reaction mixture). Figure 5CGo shows the effect of repeated thermal treatment at 90 and 120°C on the reversibility of the immobilized DHFR. Repeated thermal treatment at 90°C did not cause any loss of enzymatic activity, indicating that the thermal denaturation process is completely reversible under these conditions. On the other hand, repeated thermal treatment at 120°C caused a gradual loss of enzymatic activity, although ~95% of the activity was retained even after five autoclave treatments. Treating the immobilized enzyme with guanidine–HCl (5 M), which dissolves aggregated proteins, did not improve the recovery of the enzymatic activity lost by thermal treatment at 120°C. This suggests that chemical damage occurred in the thermally treated enzyme at 120°C that reduced the enzyme activity.



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Fig. 5. (A) Temperature dependence of the efficiency (% conversion) of the reaction catalyzed by the immobilized DHFR. The reaction column contains 100 µl gel of the immobilized DHFR (~2 nmol DHFR). The flow rate of the reaction mixture (pH 7.0) was 2 ml/min. The error bars indicate the standard deviation of triplicate measurements using the same reaction column throughout the experiment. (B) Thermal treatment of the immobilized DHFR. For the thermal treatment of the immobilized DHFR between 30 and 90°C (open circles), the reaction column containing 100 µl gel of the immobilized DHFR (~2 nmol DHFR) was kept for 30 min at the indicated temperatures, and then, the temperature was lowered to 15°C and the percentage conversion value was measured. For the thermal treatment between 100 and 120°C (closed diamonds), the reaction column was placed in an autoclave and the timer was set for 5 min at various temperatures. After autoclaving, the treated column was placed back in the flow reactor and the percentage conversion value was measured at 15°C. For the thermal treatment of free enzyme in MTEN buffer pH 7.0 (closed circles) and in reaction mixture pH 7.0 (open squares), 0.1 ml of AS-DHFR (20 nmol/ml) was incubated at the indicated temperature for 30 min, then chilled on ice for more than 20 min. Activity was determined by the standard enzyme assay mixture at 15°C. Residual activity is defined as 100x(the percentage conversion value after thermal treatment/the percentage conversion value before thermal treatment) for the immobilized enzyme and as 100x(the enzymatic activity after thermal treatment/the enzymatic activity before thermal treatment) for free enzyme. (C) Effects of repeated thermal treatment on the activity of the immobilized DHFR at 90°C (open circles) and 120°C (closed circles). The thermal treatments and activity measurements were carried using the same methods described in (B).

 
Immobilization reaction

Previously, we attached DHFR to a polymer support through a disulfide (S–S) linkage at its C-terminal end (Iwakura and Kokubu, 1993Go). Probably because the S–S linkage was not stable enough to prevent loss of protein at high temperatures, thermal denaturation of DHFR immobilized in this way was not fully reversible (Iwakura et al., unpublished observation). Although amide- and azide-bonds are much more stable than an S–S linkage (Srere and Uyeda, 1976Go), side reactions involving amino acid side-chains such as the COOH group of Asp and Glu residues and the NH2 group of Lys residues make it difficult to control the specificity of the reaction so that immobilization occurs only at the C-terminus. Our cyanocysteine-mediated reaction system (Takenawa et al., 1998Go; Ishihama et al., 1999Go) provides a stable covalent linkage between a protein and a polymer support (amide bond) and produces homogeneously immobilized enzyme. Although four reaction routes shown in Figure 1Go are involved in the cyanocysteine mediated reaction, only the route 4 reaction produces immobilized protein, allowing the other reaction products and unreacted protein to be removed by washing the resin with guanidine–HCl.

Protein design

In order to use this immobilization reaction, the amino acid sequence of the naturally occurring protein must be rendered Cys-free while still retaining its original activity. Then, a linker-polypeptide with a C-terminal sequence of Cys-Ala, that is required for an efficient cyanocysteine mediated reaction, is attached to the C-terminal end of the cysteine-free protein. In the case of DHFR, two Cys residues (Cys-85 and Cys-152) in the wild-type sequence were successfully substituted to other amino acids with no loss of activity (Iwakura et al., 1995Go). Because internal cysteine and cystine residues are not easily converted to cyanocysteine by chemical modification under native conditions and because the Cys-residue in the linker polypeptide was highly reactive (Iwakura and Kokubu, 1993Go), it may be sufficient to perform site-specific amino acid substitutions only at surface Cys-residues in the native structure. Therefore, our immobilization reaction can be widely applied to other proteins. We have successfully immobilized several proteins including xylanase; preliminary examinations showed high reversibility (Iwakura et al., unpublished observation).

Prevention of aggregation

As demonstrated by Anfinsen (1973), protein unfolding is reversible so that an amino acid sequence determines the native tertiary structure under native conditions (the last part of the central dogma). Practically speaking, however, recovery of native protein (renaturation) from unfolded protein is rarely complete. Not surprisingly, raising the efficiency of protein renaturation is an important goal of biotechnology (Volkin and Klibanov, 1989Go). Misfolding and aggregation of polypeptide chains are major reasons for the low recovery in the renaturation process (Anfinsen, 1973Go; Speed et al., 1996Go; Stempfer et al., 1996Go; Altamirano et al., 1997Go; Fink, 1998Go). Indeed, the prevention of intermolecular self-association of partially folded polypeptides by using chaperone proteins improves the recovery of renaturated protein (Altamirano, et al., 1997Go; Fenton and Horwich, 1997Go). As shown in this study, our immobilization strategy provides a simple and reliable method for preventing the intermolecular interaction of proteins. In the absence of intermolecular interactions, a unimolecular folding reaction is guaranteed. Then, the state of the protein is completely under the control of closed-system thermodynamics, and, therefore, protein denaturation in spatial isolation is fully reversible (see below).

Reversible folding

The restriction of the movement of the C-terminal part of the immobilized protein is similar to in vivo protein folding in which the C-terminus of a newly synthesized polypeptide is tethered to the ribosome during protein synthesis (Seckler and Jaenicke, 1992Go). As reported previously, the folding kinetics of DHFR that had been site-specifically immobilized through its C-terminus onto a surface plasmon resonance biosensor surface, were similar to that of the free protein (Sota et al., 1998Go). In the current study, immobilized DHFR was shown to have the same specific activity as free DHFR. Therefore, the spatial restriction at the C-terminus does not limit in vitro protein folding, and the immobilization strategy in this study is proven to be valid. As pointed out by Stempfer et al. (1996), when the denatured protein was bound to a solid surface at multiple points, a drastic decrease in renaturation was observed, underscoring the importance of single-site immobilization for reversible folding. Before our present work, several strategies for single-site immobilization such as the S–S linkage described above, glutathione and the glutathione S-transferase interaction (Frangioni and Neel, 1993Go), and His-tag affinity interactions (Shiha et al., 1994Go) had been developed. In all of these cases except the S–S linkage, the immobilization was designed to be temporary so that a target protein could be purified efficiently through specific association and dissociation.

If protein aggregation takes place primarily in a folding intermediate (Speed et al., 1996Go), then intermolecular interactions of enzyme immobilized at a low surface density can be ignored, because the radius of a protein in the molten globule phase is at most 1.5 times that of the native protein (Kataoka et al., 1995Go; Ptitsyn, 1995Go). Indeed, even when resin with a five times greater capacity (~100 µmol NH2/ml gel) was used in place of one with lower capacity, completely reversible thermal denaturation was demonstrated with immobilized DHFR (~45 nmol enzyme/ml gel).

Thermal damage

Repeated thermal treatments at 120°C resulted in a gradual loss of enzyme activity. Because guanidine–HCl treatment did not improve recovery of the activity, the activity loss was attributed to covalent modification of the polypeptide. After autoclaving free DHFR at 120°C for more than 5 h, certain fragments of the polypeptide chain could be detected by SDS–polyacrylamide electrophoresis although the fractions were small (data not shown). Therefore, hydrolysis of protein is one of the reasons for the loss of the activity, although other chemical reactions to modify side chains of polypeptide would take place. The chemical stability of peptide bond is intrinsic and may still limit the range of utilization of proteins. Nevertheless, because the loss of the activity is small in each treatment at 120°C and cannot be detected at 90°C, the chemical damages of the immobilized enzyme can be ignored in practical applications at conditions to sterilize enzyme reactors or enzyme sensor tips.


    Conclusion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Here, we have successfully demonstrated a great improvement of reversibility of DHFR denaturation against thermal treatment as high as 120°C. This improvement was achieved by taking the following factors into consideration as general problems: investigation of a new immobilization reaction, design of the protein to be compatible with the new immobilization reaction, and prevention of aggregation by immobilization. The ability of DNA to completely recover its native structure after repeated heating and cooling cycles is a form of on–off control that has been used to perform DNA amplification in a PCR (Saiki et al., 1985Go). Because we have a general approach to create a completely reversible enzyme, we can begin to consider constructing protein devices that perform on–off control by physical means. For example, in our preliminary experiments, repeat of MTX binding to and releasing from the immobilized DHFR was reproducibly carried out by temperature control and useful to recover MTX from very diluted solution as low as 1 nM.


    Notes
 
1 To whom correspondence should be addressed. E-mail: masa-iwakura{at}aist.go.jp Back


    Acknowledgments
 
We are grateful to Dr Yukio Shimura, University of Kanto-Gakuen, and Professor C. Robert Matthews and Dr Virginia F. Smith, Pennsylvania State University, for continuous discussion and encouragement and critical review of this article. This research was supported in part by New Energy and Industrial Technology Development Organization (NEDO).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
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Received January 12, 2001; revised May 10, 2001; accepted May 17, 2001.





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