National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
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Abstract |
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Keywords: complete reversibility/cyanocysteine-mediated reaction/dihydrofolate reductase/immobilized enzyme/thermal denaturation
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Introduction |
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In contrast to the efforts made to improve thermal stability through protein engineering (Liao et al., 1986; Shortle, 1992
; Malakauskas and Mayo, 1998
), few attempts have been made to improve thermal reversibility (Volkin and Klibanov, 1987; Iwakura et al., 1995
; Iwakura and Honda, 1996
), 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, 1996
), 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., 1996
; Stempfer et al., 1996
; Altamirano et al., 1997
), preventing intermolecular interactions would be a simple way to achieve reversible protein denaturation (Klibanov, 1983
; Volkin and Klibanov, 1987; Stempfer et al., 1996
) 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., 1996
). 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, 1976; Srere and Uyeda, 1976
). Almost all such reactions, however, rely on the chemical reactivity of amino acid side chains (Srere and Uyeda, 1976
), 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 1
) (Takenawa et al., 1998
; Ishihama et al., 1999
). 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, 1971
Degani and Patchornik, 1974
). 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., 1999
). 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 guanidineHCl. To examine the effectiveness of the route 4 reaction, dihydrofolate reductase (DHFR) was used as a model protein.
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Materials and methods |
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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., 1995). 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 I
and the plasmid pDFHR20 (Iwakura et al., 1995
) 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., 1983
). 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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Enzyme assay
The activity of free DHFR was determined spectrophotometrically at 15°C by following the disappearance of NADPH and DHF at 340 nm (340 = 11 800 M1 cm1; Hillcoat et al., 1967
). 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.
LCMS measurements
Liquid chromatographyelectrospray mass spectrometric (LCMS) measurements were performed as previously described (Takenawa et al., 1998). 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.
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Results and discussion |
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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 2. The sole Cys-SH residue in the tethered DHFRs was converted to S-cyanocysteine by a treatment with NTCB.
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The efficiency of the route 4 reaction with 10 mM octylamine was studied (reaction with [1] in Figure 2). 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 LCMS measurement, probably because of the increased hydrophobicity of the attached octylamine. Figure 3A
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 2B
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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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 guanidineHCl, 1 mM EDTA and 14 mM 2-mercaptoethanol to remove non-covalently bound protein, then equilibrated with the same buffer without guanidineHCl. 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 guanidineHCl and the concentration of the eluted MTX was determined spectrophotometrically at 302 nm at pH 12 (302 = 22 100 M1 cm1). 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 4 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 4
, 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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Thermal treatment of immobilized DHFR
Temperature dependence of the activity of the immobilized enzyme is shown in Figure 5A. 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 3
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 5B
). 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 5C
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 guanidineHCl (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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Previously, we attached DHFR to a polymer support through a disulfide (SS) linkage at its C-terminal end (Iwakura and Kokubu, 1993). Probably because the SS 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 SS linkage (Srere and Uyeda, 1976
), 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., 1998
; Ishihama et al., 1999
) 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 1
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 guanidineHCl.
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., 1995). 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, 1993
), 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, 1989). Misfolding and aggregation of polypeptide chains are major reasons for the low recovery in the renaturation process (Anfinsen, 1973
; Speed et al., 1996
; Stempfer et al., 1996
; Altamirano et al., 1997
; Fink, 1998
). Indeed, the prevention of intermolecular self-association of partially folded polypeptides by using chaperone proteins improves the recovery of renaturated protein (Altamirano, et al., 1997
; Fenton and Horwich, 1997
). 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, 1992). 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., 1998
). 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 SS linkage described above, glutathione and the glutathione S-transferase interaction (Frangioni and Neel, 1993
), and His-tag affinity interactions (Shiha et al., 1994
) had been developed. In all of these cases except the SS 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., 1996), 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., 1995
; Ptitsyn, 1995
). 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 guanidineHCl 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 SDSpolyacrylamide 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.
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Conclusion |
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Notes |
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Acknowledgments |
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References |
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Altamirano,M.M., Golbik,R., Zahn,R., Buckle,A.M. and Fersht,A.R. (1997) Proc. Natl Acad. Sci. USA, 94, 35763578.
Blakley,R.L. (1984) Dihydrofolate reductase. In Blakley,R.L. and Benkovic,S.J. (eds), Folates and Pteridines, Vol. 1. John Wiley, New York, USA, pp. 191253.
Catsimpoolas,N. and Wood,J.L. (1966) J. Biol. Chem., 241, 17901796.
Degani,Y. and Patchornik,A. (1971) J. Org. Chem., 36, 27272728.[ISI]
Degani,Y. and Patchornik,A. (1974) Biochemistry, 13, 111.[ISI][Medline]
Fink,A.L. (1998) Fold. Des., 3, R9R23.[ISI][Medline]
Frangioni,J.V. and Neel,B.G. (1993) Anal. Biochem. 210, 179187.[ISI][Medline]
Fenton,W.A. and Horwich,A.L. (1997) Protein Sci., 6, 743760.
Hillcoat,B., Nixon,P. and Blakely,R.L. (1967) Anal. Biochem., 21, 178189.[ISI][Medline]
Ishihama,Y., Ito,O., Oda,Y., Takenawa,T. and Iwakura,M. (1999) Tetrahedron Lett., 40, 34153418.[ISI]
Iwakura,M. and Honda,S. (1996) J. Biochem., 119, 414420.[Abstract]
Iwakura,M. and Kokubu,T. (1993) J. Biochem., 114, 339343.[Abstract]
Iwakura,M., Shimura,Y. and Tsuda,K. (1983) J. Biochem., 93, 927930.[Abstract]
Iwakura,M., Furusawa,K., Kokubu,T., Ohashi,S., Tanaka,Y., Shimura,Y. and Tsuda,K. (1992) J. Biochem., 111, 3745.[Abstract]
Iwakura,M., Jones,B.E., Luo,J. and Matthews,C.R. (1995) J. Biochem., 117, 480488.[Abstract]
Jacobson,G.R., Schaffer,M.H., Stark,G.R. and Vanaman,T.V. (1973) J. Biol. Chem., 248, 65836591.
Kataoka,M., Nishii,I., Fujisawa,T., Ueki,T., Tokunaga,F. and Goto,Y. (1995) J. Mol. Biol., 249, 215228.[ISI][Medline]
Klibanov,A.M. (1983) Science, 219, 722727.[ISI]
Liao,H., McKenzie,T. and Hageman,R. (1986) Proc. Natl Acad. Sci. USA, 83, 576580.[Abstract]
Malakauskas,S.M. and Mayo,S.L. (1998) Nature Struct. Biol., 5, 470475.[ISI][Medline]
Morrison,J.F. and Stone,S.R. (1988) Biochemistry, 27, 54995506.[ISI][Medline]
Mosbach,K. (1976) Methods Enzymol., 44, 37.[Medline]
Ptitsyn,O.B. (1995) Adv. Protein Chem., 47, 83229.[ISI][Medline]
Saiki,R.K., Scharf,S., Faloona,F., Mullis,K.B., Horn,G.T., Erlich,H.A. and Arnheim,N. (1985) Science, 230, 13501354.[ISI][Medline]
Seckler,R. and Jaenicke,R. (1992) FASEB J., 6, 25452552.
Shiha,D., Bakhshi,M. and Vora,R. (1994) Biotechniques, 17, 509514.[ISI][Medline]
Shortle,D. (1992) Q. Rev. Biophys., 25, 205255.[ISI][Medline]
Sota,H., Hasegawa,Y. and Iwakura,M. (1998) Anal. Chem., 70, 20192024.[ISI][Medline]
Speed,M.A., Wang,D.I.C. and King,J. (1996) Nature Biotechnol., 14, 12831287.[ISI][Medline]
Srere,P.A. and Uyeda,K. (1976) Methods Enzymol., 44, 1119.[Medline]
Stempfer,G., Holl-Neugebauer,R. and Rudolph,B. (1996) Nature Biotechnol., 14, 329334.[ISI][Medline]
Takenawa,T., Oda,Y., Ishihama,Y. and Iwakura,M. (1998) J. Biochem., 123, 11371144.[Abstract]
Tanford,C. (1968) Adv. Protein. Chem., 23, 121282.[Medline]
Touchette,N.A., Perry,K.P. and Matthews,C.R. (1986) Biochemistry, 25, 54455452.[ISI][Medline]
Uwajima,U., Oshiro,T., Eguchi,T., Kuge,Y., Horiguchi,A., Igarashi,A., Mochida,K. and Iwakura,M. (1990) Biochem. Biophys. Res. Commun., 171, 684689.[ISI][Medline]
Villafranca,J.E., Howell,E.E., Voet,D.H., Strobel,M.S., Ogden,R.C., Abelson,J.N. and Kraut,J. (1986) Science, 222, 782788.
Volkin,D.B. and Klibanov,A.M. (1989) In Creighton,T.E. (ed.), Protein Function: a Practical Approach. IRL Press, Oxford, pp. 124.
Received January 12, 2001; revised May 10, 2001; accepted May 17, 2001.