Department of Biochemistry, Health Science Building, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5 and 2 Plant Biotechnology Institute, National Research Council of Canada,110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9, Canada 1 Present address: Cancer Immunobiology Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas Texas,7234-8576, USA
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Abstract |
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Keywords: antibody/HPr/gene synthesis/protein binding constant/protein folding/single-chain Fv
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Introduction |
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The complex of the mouse monoclonal antibody Jel42, which interacts with the small (85 amino acid) histidine-containing phosphocarrier protein, HPr, which is a component of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), has been described previously (Prasad et al., 1993, 1998
). The PTS is involved in sugar transport, sugar phosphorylation and regulation of carbohydrate metabolism (for reviews see Postma et al., 1993
, 1996
). Jel42 is one of three HPr-specific monoclonal antibodies that bind to distinct epitopes, and the epitopes for all three have been mapped by mutagenesis (Sharma et al., 1991
; Sharma 1992
). In addition to determining the effects of mutation by relative binding measurements using a solid-phase radioimmunoassay (SPRIA), a fluorescent polarization binding assay has allowed the determination of the Kd values for all three HPr-specific antibodies in solution (Smallshaw et al., 1998
). While the effects of mutation on the antigen, HPr, can be readily investigated (Sharma et al., 1991
; Sharma, 1992
; Smallshaw et al., 1998
), the effects of mutation on the antibody binding site first requires the production of the antibody in a system which can be manipulated.
One early example of an antibody that could be manipulated was the molecular construct of the Fv domain the anti-lysozyme antibody D1.3 for which a tertiary structure has been determined (Bhat et al., 1990, 1994
). The D1.3 Fv was shown to have the same binding site structure as that found for the Fab (Amit et al., 1986
). Often, the Fv or the more common constructs, single chain Fvs (scFv) (Bird et al., 1988
; Huston et al., 1988
) have binding constants that are similar (within about a 10-fold difference) to the antibody binding constants. In several investigations, mutations of scFvs have been assessed for binding efficacies, with a view to improvement (Deng et al., 1994
, 1995
; Schier et al., 1996
). As has been recently shown, care must be taken to distinguish between avidity and affinity (Schier et al., 1996
). Despite these results that show that scFvs have both function and structure that is almost indistinguishable from the binding site in the whole antibody, many scFv have not behaved ideally as they are often produced as inclusion bodies when overexpressed, renature inefficiently and show poor solubility which precludes structural approaches (some examples include: Denzin et al., 1991
; Pantoliano et al., 1991
; Anthony et al., 1992
; Lake et al., 1994
; Ayala et al., 1995
; Cho et al., 1995
; Mallender et al., 1996
).
In order to use the Jel42HPr complex to investigate further the molecular details of antibodyprotein antigen interaction, a scFv for Jel42 was needed. This paper describes the construction of the Jel42 scFv gene, its production as a refolded protein from inclusion bodies, and binding measurements with many HPr mutants that show that despite inefficient refolding, the presumably correctly refolded scFv maintains a Kd similar to the antibody and shows very similar specificity.
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Methods and materials |
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Nickel-chelating resin was obtained from Novagen. Taq DNA polymerase and ampholytes were from Pharmacia; Vent or Deep Vent DNA polymerase and DNA ligase were from New England Biolabs, and restriction endonucleases were from either Pharmacia or New England Biolabs. Carbonic anhydrase, cytochrome c (horse heart), bovine hemoglobin, chicken egg white lysozyme, chicken egg ovalbumin and bovine serum albumin were obtained from Sigma. Glucose 6-phosphate dehydrogenase (yeast) and horse heart lactate dehydrogenase were obtained from Boehringer Mannheim. GuanidineHCl, Ultrapure, was from ICN.
HPr mutants
HPr and the mutant HPrs were obtained as described previously (Smallshaw et al., 1998).
Synthesis of oligonucleotides
Synthesis of the oligonucleotides was performed using the methodology described by Mateucci and Caruthers (1981) and an Applied Biosystems Synthesizer Model 392. Two types of oligonucleotides were synthesized to construct the gene for Jel42 scFv: approximately 60mers comprising the complete 5'3' strand (reading frame), and 20mers of the 3'
5' strand consisting of overlaps between the 60mers. These sequences are described by Smallshaw (1997). The primers used to sequence the variable domain gene regions of the cDNAs of the heavy and light chains of Jel42 were described by Steeves et al. (1991).
DNA sequencing
DNA sequencing was performed using the T7 Sequenase kit (Pharmacia). The scFv gene was sequenced by creating smaller restriction fragments (Smallshaw, 1997).
Gene design
Once the amino acid sequence of the gene product had been determined by translation of the corrected DNA sequence (see Results), a DNA sequence using the codons found in highly expressed E.coli genes (Sharp and Li, 1987) was made, incorporating codon redundancy where appropriate. The sequence was then searched for potential restriction endonuclease sites, and changes were made that did not introduce unfavourable codons. Sequences were checked to ensure the lack of formation of stemloop structures and internal sequence similarities. DNA sequence design manipulations were performed on the Beckman MicroGenie® Sequence software and the DNAid+ 1.1 computer programs
In vitro gene synthesis
The annealing of the staple fragments to the ~60mer fragments was an adaptation of the site-directed mutagenesis protocol of Kunkel et al. (1987). Three gene segments (~300 bp) were constructed separately. Each of the 5' end-phosphorylated ~60mers (100 pmol) in phosphorylation buffer were combined with 200 pmol of each of the appropriate 20mer bridging staple fragments to which was added 5 µl 3 M NaCl in 300 mM sodium citrate, pH 7.0; incubated at 70°C for 10 min, then allowed to slowly cool to about 30°C. The 60mer fragments, aligned by the annealed staple fragments, were ligated together (Sambrook et al., 1989).
The ~300 bp fragments were amplified by PCR using Vent or Deep Vent DNA polymerase and appropriate primers (Smallshaw, 1997). PCR was carried out in 100 µl 1x Vent buffer (supplied with the Vent DNA polymerase) with 400 µM each dNTP, 1 µl of the ligated mixture above (a maximum of 1 pmol of template DNA plus 2 pmol each of the staple primers used in assembly), 50 pmol each PCR primer, 2 U Vent or Deep Vent DNA polymerase. The reactions were carried out in an Ericomp Single Block model TCX15 or model EZ cycler. Typically, amplification was achieved using 1520 cycles of 30 or 60 s at each of 95, 55 and 70°C, followed by 10 min at 72°C; for amplification of fragments 500 base pairs (bp) or less, 30 s cycles were used, and for larger fragments 60 s cycles were used.
Site-directed mutagenesis
The putative scFv genes were ligated into the HincII restriction endonuclease site in pUC19 (Vieira et al., 1982) and initially screened by restriction endonuclease analysis for the appropriate size of insert, and a number were sequenced. One clone was found to contain just four errors, three of which were single base deletions, all G residues within clusters of two or three Gs, (residues 113, 313 and 800), and the fourth a silent base change (residue 408, CA). These errors were common to other clones sequenced, suggesting errors that occurred early in the amplification procedures. These errors were corrected by the PCR site-directed mutagenesis by the method of Landt et al. (1990). Residues 113 and 313 were each restored by using the DNA oligonucleotides used for the gene assembly as the `mutagenic' primers (Smallshaw, 1997
). Subsequently, residue 800 was restored using the mutagenic primer (
800G, 5'-TAGTACCGCCACCGAAGGTG-3'). This generated a construct free of all sequence errors except for the single silent base change from the original design at residue 408, and the corrected scFv gene was cloned in pT7-7 for expression.
DNA isolation and manipulation
DNA fragments from the PCRs or from restriction enzyme digests of vector DNA were separated by low melting point agarose gel electrophoresis (Sambrook et al., 1989) as modified by Qian and Wilkenson (1991). DNA was recovered from regular agarose gels using the Pharmacia Sephaglas BandPrep kit. Restriction enzyme digestions, ligation reactions and phosphatase treatments were all by standard procedures (Sambrook et al., 1989
).
Expression of Jel42 scFv
The expression vector pT7-7 (Tabor and Richardson, 1985) with the Jel42 scFv gene positioned at the NdeI restriction endonuclease site downstream of the T7 RNA polymerase-specific promoter was used to transform E.coli strain BL21(DE3) (Studier and Moffatt, 1986
). To determine the optimal conditions for expression, growth in a 25 ml culture in a 250 ml side-arm flask was monitored on a Klett-Summerson photoelectric colorimeter. When the culture had reached midlog phase (OD590 0.61.0), 0.51.0 mM isopropyl ß-D-thiogalactoside (IPTG) was added. Periodically, 1 ml aliquots of cells were taken, lysed and run on SDSPAGE to determine the accumulation of the expressed gene product. For preparative scale expression, 200500 ml TB broth culture was grown.
SDSPAGE
The method of Laemmli (1970) as described by Sambrook et al. (1989) with a 12 or 15% running gel bed topped with a 3% stacking gel was used.
Isolation of the periplasmic fraction
Expressed scFv was isolated and purified from the IPTG-induced cultures described above. Cells were harvested by centrifugation at 4000 g for 10 min at 4°C. Periplasmic fractions were prepared by either the osmotic shock method (Skerra and Plückthun, 1988) or for small trials, the chloroform shock protocol (Ames et al., 1984
).
Isolation of inclusion bodies
Expressed protein was isolated from inclusion bodies formed within the cells by the protocol described by Buchner and Rudolph (1991). The inclusion body pellet was dissolved with mild agitation at room temperature for 12 h in a minimum volume of one of two denaturation buffers: for subsequent renaturation, 0.1 M TrisHCl buffer, pH 8.5, 6 M guanidineHCl, 2 mM EDTA (pH 8.0), 0.3 M dithioerythritol (DTE), or for metal chelation chromatography under denaturing conditions, 20 mM TrisHCl buffer, pH 7.9, 6 M guanidineHCl, 5 mM imidazole, 0.5 M NaCl. Insoluble material was removed by centrifugation and the supernatants stored at 70°C, if not used immediately.
Renaturation of scFv from inclusion bodies
The solubilized, denatured protein was renatured as described (Buchner et al., 1992).
Metal chelation chromatography
Metal chelation chromatography (Hochuli et al., 1988) was carried out by the procedures outlined by Novagen. All buffers contained 0.5 M NaCl, 20 mM TrisHCl buffer, pH 7.9. A column (5 ml) was washed with distilled water and charged with 15 ml 50 mM NiSO4, and then equilibrated with 15 ml buffer with 5 mM imidazole. Filtered (0.22 µm) scFv samples in binding buffer with 5 mM imidazole were applied, the column was washed with 50 ml buffer containing 60 mM imidazole, and the retained protein eluted with 30 ml buffer containing 1 M imidazole. The protein was then dialyzed against 1x phosphate-buffered saline (PBS) and concentrated by Amicon Ultrafiltration. For purification under denaturing conditions, all buffers contained 6 M guanidineHCl. The imidazole concentrations of wash and elution buffers were reduced to 20 and 300 mM respectively. The eluted sample was then renatured as described above for inclusion bodies.
Isoelectric focusing
Isoelectric focusing was performed as previously described (Waygood et al., 1987) except that the ampholytes used for the scFvHPr complex isolation were 3:2:1 of pH 3.510.0, 5.08.0 and 8.010.5.
Determination of protein concentration
Protein determinations for HPr and fluorescein 5-maleimide (F-5-M)-labeled Arg17Cys HPr were described by Smallshaw et al. (1998). Protein concentrations of scFv preparations were determined using the Bradford microprocedure (Bradford, 1976) with IgG as a standard.
Determination of binding constants
Binding constants were determined using F-5-M-labeled Arg17Cys HPr at 2 nM in 1 ml of 15 mM sodium phosphate buffer, pH 7.2, containing 0.1 mg/ml bovine serum albumin. F-5-M-labeled Arg17Cys HPr was prepared as previously described (Smallshaw, 1997; Smallshaw et al., 1998
). Binding was measured by changes in fluorescence polarization using a Beacon Fluorescence Polarization System (Panvera Corp.). Jel42 scFv was added over a concentration range of approximately (0.0130)xKd.
Determination of Ki
Ki values were determined using a concentration of Jel42 scFv that bound approximately 90% of the 2 nM F-5-M-labeled Arg17Cys HPr, and titrating with either wild-type HPr or mutant HPr. The Ki values were determined graphically using the standard equation
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where IC50 is the concentration of inhibitor necessary to displace half of the labeled ligand and FL is the free labeled ligand concentration at the titration midpoint (1 nM).
Thermodynamic treatment
Measurements were made to determine binding constants (Kd) at different temperatures under the conditions described above. From these measurements, values of the thermodynamic parameters were extracted. The change in binding enthalpy, Hbobs, was derived from the slope of the tangent to the van't Hoff plot (lnKa versus 1/T) from the equation
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where T is in Kelvin and Ka = 1/Kd. From this determination, Gbobs and T
Sbobs are calculated using
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and
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The change in heat capacity, Cp, is derived from the slope of the plot
Hbobs versus T. This plot also yields tH, the temperature at which
Hbobs is zero, while T
Sbobs versus T yields tS, the temperature at which T
Sbobs is zero.
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Results |
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The sequence of Jel42 variable regions had been determined from mRNA isolated from the hybridoma cells and part of the sequence was confirmed by protein sequencing (Steeves et al., 1991). During the determination of the tertiary structure of the Jel42FabHPr complex (Prasad et al., 1993
), it became apparent that for a number of residues the observed electron density suggested residues other than those derived from the mRNA sequencing. To resolve these differences, first, all the sequencing autoradiographs for the mRNA sequencing were re-read, and the reported sequence was confirmed. In a second approach, the cDNAs of the variable regions of both the light and heavy chains were independently cloned on two separate occasions (Barry and Lee, 1993
). The two sets of cDNA sequences agreed with each other except at one position, but one of the DNA sequences agreed with the mRNA sequence at this position. However, both cDNA sequences showed a series of differences from the mRNA sequencing. The cDNA sequences for the two chains (accession numbers M60389 and M60390) provided amino acid sequences that were compatible with the tertiary structure determination (Prasad et al., 1993
, 1998
). None of the differences occurred where protein sequence had been obtained (Steeves et al., 1991
).
Design of the Jel42 scFv gene
The design of the gene followed that described by Skerra et al. (1991): leader peptideheavy chain variablelinkerlight chain variable(His)5tail. The leader chosen was from pelB (Lei et al., 1987) and the linker was three repeats of the repetitive amino acid sequence, S-Y-S-P-T-S-P, found at the C-terminal of eukaryotic RNA polymerase II. The monoclonal antibody, Jel352, is specific for this sequence (Moyle et al., 1989
), and this linker provides a tag for an independent method of detection. The Jel42 scFv amino acid sequence and the DNA sequence for in vitro synthesis are shown in Figures 1 and 2
. The DNA sequence maximized the use of high expression codons and the creation of unique restriction sites.
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One strand of the gene was synthesized as a set of fragments (~60 bases), and parts of the other strand were synthesized as 20mers to provide linkers (staples) to join together the 60mers by specific base pairing. The 848 nucleotide sequence was initially assembled as three slightly overlapping sections of 288, 328 and 244 nucleotides. These sections (5'3') were flanked by the restriction endonuclease sites NdeI and SalI, SalI and KpnI, and KpnI and BamHI respectively which were used to produce the full-length scFv. The amplified product was cloned and sequenced in pUC19, three errors corrected by site-directed mutagensis, and subsequently cloned into pT7-7 using the NdeI and BamHI restriction sites for expression. In a first attempt at the synthesis, Taq DNA polymerase was used for the PCR amplifications. The Jel42 scFv gene that was obtained from this synthesis contained numerous errors in the sequence, presumably caused by the amplification procedure (Smallshaw, 1997). Thus in the second synthesis, Vent and Deep Vent DNA polymerases, which have greater fidelity, were used.
Expression of Jel42 scFv
The best production of the scFv was found when induction was performed in terrific broth (Sambrook et al., 1989) with the maximum amount of aeration possible by shaking. However, although the scFv was designed for export into the periplasm, no export of soluble scFv was detected using various methods of periplasmic protein isolation. In addition, neither lower temperatures (e.g. 25°C) nor varying the IPTG concentration gave soluble periplasmic expression. Rifampicin neither decreased or increased production of Jel42 scFv, but caused the overproduction of two species of the scFv (Figure 3
), and thus it was not used. Overproduction of Jel42 scFv varied between 10 and 30% of the cellular protein.
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Inclusion bodies were isolated from the majority of the other proteins in the E.coli cell to yield protein preparations that were already approaching purity; 7080% pure as judged from SDSPAGE. To obtain active Jel42 scFv, a standard renaturation procedure was used as described in the Materials and methods. The renaturation procedure resulted in minimal improvement in purity, and it is estimated that 310% of the Jel42 scFv was correctly folded depending upon the preparation. The (His)5-tail can be used to produce homogeneous denatured Jel42 scFv using Ni-chelating chromatography; however, the homogenous Jel42 scFv had the same poor efficiency in the renaturation process. If renatured scFv was chromatographed on the Ni-chelating column, the scFv precipitated on the column during elution. Initial binding studies with renatured Jel42 scFv obtained directly from the renaturation of inclusion bodies gave satisfactory results with respect to binding properties (see later); thus the Ni-chelating chromatography step was not routinely used.
Jel42 scFv isolated from inclusion bodies and denatured in 0.3 M DTE and 6 M guanidineHCl was stable at 70°C for at least several weeks. The renatured Jel42 scFv (0.22 mg/ml) dialyzed against a 10-fold volume of PBS stored on ice could be used for about 56 days. The renatured Jel42 scFv preparation had limited solubility, and further concentration by ultrafiltration caused precipitation.
Binding assay for Jel42 scFv
The Jel42 scFv was designed to allow for the use of the solid phase radioimmune assay (SPRIA) that had been used to characterize the HPr-specific antibodies (Waygood et al., 1987; Sharma et al., 1991
). The linker was the epitope for monoclonal antibody Jel352, and thus Jel42 scFv bound to surface-adhered HPr would be bound by the antibody Jel352, and this second antibody could be detected by anti-murine antibody used in the standard procedure. Jel42 scFv was detected bound to HPr-coated plates by use of Jel352, and preparation appeared to titrate in a normal manner. However, control experiments showed that the scFv preparations bound to other non-related proteins (cytochrome c, glucose-6-phosphate dehydrogenase, hemoglobin, lactate dehydrogense, lysozyme and ovalbumin) when they were attached to microtiter plates. These results suggested that the Jel42 scFv preparation, in which it was later determined only a small percentage was properly refolded, bound non-specifically to surface-adhered protein.
Another binding assay based upon fluorescent polarization has been developed to obtain binding constant values for both the antibodies and Fab fragments specific to HPr (Smallshaw, 1997; Smallshaw et al., 1998
). The assay uses Arg17Cys HPr which is specifically labeled at the unique cysteine residue using F-5-M. Binding of the antibody, Fab or scFv, reduces the tumbling time of the HPr molecule which is detected by a change in the polarization of the fluorescent signal. Using this assay, binding of the Jel42 scFv could be detected and titrated. Moreover, in competition assays, the non-specific proteins to which the scFv bound to in the SPRIA assays, did not cause any changes in polarization until high concentrations (greater than micromolar) were reached.
Determination of Kd
Kd was determined (Figure 4) for preparations of renatured Jel42 scFv that were usually 7080% pure, but as low as 30% pure as estimated by SDSPAGE. Kd varied between 20 and 200 nM reflecting the variation in purity and renaturation (Table I
). The concentration of scFv was determined by the measurement of total protein, and the value of the Kd determined could be refined by considering the purity of the preparation by SDSPAGE gel analysis (Table I
). Further, if the assumption is made that the active, correctly-refolded scFv has a Kd similar to the antibody, the refolding efficiency was about 6% except for one preparation (Table I
). However, despite the variation in purity of the scFv, when the data used to calculate Kd values was treated using the algorithm that fits to a single binding site as described by Smallshaw et al. (1998), the separate determinations gave regression analysis values of 0.920.99, which indicate that the detectable active scFv had consistent properties.
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The relative effects of mutation to HPr on binding have been determined for the Jel42 antibody and Fab fragment (Smallshaw et al., 1998). Identical measurements were performed with the Jel42 scFv preparation, and the results of these determinations are presented in Table III
. Jel42 scFv showed very similar properties.
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The temperature dependency of the Kd value has been determined for both Jel42 antibody and Fab fragment (Smallshaw et al., 1998). In these experiments, the same set of experimental samples used to measure the mP values was equilibrated at different temperatures to avoid errors associated with preparing different sets of samples. This experiment was done twice with a single scFv preparation, and the results are shown in Table IV
. The dependency of the Kd and the thermodynamic parameters on temperature for Jel42 scFv were similar to results obtained with both the Jel42 antibody and the Fab fragment.
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Discussion |
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In vitro assembly of genes from fragments as described here has been used by others in a similar manner (Plückthun et al., 1987; Huston et al., 1988; Daugherty et al., 1991
). In the conception of this work, it had been hoped that Jel42 scFv would be exported into the periplasm and be soluble. As the signal peptide is cleaved except when rifampicin is added (Figure 3
), Jel42 scFv is probably exported to the periplasm where inclusion bodies can form. Jel42 scFv exhibits the usual poor efficiency of renaturation of scFvs from inclusion bodies, and has poor solubility when renatured. Plückthun and co-workers suggest several solutions with respect to scFv stability and solubility (Steipe et al., 1994;
Knappik and Plückthun, 1995; Jung and Plückthun, 1997; Nieba et al., 1997) some which would radically change the protein sequence, and thus make comparison to the whole antibody less explicit. However, very high rates of scFv expression in soluble form are possible and improvements in solubility can involve changes to hydrophobic residues located in the scFv at the site that normally forms the interface between the constant and variable domains in the Fab fragment (Nieba et al., 1997). This interfacial site in Jel42 scFv has large areas of hydrophobic residues, which will be investigated further.
It is probable that only a small percentage of the protein correctly folded (Table I). However, the binding data was fitted to a single site binding model with very high correlation values. The specificity of the scFv for mutant HPrs (Table III
) was similar to the antibody and Fab fragment. These measurements suggest that the renatured preparation of the scFv contains two distinct populations; one that is correctly folded and is a true mimic of the binding domain in the whole antibody and the other which is comprised of incorrectly folded proteins with antigen binding potentials at least a few orders of magnitude less than the correctly folded protein. These results emphasize the similarity of the active scFv to the antibody; however, given the fact that 23-fold differences in binding are within the experimental error, small changes, such as a loss of a van der Waals contacts, may not be detected. Moreover, while most preparations were in the 7080% range of purity from inclusion body preparations, some preparations that were only 30% pure yielded results that were compatible with purer preparations.
The temperature dependence of the scFv was similar but not identical with that of either the Fab fragment or antibody (Smallshaw et al., 1998). The higher ts value for the scFv compared with the antibody and Fab fragment (Table IVb
) is possibly an indication that the additional constant regions of the Fab fragment have a stabilizing effect upon the variable region that makes up the Fv (i.e. the scFv is more dynamic). The free energy shows enthalpy compensation with increasing temperature.
It would appear that the determination of the binding constants was successful even in the poorly renatured and incompletely purified preparation of Jel42 scFv for two reasons. First, the binding assay employed did not use any form of surface interaction, and thus was not susceptible to non-specific absorption that afflicted the SPRIA assay. Other methods such as the surface plasma resonance technology can be affected by this adsorption (Kortt et al., 1997). Secondly, the detection method relied on changing the property of the antigen, F-5-M-labeled Arg17Cys HPr, which was homogeneous and well characterized (Smallshaw, 1997
; Smallshaw et al., 1998
).
In principle, the binding assay used here could be used for many other scFv preparations. Can the observations and conclusions that have been made with respect to Jel42 scFv be extrapolated to other scFvs? Because many scFv have the same general gene construction, and the antibody chains share common features of structure, it is likely that the renaturation should result in the same two classes of refolded protein: very precise mimics of the binding domain or very poor mimics whose binding properties are undetectable.
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Acknowledgments |
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Notes |
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Received July 9, 1998; revised March 9, 1999; accepted March 22, 1999.