Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

Joan E. Smallshaw1, Fawzy Georges2, Jeremy S. Lee and E.Bruce Waygood3

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


    Abstract
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 References
 
The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide–heavy chain–linker–light chain–(His)5 tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10–30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with Kd values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 ± 2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage (~6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.

Keywords: antibody/HPr/gene synthesis/protein binding constant/protein folding/single-chain Fv


    Introduction
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 References
 
Antibody diversity and specificity are essential to the immune response and for many experimental procedures. Many of the interactions of antibodies involve protein antigens, and a number of tertiary structures of complexes between antibodies and proteins have been described (see review Davies and Cohen, 1996Go). The existence of these structures offers opportunities to investigate more closely the relationship between structure and function in the interaction between antibodies and protein antigens. Antibodies to hen egg white lysozyme (HEL) have been studied extensively (Amit et al., 1986Go; Ward et al., 1989Go; Bhat et al., 1990Go, 1994Go; Tello et al., 1993Go; Braden and Poljak, 1995Go; Schwarz et al., 1995Go), and with the aid of molecular constructs of the variable domain (Fv), several reports about mutations of the Fv binding site have been presented (Hawkins et al., 1993Go; Ito et al., 1993Go). The effect of mutations in lysozyme have been studied using lysozymes from closely-related species (Lavoie et al., 1992Go; Chacko et al., 1995Go; Dall'Acqua et al., 1996Go; Schick et al., 1997Go) and by site-directed mutagenesis (Kam-Morgan et al., 1993Go). Other well described complexes involve the protein antigen influenza virus N9 neuriminidase (Gruen et al., 1993Go; Nuss et al., 1993Go; Tulip et al., 1994Go).

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., 1993Go, 1998Go). The PTS is involved in sugar transport, sugar phosphorylation and regulation of carbohydrate metabolism (for reviews see Postma et al., 1993Go, 1996Go). 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., 1991Go; Sharma 1992Go). 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., 1998Go). While the effects of mutation on the antigen, HPr, can be readily investigated (Sharma et al., 1991Go; Sharma, 1992Go; Smallshaw et al., 1998Go), 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., 1990Go, 1994Go). The D1.3 Fv was shown to have the same binding site structure as that found for the Fab (Amit et al., 1986Go). Often, the Fv or the more common constructs, single chain Fvs (scFv) (Bird et al., 1988Go; Huston et al., 1988Go) 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., 1994Go, 1995Go; Schier et al., 1996Go). As has been recently shown, care must be taken to distinguish between avidity and affinity (Schier et al., 1996Go). 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., 1991Go; Pantoliano et al., 1991Go; Anthony et al., 1992Go; Lake et al., 1994Go; Ayala et al., 1995Go; Cho et al., 1995Go; Mallender et al., 1996Go).

In order to use the Jel42–HPr complex to investigate further the molecular details of antibody–protein 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.


    Methods and materials
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 References
 
Materials

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. Guanidine–HCl, Ultrapure, was from ICN.

HPr mutants

HPr and the mutant HPrs were obtained as described previously (Smallshaw et al., 1998Go).

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, 1997Go).

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, 1987Go) 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 stem–loop 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., 1989Go).

The ~300 bp fragments were amplified by PCR using Vent or Deep Vent DNA polymerase and appropriate primers (Smallshaw, 1997Go). 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 15–20 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, C->A). 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, 1997Go). Subsequently, residue 800 was restored using the mutagenic primer ({Delta}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., 1989Go) 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., 1989Go).

Expression of Jel42 scFv

The expression vector pT7-7 (Tabor and Richardson, 1985Go) 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, 1986Go). 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 0.6–1.0), 0.5–1.0 mM isopropyl ß-D-thiogalactoside (IPTG) was added. Periodically, 1 ml aliquots of cells were taken, lysed and run on SDS–PAGE to determine the accumulation of the expressed gene product. For preparative scale expression, 200–500 ml TB broth culture was grown.

SDS–PAGE

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, 1988Go) or for small trials, the chloroform shock protocol (Ames et al., 1984Go).

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 1–2 h in a minimum volume of one of two denaturation buffers: for subsequent renaturation, 0.1 M Tris–HCl buffer, pH 8.5, 6 M guanidine–HCl, 2 mM EDTA (pH 8.0), 0.3 M dithioerythritol (DTE), or for metal chelation chromatography under denaturing conditions, 20 mM Tris–HCl buffer, pH 7.9, 6 M guanidine–HCl, 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., 1992Go).

Metal chelation chromatography

Metal chelation chromatography (Hochuli et al., 1988Go) was carried out by the procedures outlined by Novagen. All buffers contained 0.5 M NaCl, 20 mM Tris–HCl 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 guanidine–HCl. 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., 1987Go) except that the ampholytes used for the scFv–HPr complex isolation were 3:2:1 of pH 3.5–10.0, 5.0–8.0 and 8.0–10.5.

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, 1976Go) 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, 1997Go; Smallshaw et al., 1998Go). 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 (0.01–30)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


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, {Delta}Hbobs, was derived from the slope of the tangent to the van't Hoff plot (lnKa versus 1/T) from the equation


where T is in Kelvin and Ka = 1/Kd. From this determination, {Delta}Gbobs and T{Delta}Sbobs are calculated using


and


The change in heat capacity, {Delta}Cp, is derived from the slope of the plot {Delta}Hbobs versus T. This plot also yields tH, the temperature at which {Delta}Hbobs is zero, while T{Delta}Sbobs versus T yields tS, the temperature at which T{Delta}Sbobs is zero.


    Results
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 References
 
Sequencing Jel42 cDNA

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., 1991Go). During the determination of the tertiary structure of the Jel42Fab–HPr complex (Prasad et al., 1993Go), 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, 1993Go). 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., 1993Go, 1998Go). None of the differences occurred where protein sequence had been obtained (Steeves et al., 1991Go).

Design of the Jel42 scFv gene

The design of the gene followed that described by Skerra et al. (1991): leader peptide–heavy chain variable–linker–light chain variable–(His)5–tail. The leader chosen was from pelB (Lei et al., 1987Go) 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., 1989Go), 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 2GoGo. The DNA sequence maximized the use of high expression codons and the creation of unique restriction sites.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Amino acid sequence of Jel42 scFv. The leader sequence is residues 1–22; heavy chain, 23–140; linker, 141–161; light chain, 162–273; followed by (His)5. The CDRs are boxed.

 


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2. DNA sequence of Jel42 scFv. The DNA sequence for the Jel42 scFv gene and unique restriction endonuclease sites. The bolded restriction sites were used in the assembly of the scFv gene.

 
Synthesis and assembly of the Jel42 scFv gene

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, 1997Go). 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., 1989Go) 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 3Go), and thus it was not used. Overproduction of Jel42 scFv varied between 10 and 30% of the cellular protein.



View larger version (110K):
[in this window]
[in a new window]
 
Fig. 3. Expression of Jel42 scFv. (A) The effect of rifampicin on expression. Inclusion of rifampicin led to the overexpression of two protein species whose molecular weights were compatible with scFv with and without the leader sequence. SDS–PAGE showing the effects of rifamipin treatment on IPTG-induced scFv expression. Whole cell extracts taken 3 h following induction with 1 mM IPTG; 200 µg/ml rifampicin was added to cultures at intervals following induction: lane 2, 30 min; lane 3, 60 min; lane 4, 120 min; lane 5, no induction; lanes 1 and 6 molecular weight standards: ovalbumin 45 kDa; carbonic anhydrase, 31 kDa.

 
Purification of Jel42 scFv

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; 70–80% pure as judged from SDS–PAGE. 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 3–10% 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 guanidine–HCl was stable at –70°C for at least several weeks. The renatured Jel42 scFv (0.2–2 mg/ml) dialyzed against a 10-fold volume of PBS stored on ice could be used for about 5–6 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., 1987Go; Sharma et al., 1991Go). 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, 1997Go; Smallshaw et al., 1998Go). 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 4Go) for preparations of renatured Jel42 scFv that were usually 70–80% pure, but as low as 30% pure as estimated by SDS–PAGE. Kd varied between 20 and 200 nM reflecting the variation in purity and renaturation (Table IGo). 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 SDS–PAGE gel analysis (Table IGo). 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 IGo). 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 0.92–0.99, which indicate that the detectable active scFv had consistent properties.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Determination of Kd. The binding of Jel42 scFv to HPr was measured by fluorescent polarization. The millipolarization (mP) value of F-5-M-labeled Arg17Cys HPr increases with increased binding of the scFv, and a titration (Klotz plot) is shown. The data was fitted to an algorithm for single site binding (Smallshaw et al., 1998Go) with a regression analysis of 0.99.

 

View this table:
[in this window]
[in a new window]
 
Table I. Determination of Jel42 scFv dissociation constant, Kd
 
The preparations of renatured Jel42 scFv gave Kd values that were about 7–60-fold greater than the 2.8 ± 1.6 nM Kd for the antibody (Table IGo). Kd determination was dependent upon protein concentration that did not distinguish between properly and improperly refolded scFv. An assessment of the concentration of active scFv (properly refolded) was based upon the observation that Jel42–HPr and Jel44–HPr complexes can be isolated by isoelectric focusing. If a mixture of antibody and saturating HPr is loaded onto an isoelectric focusing gel, no free antibody is found (Waygood et al., 1987Go). Therefore, the F-5-M-labeled Arg17Cys HPr was added in excess to samples of the preparations of renatured Jel42 scFv which were used for the binding assay. The complex was detected following isoelectric focusing as a fluorescent band(s) with a higher pI than the labeled HPr (Figure 5Go). The area of the gel with fluorescent bands of the scFv–HPr complex was cut out, and the fluorescently-labeled HPr (or complex) allowed to elute by diffusion into 1 ml PBS with 0.1 mg/ml bovine serum albumin. The fluorescent intensity was determined over time (~12 h) by the removal of 10 µl aliquots, and ended when no more equilibration of the fluorescein-labeled HPr into the buffer was evident. The complex concentration was calculated from the fluorescent intensity (Smallshaw, 1997Go; Smallshaw et al., 1998Go). An explanation of Figure 5Go which is a demonstration follows. First, when antibody preparations such as Jel42 are characterized by isoelectric focusing, multiple bands all with high pIs are found (Waygood et al., 1987Go). All these antibody species when saturated with HPr showed a gel shift on isoelectric focusing gels (Waygood et al., 1987Go). Furthermore, Fab fragment preparations generated by papain contain multiple pI species (for example, Prasad et al., 1988Go). In Figure 5Go, the Jel42 antibody bound to the F-5-M-labeled Arg17Cys HPr, undergoes a gel shift as evident from the protein stain, but the multiple bands were not resolved on this gel, while the Fab fragment complex shows two discrete bands. In Figure 5Go, the Jel42 scFv that is bound to F-5-M-labeled Arg17Cys HPr, gave two bands. scFv preparations very often contain a mixture of monomers and dimers. Experience with various cysteine mutants of HPr which can be isolated as both dimers and monomers has demonstrated that on native isoelectric focusing gels, significant differences in pIs are found for some monomers and dimers (Smallshaw, 1997Go). Such may also be the case for Jel42 scFv monomers and dimers. It should be noted that the ability to detect the complexed forms on an isoelectric focusing gel is due to the great sensitivity of the fluorescent detection compared with the protein staining method.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5. Detection of scFv complexes on isoelectric focusing gels. This is a demonstration of the gel shift of the F-5-M-labeled HPr caused by binding to antibody (lanes 1 and 2), Fab fragment (lanes 3 and 4) and scFv (lanes 5 and 6). The isoelectric focusing gel was carried out as described in Materials and methods. (A) Fluorescent bands visualized using a standard UV box (365 nm); (B) protein staining bands of the same isoelectric focusing gel. Protein staining did not detect the scFv. Lane 1 is F-5-M-labeled Arg17Cys HPr; lane 2, Jel42 antibody; lane 3, Jel42 antibody mixed with the F-5-M-labeled Arg17Cys HPr, note the appearence of the fluorescently-labeled antibody complex with a lower pI than Jel42; lane 4, the Jel42 Fab fragment; lane 5, Jel42 Fab fragment with F-5-M-labeled Arg17Cys HPr; lane 6, renatured Jel42 scFv preparation; lane 7, the Jel42 scFv with F-5-M-labeled Arg17Cys HPr. The scFv preparations did not yield a major discrete protein staining band on isoelectric focusing gels. The amount of the F-5-M-labeled Arg17Cys HPr added was not saturating for the antibody or Fab fragment samples, but was for the amount of scFv. The gel photographed under the two conditions was not of the same size as the protein staining procedure expands the gel; the photographic enlargement has been adjusted to make the gels approximately the same size.

 
When the Kds were calculated using the concentration of active scFv as determined by this method the average value was 2.7 ± 2.0 nM from five determinations on separate preparations in agreement with Kd's determined for Jel42 antibody and Fab (Table IIGo). Ki values, with wild type HPr as the competitor, were also determined for four of the five scFv preparations.


View this table:
[in this window]
[in a new window]
 
Table II. Binding constants of Jel42 antibody, Fab fragment and scFv
 
Binding to HPr mutants

The relative effects of mutation to HPr on binding have been determined for the Jel42 antibody and Fab fragment (Smallshaw et al., 1998Go). Identical measurements were performed with the Jel42 scFv preparation, and the results of these determinations are presented in Table IIIGo. Jel42 scFv showed very similar properties.


View this table:
[in this window]
[in a new window]
 
Table III. Summary of Jel42 competition experiments
 
Dependence of binding upon temperature

The temperature dependency of the Kd value has been determined for both Jel42 antibody and Fab fragment (Smallshaw et al., 1998Go). 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 IVGo. 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.


View this table:
[in this window]
[in a new window]
 
Table IV. Thermodynamic parameters
 

    Discussion
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 References
 
The assembly of Jel42 was initially compromised by errors in the mRNA sequence and the re-examination of the mRNA sequencing autoradiographs gave no reason to question the sequence, but both the tertiary structure determination of the Jel42–HPr complex (Prasad et al., 1993Go, 1998Go) and the cDNA sequencing yielded sequence differences of some consequence. Many of the sequence differences occurred in the CDRs. The mRNA sequences of a number of other scFvs were determined at the same time and have been found to be essentially correct when the cDNA was sequenced (Barry and Lee, 1993Go; Kuderova et al., 1994Go; Tanha et al, 1997).

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., 1988Go; Daugherty et al., 1991Go). 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 3Go), 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; GoKnappik 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 IGo). 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 IIIGo) 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 2–3-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 70–80% 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., 1998Go). The higher ts value for the scFv compared with the antibody and Fab fragment (Table IVbGo) 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., 1997Go). 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, 1997Go; Smallshaw et al., 1998Go).

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.


    Acknowledgments
 
Laura Latimer and Michele Barry are thanked for the cloning of the cDNA for Jel42 variable regions. Alena Kuderova is thanked for her advice with respect to expression of the scFv. The nucleotide sequence corrections for Jel42 light and heavy chains have been deposited: accession numbers M60389 and M60390. This work was supported by the Medical Research Council of Canada operating grants MT6147 (E.B.W.) and MT9098 (J.S.L.). J.E.S. was the recipient of a Medical Research Council Studentship and a University of Saskatchewan Graduate Student Scholarship.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 References
 
Ames,G.F.-L., Prody,C. and Kustu,S. (1984) J. Bacteriol. 160, 1181–1183.[ISI][Medline]

Amit,A.G., Mariuzza,R.A., Phillips,S.E.V. and Poljak,R.J. (1986) Science 233, 747–753.[ISI][Medline]

Anthony,J., Near,R., Wong,S.-L., Iida,E., Ernst,E., Wittekind,M., Haber,E. and Ng.,S.-C. (1992) Mol. Immunol. 29, 1237–1247.[ISI][Medline]

Ayala,M., Balint,R.F., Fernández-de-Cossío,M.E., Canaán-Haden,L., Larrick,J.W. and Gavilondo,J.V. (1995) Biotechniques 18, 832–842.[ISI][Medline]

Barry,M.M. and Lee,J.S. (1993) Molecular Immunol. 30, 833–840.[ISI][Medline]

Bhat,T.N., Bentley,G.A., Fischmann,T.O., Boulot,G. and Poljak,R.J. (1990) Nature 347, 483–485.[ISI][Medline]

Bhat,T.N., Bentley,G.A., Boulot,G., Greene,M.I., Tello,D., Dall'Acqua,W., Souchon,H., Schwarz,F.P., Mariuzza,R.A. and Poljak,R.J. (1994) Proc. Natl. Acad. Sci. USA 91, 1089–1093[Abstract]

Bird,R.E., Hardman,K.D., Jacobson,J.W., Johnson,S., Kaufman,B.M., Lee,S.-M., Lee,T., Pope,S.H., Riordan,G.S. and Whitlow,M. (1988) Science 242, 423–426.[ISI][Medline]

Braden,B.C. and Poljak,R.J. (1995) FASEB J. 9, 9–16.[Abstract/Free Full Text]

Bradford,M.M. Braden,B.C. and Poljak,R.J. (1976) Anal. Biochem. 72, 248–254.[ISI][Medline]

Buchner,J. and Rudolph,R. (1991) Bio/Technology 9, 157–162.[Medline]

Buchner,J., Pastan,I. and Brinkmann,U. (1992) Anal. Biochem. 205, 263–270.[ISI][Medline]

Chacko,S. Silverton,E., Kam-Morgan,L., Smith-Gill,S., Cohen,G. and Davies,D. (1995) J. Mol. Biol. 245, 261–274.[ISI][Medline]

Cho,B.K., Schodin,B.A. and Kranz,D.M. (1995) J. Biol. Chem. 270, 25819–25826.[Abstract/Free Full Text]

Dall'Acqua,W., Goldman,E.R., Eisenstein,E. and Mariuzza,R.A. (1996) Biochemistry 35, 9667–9676.[ISI][Medline]

Daugherty,B.L., DeMartino,J.A., Law,M.-F., Kawka,D.W., Singer,I.I. and Mark,G.E. (1991) Nucleic Acids Res. 19, 2471–2476.[Abstract]

Davies,D.R. and Cohen,G.H. (1996) Proc. Natl. Acad. Sci. USA 93, 7–12.[Abstract/Free Full Text]

Deng,S., MacKenzie,C.R., Sadowska,J., Michniewicz,J., Young,N.M., Bundle,D.R. and Narang,S.A. (1994) J. Biol. Chem. 269, 9533–9538[Abstract/Free Full Text]

Deng,S., MacKenzie,C.R., Hirama,T., Brousseau,R., Lowary,T.L., Young,N.M., Bundle,D.R. and Narang,S.A. (1995) Proc. Natl Acad. Sci. USA 92, 4992–4996[Abstract]

Denzin,L.K., Whitlow,M. and Voss,E.W.,Jr (1991) J. Biol. Chem. 266, 14095–14103.[Abstract/Free Full Text]

Gruen,L.C., McInerney,T.L., Webster,R.G. and Jackson,D.C. (1993) J. Protein Chem. 12, 255–259.[ISI][Medline]

Hawkins,R.W., Russell,S.J. Baier,M. and Winter,G. (1993) J. Mol. Biol. 234, 958–964.[ISI][Medline]

Hochuli,E., Bannwarth,W., Dobeli,H., Gentz,R. and Stuber,D. (1988) Bio/Technology 6, 1321–1325.[ISI]

Huston,J.S., Levinson,D., Mudgett-Hunter,M., Tai,M.-S., Novotny,J., Margolies,M.N., Ridge,R.J., Bruccoleri,R.E., Haber,E., Crea,R. and Oppermann,H. (1988) Proc. Natl Acad. Sci. USA 85, 5879–5883.[Abstract]

Ito,W., Iba,Y. and Kurasawa,Y. (1993) J. Biol. Chem. 268, 16639–16647.[Abstract/Free Full Text]

Jung,S. and Plünkthun,A. (1997) Protein Engng 10, 959–966[Abstract]

Kam-Morgan,L.N.W., Smith-Gill.S.J., Taylor,M.G., Zhang,L., Wilson,A.C. and Kirsch,J.F. (1993) Proc. Natl Acad. Sci. USA 90, 3958–3962.[Abstract]

Knappik,A. and Plünkthun, A (1995) Protein Engng 8, 81–89.[Abstract]

Kortt,A.A., Oddie,G.W., Iliades,P., Gruen,L.C. and Hudson,R.J. (1997) Anal. Biochem. 253, 103–111.[ISI][Medline]

Kuderova,A., Tanha,J. and Lee,J.S. (1994) J. Biol. Chem. 269, 32957–32962.[Abstract/Free Full Text]

Kunkel,T.A., Roberts,J.D. and Zakour,R.A. (1987) Methods Enzymol. 54, 367–382.

Laemmli,U.K. (1970) Nature 227, 680–685.[ISI][Medline]

Lake,D.F., Lam,K.S., Peng,L. and Hersh,E.M. (1994) Mol. Immunol. 31, 845–856.[ISI][Medline]

Landt,O., Grunert,H.-P. and Hahn,U. (1990) Gene 96, 125–128.[ISI][Medline]

Lavoie,T.B., Drohan,W.N. and Smith-Gill,S.J. (1992) J. Immunol. 148, 503–513.[Abstract/Free Full Text]

Lei,S.-P., Lin,H.-C., Wang,S.-S., Callaway,J. and Wilcox,G. (1987) J. Bacteriol. 169, 4379–4383.[ISI][Medline]

Mallender,W.D., Carrero,J. and Voss,E.W.,Jr (1996) J. Biol. Chem. 271, 5338–5346.[Abstract/Free Full Text]

Mateucci,M.D. and Caruthers,M.H. (1981) J. Am. Chem. Soc. 103, 3186–3191.

Moyle,M., Lee,J.S., Anderson,W.F. and Ingles,C.J. (1989) Mol. Cell. Biol. 9, 5750–5753.[ISI][Medline]

Neiba,L., Honegger,A., Krebber,C. and Plünkthun, A (1997) Protein Engng 10, 435–444[Abstract]

Nuss,J.M., Bossart-Whitaker,P. and Air,G.M. (1993) Proteins Struct. Funct. Genet. 15, 121–132.[ISI][Medline]

Pantoliano,M.W., Bird,R.E., Johnson,S., Asel,E.D., Dodd,S.W., Wood,J.F. and Hardman,K.D. (1991) Biochemistry 30, 10117–10125.[ISI][Medline]

Postma,P.W., Lengler,J.W. and Jacobson,G.R. (1993) Microbiol. Rev. 57, 543–594.[Abstract]

Postma,P.W., Lengeler,J.W. and Jacobson,G.R. (1996) In Neidhardt,F.C. et al. (eds), Escherichia coli and Salmonella. Cellular and Molecular Biology. American Society for Microbiology Press, Washington, DC.

Prasad,L., Vandonselaar,M., Lee,J.S. and Delbaere,L.T.J. (1988) J. Biol. Chem. 263, 2571–2574[Abstract/Free Full Text]

Prasad,L., Sharma,S., Vandonselaar,M., Quail,J.W., Lee,J.S., Waygood,E.B., Wilson,K.S., Dauter,Z. and Delbaere,L.T.J. (1993) J. Biol. Chem. 268, 10705–10708.[Abstract/Free Full Text]

Prasad,L., Waygood,E.B., Lee,J.S. and Delbaere,L.T.J. (1998) J. Mol. Biol. 280, 829–845.[ISI][Medline]

Qian,L. and Wilkenson,M. (1991) Biotechniques 10, 736–738.[ISI][Medline]

Sambrook,J., Firtsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory manual, 2nd Edn. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY.

Schick,K.A., Xavier,K.A., Rajpal,A., Smith-Gill,S.J. and Willson,R.C. (1997) Biochim. Biophys. Acta. 1340, 205–214.[ISI][Medline]

Schier,R., Bye,J., Apell,G., McCall,A., Adams,G.P., Malmqvist,M., Weiner,L.M. and Marks,J.D. (1996) J. Mol. Biol. 255, 28–43.[ISI][Medline]

Schwarz,F.P., Tello,D., Goldbaum,F.A., Marriuzza,R.A. and Poljak,R.J. (1995) Eur. J. Bioch. 228, 388–394.[Abstract]

Sharma,S., Georges,F., Delbaere,L.T.J., Lee,J.S., Klevit,R.E. and Waygood,E.B. (1991) Proc. Natl Acad. Sci. USA 88, 4877–4881.[Abstract]

Sharma,S. (1992) Epitope Mapping of Monoclonal Antibodies Specific for the Histidine-Containing Protein, HPr, by Site-Directed Mutagenesis. Ph.D. Thesis, University of Saskatchewan.

Sharp,P.M. and Li,W.-H. (1987) Nucleic Acid. Res. 15, 1281–1295.[Abstract]

Skerra,A. and Plückthun,A. (1988) Science 240, 1038–1041.[ISI][Medline]

Skerra,A., Pfitzinger,I. and Plückthun,A. (1991) Bio/Technology 9, 273–278.[ISI][Medline]

Smallshaw,J.E. (1997) Ph.D. thesis, University of Saskatchewan.

Smallshaw,J.E., Brokx,S., Lee,J.S. and Waygood,E.B. (1998) J. Mol. Biol. 280, 765–774[ISI][Medline]

Steeves,T., Barry,M.M., Duckworth,H.W., Waygood,E.B. and Lee.,J.S. (1991) Biochem. Cell Biol. 69, 297–302.[ISI][Medline]

Steipe,B., Schiller,B., Plünkthun,A. and Steinbauer,S. (1994) J. Mol. Biol. 240, 188–192.[ISI][Medline]

Studier,F.W. and Moffatt,B.A. (1986) J. Mol. Biol. 189, 113–130.[ISI][Medline]

Tabor,S. and Richardson,C.C. (1985) Proc. Natl Acad. Sci. USA 82, 1074–1078.[Abstract]

Tanha,J. and Lee,J.S. (1997) Nucleic Acids Res., 25, 1442–1449.[Abstract/Free Full Text]

Tello,D., Goldbaum,F.A., Mariuzza,R.A., Ysern,X., Schwarz,F.P. and Poljak,R.J. (1993) Biochem. Soc. Trans. 21, 943–946.[ISI][Medline]

Tulip,W.R., Harley,V.R., Webster,R.G. and Novotny,J. (1994) Biochemistry 33, 7986–7997.[ISI][Medline]

Vieira,J. and Messing,J. (1982) Gene 19, 259–268.[ISI][Medline]

Ward,E.S., Gussow,D., Griffiths,A.K., Jones,P.T. and Winter,G. (1989) Nature 341, 544–546.[ISI][Medline]

Waygood,E.B., Reiche,B., Hengstenberg,W. and Lee,J.S. (1987) J. Bacteriol. 169, 2810–2818.[ISI][Medline]

Received July 9, 1998; revised March 9, 1999; accepted March 22, 1999.