In Vitro Enzymatic Biotinylation of Recombinant Fab Fragments

Bioconjugate Chem. 1998, 9, 725−735

725

In Vitro Enzymatic Biotinylation of Recombinant Fab Fragments through a Peptide Acceptor Tail Petri Saviranta,* Tapio Haavisto, Pekka Rappu, Matti Karp, and Timo Lo¨vgren Department

of

Biotechnology,

University

of

Turku,

Tykisto¨katu

6,

FIN-20520

Turku,

Finland.

Received February 19, 1998; Revised Manuscript Received July 2, 1998

We describe the site-specific enzymatic biotinylation of recombinant anti-estradiol Fab fragments through a 13 amino acid acceptor peptide translationally fused to the C-terminus of the Fd chain. The Fab-peptide fusion proteins were secreted to the periplasm of Escherichia coli, purified, and biotinylated in vitro using biotin ligase, biotin, and ATP. The E. coli biotin ligase (the BirA protein) was produced as a novel N-terminal fusion protein with glutathione S-transferase (GST) and purified in one step from bacterial cell lysate using a Glutathione Sepharose affinity column. The purified fusion protein worked as such (without cleavage of the GST part) for the in vitro biotinylation of the Fab fragments. After the removal of nonbiotinylated Fab fragments by monomeric avidin chromatography, the overall yield of biotinylated Fab was 40%. The site-specifically biotinylated Fab fragments (BioFab) were tested in streptavidin-coated microtitration wells, to which they were shown to bind linearly with respect to the amount of BioFab added, specifically as indicated by biotin inhibition, and tightly with a half-life of several days. Moreover, the enzymatic BioFab exhibited uniform antigen binding affinity unlike the same recombinant Fab fragments biotinylated through random chemical conjugation to surface lysines. Finally, the BioFab demonstrated its potential as a well-behaving immunoassay reagent in a model competitive assay for estradiol.

INTRODUCTION

The biotin-avidin/streptavidin system is used in a variety of biotechnological and diagnostic applications owing to the exceptionally high affinity of this noncovalent interaction between a protein and a small organic molecule (Wilchek and Bayer, 1990a). In in vitro diagnostics, streptavidin-coated tubes, microtitration wells, or spherical particles are used as universal surfaces for the capture of biotinylated antibodies in immunoassays of various formats (Diamandis and Christopoulos, 1991). The biotinylation of antibodies can be done by several well-established chemical methods in which an activated biotin derivative is conjugated to the protein surface residues (most often lysine) to the hinge region cysteines or to a carbohydrate moeity (Bayer and Wilchek, 1990; Diamandis and Christopoulos, 1991; Hermanson, 1996). The most widely used biotin derivative, biotinyl-Nhydroxysuccinimide ester (BNHS),1 reacts mainly with the -amino groups of surface lysines and, under optimal conditions, generally causes no serious damage to the antigen binding activity (Bayer and Wilchek, 1990). Mukkala et al. (1993) synthesized an isothiocyanate derivative of biotin (BITC), which is, like BNHS, prima* Author to whom correspondence should be addressed. Tel: 358-2-333 8083. Fax: 358-2-333 8050. E-mail: Petri.saviranta@ utu.fi. 1 Abbreviations: apo-Fab, nonbiotinylated form of the Fabbiotin acceptor peptide fusion protein; BioFab, Fab fragment that is biotinylated through the 13 amino acid peptide tail; BITC, biotin isothiocyanate; BirA, the bifunctional biotinholocarboxylase synthetase/biotin operon repressor protein of Escherichia coli; BNHS, biotinyl-N-hydroxysuccinimide ester; BSA, bovine serum albumin; dNTP, deoxynucleoside triphosphate; GST, glutathione S-transferase of Schistosoma japonicum; IPTG, isopropyl β-D-thiogalactopyranoside; OD600, optical density at the wavelength of 600 nm.

rily reactive with amino groups. The BITC reagent has since been used in our laboratory for the biotinylation of a number of different purified mAbs and has proven to be a viable complement to BNHS and other commercially available biotin reagents. The recent advances in antibody engineering (for a review, see Hayden et al., 1997) and the availability of bacterially produced recombinant Fab fragments has led to a situation in which increasingly more antibodies will be biotinylated in the Fab form. As the Fab fragment is only one-third of the size of the whole immunoglobulin, the chemical biotinylation at surface residues even at low degrees of conjugation will more often result in the destruction or modification of the antigen-binding site. The recombinant Fab fragments come with an advantage that they can be modified by protein engineering. For example, new functionalities useful in specific applications can be added to the carboxyl ends of the constant domains while leaving the variable regions intact (Rapley, 1995). Therefore, we sought for a protein engineering solution to aid in the safe biotinylation of recombinant Fab fragments. In the living cells, a few proteins (depending on the organism) are naturally biotinylated through the covalent linkage of biotin to a unique lysine residue (Wood, 1977). This posttranslational modification is catalyzed by a biotin ligase and involves the formation of an amide bond between the carboxyl group of biotin and the -amino group of the lysine residue (Figure 4A; Shenoy and Wood, 1988). In Escherichia coli, the only biotinylated protein is the biotin carboxyl carrier protein (BCCP), a subunit of the acetyl-CoA carboxylase. The C-terminal 84-88 amino acids of BCCP form an independent domain that can be fused to recombinant proteins and biotinylated in vivo by the E. coli endogenous biotin ligase (Cronan, 1990; Li and Cronan, 1992). Although the biotinylation

10.1021/bc9800217 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/03/1998

726 Bioconjugate Chem., Vol. 9, No. 6, 1998

takes place in the cytoplasm, also fusions of the biotinyl domain with periplasmic proteins such as alkaline phosphatase and β-lactamase can be biotinylated to some extent (Reed and Cronan, 1991). The biotinylation of secreted fusion proteins can be enhanced by slowing down the secretory process (using sec- strains) so as to win extra time for the action of the biotin ligase and/or by coexpressing the biotin ligase so as to increase the biotinylation rate. (Reed and Cronan, 1991; Jander et al., 1996). However, we did not want to compromise the secretion of the Fab fragments and, therefore, considered an alternative approach to achieve higher biotinylation yields. We chose to perform enzymatic biotinylation in vitro after the production of the recombinant Fab fusion protein, which enabled us to control the biotinylation conditions independently of the various cellular factors. Furthermore, to possibly improve the secretion and periplasmic stability of the fusion protein, we aimed at minimizing the size of the fusion partner. Schatz (1993) used an elegant selection strategy to find novel peptide substrates for the biotin ligase from plasmid-encoded random peptide libraries. He defined a consensus sequence of 13 amino acids, containing an internal lysine residue, sufficient for in vivo biotinylation in E. coli. We here describe the construction of a Fab fusion vector in which the coding sequence for such a biotin acceptor peptide is inserted at the C-terminus of the Fd chain. The biotin ligase (biotin holoenzyme synthetase) of E. coli is a bifunctional protein coded by the birA gene (Barker and Campbell, 1981). In addition to being biotin ligase, the BirA protein acts as the repressor of the bio operon responsible for biotin biosynthesis (Cronan, 1989). The BirA protein can be overexpressed in E. coli up to 1000-fold with apparently no toxic effects (Buoncristiani and Otsuka, 1988) thus facilitating the production of large amounts of biotin ligase for in vitro studies. To simplify the enzyme purification, we constructed a fusion protein between BirA and the glutathione S-transferase (GST) of Schistosoma japonicum (Smith and Johnson, 1988), which allowed us to use Glutathione-Sepharose for the one step purification of the enzyme from crude cell lysate. In this study, we show that the purified fusion protein is capable of in vitro biotinylation of the 13 amino acid peptide tail in the presence of the substrates biotin and ATP. Recently, we cloned and expressed in E. coli the recombinant Fab fragment of the monoclonal anti-estradiol antibody 57-2 (Pajunen et al., 1997). In this paper, we describe the construction of the biotin acceptor peptide fusion protein of Fab 57-2 as well as the in vitro biotinylation of the apo-Fab using the GST-BirA fusion protein. The immunochemical properties of the enzymatically biotinylated Fab fragment relevant to immunoassay applications will be characterized and discussed. Finally, the functionality of the novel biotinylated Fab fragment is demonstrated by setting up a competitive estradiol immunoassay. MATERIALS AND METHODS

Strains and Plasmids. E. coli MC1061 [cI+, ∆(ara, leu)7697, ∆lacX74, galU-, galK-, hsr-, hsm+, strA, araD139; Casadaban and Cohen, 1980] was used for DNA cloning and for the overproduction of the GST-BirA fusion protein. The strain XL-1 Blue [recA1, endA1, gyrA96, thi1, hsdR17, supE44, relA1, lac (F′ proAB, lacIqZ∆M15, Tn10 (tetr)), Stratagene] was used for Fab expression. The plasmid pJMR1 containing the birA gene from the E. coli biotin biosynthetic operon was kindly provided by

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Dr. Anthony J. Otsuka. The plasmid pGEX-1λT with the glutathione S-transferase gene of Schistosoma japonicum was purchased from Pharmacia Biotech (Uppsala, Sweden). Construction of Biotin Acceptor Peptide:Fab Genetic Fusion. The plasmid p57-2 (Pajunen et al., 1997) was used as the vector for the insertion of the biotin acceptor peptide coding sequence. The vector was digested with the restriction enzymes SpeI and NheI and dephosphorylated with calf intestinal phosphatase (Pharmacia) followed by agarose gel purification of the 4.6 kb fragment. The insert was prepared from two partially complementary (15 nucleotide overlap at the 3′-ends) synthetic oligonucleotides: the forward 43-mer 5′-GTAC ACT AGT GG C GGT GGC CCA GGT GGC CTG GTT TCT ATC TT-3′ and the reverse 53-mer 5′-CTAG GCT AGC TTA GTG CCA TTC GAT TTT CTG AGC TTC GAA GAT AGA AAC CAG-3′. The forward and reverse oligonucleotides (50 pmol each) were annealed together by heating to 90 °C for 5 min and slowly cooling to room temperature. The single-stranded ends were filled in with the Klenow fragment of DNA polymerase I (Boehringer Mannheim) for 15 min at room temperature in the presence of 1 mM dNTPs. The double-stranded 80mer was digested with SpeI and NheI (the recognition sites are underlined in the oligonucleotide sequences above) followed by agarose gel purification with the Glass Milk procedure (Bio101). The purified fragment was ligated to the vector with T4 DNA ligase and electroporated into E. coli MC1061 cells. The presence of the insert was tested from plasmid minipreps by digestion with SfiI, and the correct coding sequence was verified by nucleotide sequencing over the whole insert region. The plasmid construct (p572Bio) was later transferred to the strain XL-1 Blue for Fab production. An otherwise identical construct but lacking the biotin tail was produced for the chemical biotinylation experiments (see below) by self-ligating the compatible cohesive ends of the SpeI-NheI digested vector (the open reading frame ends right after the NheI site at the tandem stop codons TAG TAA). Construction of the GST-BirA Genetic Fusion. The plasmid pGEX-1λT was used as the vector for the construction of the GST-BirA genetic fusion. The vector was linearized by digestion with EcoRI followed by filling in the cohesive ends with the Klenow fragment of DNA polymerase I for 15 min in the presence of 1 mM dNTPs. The birA gene was cut from the plasmid pJMR1 by double digestion with BspHI and XbaI and was made blunt ended similarly as the vector. The insert and the vector were gel purified, blunt ligated with T4 DNA ligase, and electroporated into MC1061 cells. The orientation of the insert as well as the integrity of the joints in the resulting plasmid were checked by test digestions with the appropriate restriction enzymes (in the correct construct all the restriction sites were regenerated as a result of the ligation of the blunt-ended fragments). Overproduction and Purification of the GSTBirA Fusion Protein. E. coli MC1061 strain containing the plasmid pGEX-BirA was inoculated in two 1-L shake flasks each containing 250 mL of 2× YT (16 g/L Bacto tryptone, 10 g/L Yeast extract, and 5 g/L NaCl) supplemented with 0.2% glucose and 100 mg/mL ampicillin. The cells were grown at 30 °C at 240 rpm until the OD600 of 1 was reached. The fusion protein expression was induced by the addition of 0.1 mM IPTG, after which the cells were grown for an additional 6 h and harvested by centrifugation. The cell pellet was resuspended in 15 mL of PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2-

In Vitro Enzymatic Biotinylation of Fab Fragments

HPO4, and 1.8 mM KH2PO4, pH 7.4) containing 5% glycerol, 0.1 mM DTT, and 10 mM MgCl2. The cells were disrupted with the French press (Aminco Corp.) after which DNaseI was added into the lysate (final concentration 1 µg/mL) and the cell debris was removed by centrifugation. Triton X-100 was added to a final concentration of 1% (v/v), and the extract was filtered with a 0.45 µm filter. GST-BirA fusion protein was purified from the cell extract by affinity chromatography through Glutathione Sepharose 4B (Pharmacia Biotech). The column (bed volume 5 mL) was equilibrated with PBS buffer containing 5% glycerol, 0.1 mM DTT, 10 mM MgCl2, and 1% Triton X-100. The sample (15 mL) was loaded on the column at a flow rate of 0.5 mL/min, followed by washing with 10 vol of the equilibration buffer except that the concentration of Triton X-100 was reduced to 0.01%. The purified product was eluated with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, containing 5% glycerol and 10 mM MgCl2. The pooled fractions were concentrated, and the protein concentration was determined by Bradford’s protein determination assay (Bradford, 1976) using BSA as a standard. The purified GSTBirA protein was divided into aliquots and stored at -20 °C in a buffer containing 52% glycerol, 50 mM NaCl, 24 mM Tris-HCl, pH 8.0, 4.7 mM reduced glutathione, and 4.7 mM MgCl2. Fab Production and Purification. A fresh colony of E. coli XL-1 Blue cells containing the plasmid p572Bio was inoculated in 5 mL of SB medium (30 g/L tryptone, 20 g/L Yeast extract, and 10 g/L MOPS, pH 7.0) supplemented with glucose (0.2%), ampicillin (100 µg/mL) and tetracycline (10 µg/mL) and grown at 300 rpm, 37 °C until the OD600 of approximately 0.5 was reached. One milliliter of the preculture was inoculated in 50 mL of SB medium supplemented with 0.05% glucose, 50 µg/mL ampicillin, 5 µg/mL tetracycline, and 100 µM IPTG and shaken at 240 rpm and 30 °C for 14 h. The cells were collected by centrifugation, resuspended in 10 mL of TBS buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl), and disrupted by sonication (model Labsonic U fitted with the needle probe 40T, B. Braun, Germany). The lysate was clarified by centrifugation (20000g, 4 °C, 15 min), the supernatant was recovered and stored frozen (-20 °C) until the purification. The Fab was purified from the clarified lysate with a prepacked 1 mL HiTrap Protein G column (Pharmacia Biotech) according to the manufacturer’s instructions. Briefly, the thawed lysate was filtered through a 0.22 µm filter and loaded on the column which was preequilibrated with TBS buffer. After washing with 5 column volumes of TBS, the Fab was eluted with 100 mM glycine-HCl buffer, pH 2.5, containing 0.01% Tween20, at a flow rate of 0.5 mL/min. Fractions (1 mL) were collected in Eppendorf tubes that contained 30 µL of neutralization buffer (2 M Tris, pH 9.0, and 0.33% BSA). After the determination of the immunoreactive Fab concentrations, the peak fractions were pooled and stored at +4 °C. Immunoassay Reagents and Equipment. All immunochemical measurements were performed using the DELFIA technology (Wallac, Finland), which is based on the time-resolved measurement of fluorescent lanthanide chelates formed in the enhancement solution after the lanthanide ion has been dissociated from the original, nonfluorescent chelate conjugated to the biomolecule. The following DELFIA assay reagents and equipment were used: (rabbit) anti-mouse IgG-coated microtitration strips, streptavidin-coated microtitration strips, europiumlabeled streptavidin, Assay buffer, Washing solution,

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Enhancement solution, and Plate Fluorometer (model 1232). Two europium-labeled estradiol derivatives, conjugated either from C4 or C6 of the steroid skeleton, were used as immunoassay tracers. The position 6 conjugate (estradiol-6-carboxymethyl oxime conjugated to an europium chelate; Pajunen et al., 1997) was kindly provided by H. Mikola while the position 4 conjugate (unpublished material) was obtained from N. Meltola. Determination of Immunoreactive and Biotinylated Fab Fragments. Immunoreactive Fab concentrations were determined with a microtitration plate assay using europium-labeled estradiol (C4 conjugate) as the detection marker. Purified, nontailed recombinant Fab 57-2 (Pajunen et al., 1997) was used as the calibration standard. All dilutions were made in the Assay buffer. Samples and standards (200 µL) were added to prewashed anti-mouse IgG-coated microtitration strips, shaken for 60 min at room temperature, followed by four washes after which 200 µL of europium-labeled estradiol (2 nM) was added to each well. The incubation was continued for 60 min, after which the strips were washed four times and Enhancement solution (200 µL) was added. The time-resolved fluorescence signal was measured with the Plate Fluorometer, controlled by the MultiCalc immunoassay program (1224-310) running on a personal computer. The program automatically produced a spline-fitted standard curve and calculated the unknown Fab concentrations. The detection of biotinylated Fab fragments was performed as above, except that Eu-labeled streptavidin at 100 ng/mL instead of Eu-labeled estradiol was used. The degree of biotinylation was estimated by comparing the signals obtained from the same sample detected with either Eu-labeled estradiol or Eu-labeled streptavidin. Enzymatic Biotinylation. The Fab sample (3 mL) was changed into the biotinylation buffer (100 mM potassium phosphate, 12 mM MgCl2, and 0.06% Tween20) using a Centricon-30 concentrator (Amicon) with two cycles of concentration and dilution. Buffer-exchanged Fab (50 µg) was used for a 500 µL biotinylation reaction which contained 10 mM ATP, 100 µM D-biotin, 1 µg/mL pyrophosphatase, and 100 µg/mL of the GST-BirA fusion protein in the biotinylation buffer. The reaction was kept at 37 °C for 4 h, after which the unreacted biotin was removed by gel filtration using a prepacked Sephadex G-25 column (NAP-5, Pharmacia Biotech). The biotinylated and unbiotinylated Fab fragments were separated using monomeric avidin agarose (the Softlink resin, Promega) according to the manufacturer’s instructions. Briefly, 1 mL of the Softlink resin was packed into a disposable minicolumn, regenerated with 10% acetic acid and equilibrated with TBS-Tween (TBS, pH 7.5, containing 0.05% Tween20). The Fab sample was passed through the column at a flow rate of 0.5 mL/min, followed by washing with 5 mL of TBS-Tween, after which the biotinylated Fab was eluted with 5 mM D-biotin in TBSTween supplemented with 0.01% BSA. Fractions (0.51.0 mL) were collected and analyzed for the immunoreactive Fab concentrations. The peak fractions eluted with biotin were pooled, and the free biotin was removed by repeated concentration with Centricon-30 followed by dilution with TBS-Tween. After seven cycles of concentration/dilution, the final volume was 0.3 mL and the estimated residual free biotin concentration was 0.3 µM. Chemical Biotinylation with Biotin Isothiocyanate. Biotin isothiocyanate (BITC) was obtained as a gift from V.-M. Mukkala. BITC is reactive mainly with the primary amino groups in the proteins (Mukkala et al., 1993) and is used the same way as the commonly used


N-hydroxysuccinimide derivatives of biotin. The chemistry of the biotinylation reaction is described in Figure 4B. The recombinant Fab 57-2 fragments used for the chemical biotinylation were produced using an expression vector that did not contain the biotin acceptor tail at the end of the Fd chain. Purified Fab, originally stored in 10 mM MES, pH 6.0, was buffer exchanged into 0.9% NaCl using a NAP5 column (Pharmacia Biotech). The biotinylation was performed in a reaction volume of 100 µL containing 100 µg (2 nmol) of Fab, either 30 or 50 nmol of freshly dissolved BITC, and 50 mM sodium carbonate buffer (pH 9.8). The BITC reagent was used in either 15- or 25-fold molar excess, known from previous experience to be near optimal for Fab fragments. For a number of reasons, the conditions used do not lead to the stoichiometric conjugation of biotin and therefore we prefer to state the molar reagent concentrations (300 and 500 µM) instead of the reagent excess per se. The biotinylation reaction was incubated at room temperature for 4 h after which the unreacted BITC was separated by passage through two buffer exchange columns (NAP5, followed by NAP10, Pharmacia Biotech), equilibrated with TSA (50 mM Tris-HCl, pH 7.75, 150 mM NaCl, and 0.02% NaN3). After the buffer exchange, BSA (0.1%) and NaN3 (0.05%) were added and the sample was filtered through a 0.22 µm filter. The biotinylated fraction (>90%) was purified using the monomeric avidin resin similarly as described for the purification of the enzymatically biotinylated Fab. Immunochemical Characterization of Biotinylated Fab Fragments. The biotinylated Fab fragments were tested in streptavidin-coated microtitration strips for binding capacity, biotin inhibition, binding stability, and antigen affinity constant. All incubations were done at room temperature, the strips covered with an adhesive tape. All dilutions were made in the Assay buffer. To determine the binding capacity of the streptavidincoated wells for BioFab, a series of BioFab samples (0.03-60 ng/200 µL) was added to the wells and shaken for 60 min at room temperature, followed by four washes. The bound BioFab was then detected with europiumlabeled estradiol (10 nM C6 conjugate/200 µL) which was shaken for 30 min, followed by four washes. Finally, the europium content in the wells was determined by adding 200 µL of Enhancement solution, shaking for 30 min to develop the fluorescent chelates, and measuring the europium signals with the Plate Fluorometer. In the biotin inhibition test, the samples (200 µL) contained fixed amounts of Fab (5.4 ng) and europiumlabeled estradiol (2 nM C6 conjugate), while each sample contained a different concentration of biotin (0-10 µM). The premixed samples were added to the streptavidincoated wells, shaken for 60 min, and washed four times. Since the europium-labeled estradiol was already present from the beginning, there was no need for a separate tracer incubation for the detection of the bound BioFab. The europium signals were measured as above. The binding stability test was started by preattaching BioFab to the streptavidin-coated microtitration wells (5.4 ng/200 µL, 120 min), after which further binding and rebinding was stopped by the addition of 20 µL of 33 µM biotin (final concentration 3 µM). The incubation was continued for varying times (15 min-24 h), followed by the removal of unbound/dissociated BioFab with four washes. The bound BioFab was detected with europiumlabeled estradiol (2 nM C6 conjugate, 30 min, four washes); the europium signals were measured as described above.

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In the affinity constant determination, the BioFab samples (2 ng/200 µL) were preattached to the streptavidin-coated wells for 120 min, followed by four washes. Europium-labeled estradiol (200 µL) (C4 tracer) was added at varying concentrations (0.08-8 nM) to each well and incubated for 2 h, followed by the removal of the unbound tracer with four washes. Finally, the bound tracer concentrations were determined by the measurement of the europium signals as before. Calculation of Affinities. The affinities (Kd constants) were calculated with the KELL RADLIG program version 5.0.2 (Biosoft, Cambridge, U.K.). The analysis was performed with the hot saturation mode; binding data were input as molar concentrations; free ligand was calculated from specific bound. The multisite saturation models (1, 2, or 3 binding sites) were fitted with the 1/Y2 weighting; the fits were statistically compared using a built-in approximate F test included in the RADLIG program. In essence, the test determined whether there was a significant decrease in the weighted sum of squared deviations, given the increase in the model parameters when assuming more binding sites. Competitive Immunoassay for Estradiol. BioFab (2 ng in 200 µL of assay buffer) was preattached to streptavidin-coated microtitration wells for 60 min followed by four washes. Estradiol standards (0-100 nM) were prepared directly in the tracer solution (0.2 nM C6 conjugate in assay buffer). Each standard (200 µL) (i.e., mixture of estradiol and tracer) was added to three replicate wells, incubated for 60 min, and washed four times, after which the measurement of the bound tracer was performed as above. The MultiCalc immunoassay program was used for the data management and the following calculations. A four-parameter logistic fitting algorithm was used to produce the standard curve and the associated parameters: ED50, i.e., the concentration corresponding to the half-maximal signal and the slope factor, i.e., the measure of the steepness of the sigmoidal curve. The estradiol concentrations in the individual wells were back-calculated from the fitted standard curve using the measured signals, after which the coefficient of variation (CV%) was calculated for each set of triplicate wells. RESULTS

Design of the Fab-Biotin Acceptor Peptide Fusion Protein. To produce the Fab-biotin acceptor peptide fusion protein, we constructed the plasmid p572Bio (Figure 1). The Fd and light chain genes of the original anti-estradiol mAb 57-2 (Pajunen et al., 1997) were previously cloned into the phage display vector pComb3 (Barbas et al., 1991), where the Fd chain was fused to the carboxyl-terminal domain of the gene III protein of the filamentous phage M13. We excised the gene III fragment using the unique flanking restriction sites SpeI and NheI and replaced it by a synthetic DNA cassette containing the coding sequence for a 13 amino acid biotinylation signal, adapted from Schatz (1993). To increase the availability of the biotin acceptor peptide for the BirA protein, we inserted a flexible linker of eight amino acids between the peptide and the carboxyl end of the Fd chain. The cassette was constructed from two overlapping synthetic oligonucleotides which also included the restriction sites SpeI and NheI for cloning into the pComb3 vector and an internal unique SfiI site for the checking of the presence of the insert. The nucleotide sequence of the cassette is shown in the upper part of Figure 1.


Figure 1. Map of the plasmid p572Bio. The expression of the heavy (Fd) and the light chains is controlled by the lac promoteroperator (lacPO) in front of each gene. pelB signal sequences, fused to the amino termini of each chain, direct the synthesized polypeptides to the periplasmic space. The biotin acceptor peptide coding region is cloned in-frame to the carboxyl terminus of the Fd chain which ends right before the SpeI site. The nucleotide sequence between the SpeI and NheI sites as well the translated amino acid sequence are shown above the plasmid map. The biotinylated lysine residue is indicated by a circle. Labels: ColE1 ori, the region containing the plasmid origin of replication; f1 ori, the region containing a filamentous phage origin of replication; ApR, amipicillin resistance (beta-lactamase) gene.

Construction, Expression and Purification of the GST-BirA Fusion Protein. The translational fusion between the GST and the BirA protein was carried out by cloning the birA gene of E. coli to the GST gene fusion vector pGEX-1λT containing the gene for the glutahione S-transferase of S. japonicum (Smith and Johnson, 1988). In the resulting plasmid pGEX-BirA, the GST coding region, followed by a thrombin cleavage site, is fused inframe to the first methionine codon of the full-length birA gene (Figure 2). The overexpression of the fusion protein was achieved by IPTG induction of the strong tac promoter, resulting in the accumulation of approximately 10 mg/L of the fusion protein in the cells. Comparison of the SDS-PAGE samples of the whole cell lysate before and after centrifugation suggests that the fraction of insoluble protein was less than 30% of the total recombinant protein produced (Figure 3, lanes 2-3). The GST-BirA protein was purified from the clarified cell lysate by one-step affinity chromatography using a glutathione Sepharose 4B column. The purification procedure was highly efficient as judged by the SDSPAGE of the samples taken before the column, from the flow-through fraction and from the eluted material (Figure 3, lanes 3-5). The eluted fusion protein (lane 5) appeared to be over 90% pure, migrated roughly according to the expected size (62 kDa), and showed no signs of proteolytic degradation into truncated fragments such as the mere GST part (∼26 kDa). The final yield of the purified GST-BirA from the 500 mL shake flask culture was 3.5 mg. Purification and Biotinylation of apo-Fab 57-2. The total production level of apo-Fab in a shake flask


Figure 2. Map of the plasmid pGEX-BirA. A BspHI-XbaI fragment containing the birA gene was excised form the plasmid pJMR1 (a gift from A. Otsuka) and cloned into the EcoRI site of the vector pGEX-1λT. The cohesive ends of the insert and the linearized vector were filled in with the Klenow fragment of DNA polymerase I and ligated as blunt-ended fragments. The ligation of the insert in the correct orientation resulted in the regeneration of all the original restiriction sites. The GST-BirA fusion gene is controlled by the strong tac promoter (tacP) which is in turn repressed by the plasmid-encoded lac repressor (lacIq). Fusion protein expression is induced by IPTG which derepresses the tac promoter. The joining region from the end of the glutathione S-transferase gene to the start of the birA gene is shown above the plasmid map. The thrombin site is indicated above the amino acid translation. For other labels, see legend to Figure 1.

Figure 3. SDS-PAGE analysis of the purification of the GSTBirA fusion protein. Samples were diluted in PBS, mixed with equal volume of 2× reducing sample buffer and heated to 95 °C for 5 min. After a brief centrifugation, 1 µL of each sample was loaded on a PhastGel 8-25% polyacrylamide gel which was run with SDS buffer strips using the PhastSystem electrophoresis apparatus (Pharmacia Biotech). Samples: molecular weight markers (1); cell lysate before (2) and after (3) the removal of insoluble material by centrifugation; flow through fraction (4) and eluate (5) of the glutathione Sepharose 4B chromatography.

culture after overnight induction at 30 °C was 800 µg/L, of which 80% remained in the periplasm. The cells were disrupted by sonication after which the apo-Fab was purified from the total cell lysate by protein G chromatography. This simple miniscale purification procedure


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Figure 4. Protein biotinylation via the -amino group of a lysyl residue. A three-residue fragment of a larger polypeptide chain is shown with the lysyl residue in the middle. R-1 and R+1 denote the side chains of the residues preceding and following the lysyl residue. (A) The enzymatic biotinylation reaction catalyzed by the biotin ligase. The reaction proceeds through a biotinyl-AMP intermediate from which the biotinyl moeity is transferred to the acceptor protein. (B) The chemical biotinylation reaction using BITC. The nucleophilic amine of the lysyl residue attacks the central, electrophilic carbon of the isothiocyanate group. An electron shift and a proton loss create a stable thiourea bond. Table 1. Purification and Biotinylation of the apo-Fab yield (%)a

procedural step initial purification clarified lysate after freeze and thaw protein G pool biotinylationb reaction mixture after buffer exchange monomeric avidin columnc flow-through + washes eluted fractions (pool) total out concentrated pool

Fab Fab volume concn mass step- over(mL) (µg/mL) (µg) wise all 10 10 3 0.5 1.1 5.5 2.5 0.3

21 16 26

207 160 79

100 77 50

100 77 38

100 48

50 53

100 106

100 106

29 16 45 19

55 29 85 123

58 31 90 38

5.3 6.2 64

a

Percentage of Fab remaining after each step, expressed with respect to the preceding step (stepwise yield) as well as with respect to the starting point of the procedure (overall yield). b The complete biotinylation procedure consisted of the enzymatic reaction, buffer exchange with NAP5 column, and separation of biotinylated and nonbiotinylated Fab with monomeric avidin column and the final concentration of the pooled fractions with a Centricon 30 concentrator. c All the buffer-exchanged material (1.1 mL) was loaded on the monomeric avidin column.

gave a relatively low overall yield (38%), since there were losses both in the intermediate frozen strorage of the clarified lysate as well as in the protein G chromatography (Table 1). No optimization of the purification method was attempted. GST-BirA fusion protein was used to biotinylate 50 µg of the purified apo-Fab. After the biotinylation reaction and a buffer change to remove free biotin, the biotinylated and nonbiotinylated Fab fragments were separated with monomeric avidin chromatography (SoftLink resin); the bound Fab fragments were eluted from the column with 5 mM free biotin. All fractions (flow-through, wash as well as elution) were recovered and subjected for the determination of immunoreactive Fab concentrations (Table 1). The total sum of the collected fractions was 85% of the input, consisting of 55% in the flow-through

and wash fractions (representing the apparently nonbiotinylated Fab fragments) and 29% in those fractions eluted with biotin (representing the biotinylated Fab fragments). Thus, the degree of biotinylation could be estimated to be about 29/85 ≈ 34%. The fact that apparently 15% of the input Fab was not recovered at all may be partly explained by minor underestimation of the individual fraction volumes. After the pooling and final concentration steps, we had 19 µg of purified BioFab, which constituted 38% of the initial 50 µg used for the biotinylation procedure. Thus the net efficiency of the whole procedure was estimated to be 38% (bottom line of Table 1). The degree of biotinylation was also estimated by another method in which the Fab samples were first immobilized on secondary antibody-coated wells and then detected either with europium-labeled streptavidin or with europium-labeled estradiol. For each sample, the ratio of the signals obtained with europium-labeled streptavidin versus europium-labeled estradiol was taken as an indicator of the degree of biotinylation. The analysis of the Fab samples before and after the enzymatic reaction as well as after the final purification is described in Table 2. Assuming that the signal ratio of the purified BioFab (4.68) represents a fully biotinylated population, the efficiency of the biotinylation reaction was calculated to be 42%, agreeing well with the previous estimate based on the separation of the biotinylated and nonbiotinylated forms with the monomeric avidin resin (above). The degree of biotinylation in vivo (i.e., before the enzymatic in vitro reaction) was approximately 2%. Binding of BioFab to Streptavidin-Coated Wells. The interaction of the enzymatically biotinylated Fab 57-2 with coated streptavidin was characterized in terms of the binding capacity, specificity, and stability. To study the binding capacity, varying amounts of BioFab (0.003-160 ng) were incubated in the streptavidin-coated wells. After washes, the bound BioFab was detected using europium-labeled estradiol as the tracer. The



Table 2. Degree of Biotinylationa

process stage purified apo-Fab after enzyme reaction purified BioFab

streptavidin estradiol signal signal proportionc (CPS) (CPS) ratiob biotinylated 5460 129 680 422 192

54 398 65 403 90 294

0.10 1.98 4.68

2% 42% 100%

a Fab samples were captured to anti-mouse IgG coated microtitration strips and detected with either europium-labeled streptavidin or europium-labeled estradiol. The streptavidin signal is proportional to the amount of biotinylated fragments in the sample while the estradiol signal is proportional to the amount of Fab fragments containing intact binding sites. b The ratio of the signals obtained with labeled streptavidin and estradiol. c The extent of biotinylation was estimated by comparing the streptavidin/estradiol signal ratio at each stage with that of the purified BioFab. It was assumed that the BioFab fragments purified with the monomeric avidin resin were completely biotinylated.

dependence of the measured europium signals on the amount of BioFab added is shown in Figure 5A. The response stays virtually linear for almost 5 orders of magnitude, indicating that the binding of the BioFab is not dependent on its own concentration. No saturation of the streptavidin surface could be demonstrated within the range of BioFab concentrations tested; therefore, the binding capacity is at least 160 ng/well. As a control for the specificity of the attachment of BioFab to the streptavidin-coated wells, we tested whether the binding could be inhibited by free biotin. Figure 5B shows how the BioFab binding decreased rapidly as the free biotin concentration went from 40 to 100 nM, indicating that BioFab and biotin competed for the same binding sites. Consequently, the observed binding of the BioFab to the surface was not caused by general nonspecific forces such as the stickiness of the protein. The inhibition experiment also revealed that 3 µM biotin concentration blocked practically all BioFab binding. Consequently, this concentration was used in the stability test (below) to prevent rebinding of dissociated BioFab fragments. The stability of the complex between BioFab and the coated streptavidin in the microtitration well was studied by incubating the preformed complexes in 3 µM biotin solution for varying times, followed by washes and detection of the bound BioFab with labeled antigen. Although 3 µM biotin effectively blocked the binding of BioFab when added simultaneously (Figure 5B), it could not displace BioFab already bound to streptavidin (Figure 5C). In the time period of 24 h, less than 10% of the bound BioFab dissociated from streptavidin, indicating that the half-life of the complex must be several days. Affinity Comparison of Enzymatically and Chemically Biotinylated Fab Fragments. To study whether the enzymatic and chemical biotinylation methods differed in their effects on the antigen-binding affinity, we made a binding site titration using labeled antigen. Biotinylated Fab fragments were immobilized on streptavidin-coated wells, after which varying concentrations of europium-labeled estradiol (C4 conjugate) were added and allowed to equilibrate between the bound and free states. Figure 6A shows that the binding sites were efficiently titrated as the signal levels of the bound labeled antigen were approaching saturation at the higher total concentrations. To determine the affinity constants, the data were initially plotted as bound/free vs bound (the Scatchard’s plot) so as to calculate the Kas from the slopes of the plots by linear regression (Figure 6B). However, the plots for the chemically biotinylated Fab fragments were clearly curved, suggesting the pres-

Figure 5. Testing the binding of BioFab to streptavidin-coated microtitration wells. The amount of BioFab bound to the wells was detected with europium-labeled antigen (C6 conjugate of estradiol). Each symbol in the plots represents the average of two replicate wells. (A) Capacity. The signal of the bound labeled antigen is linearly dependent on the amount of BioFab added throughout the tested range. (B) Specificity. BioFab binding is inhibited by increasing concentrations of free biotin. (C) Stability. The dissociation of bound BioFab from the coated streptavidin was monitored for 24 h. Excess free biotin was used to block the rebinding of dissociated BioFab.

ence of two or more subpopulations of binders with different affinity constants. Therefore, the binding data were nonlinearly fitted to different models involving one


Saviranta et al. Table 3. Affinities for Europium-Labeled Estradiola biotinylation methodb

percentage of major binderc

Bmax (pM ( SD)

Kd (nM ( SD)

enzymatic BITC 300 µM BITC 500 µM

98.6% 94.5% 93.5%

66.0 ( 1.5 78.2 ( 9.5 74.2 ( 9.4

1.57 ( 0.16 4.11 ( 1.34 5.70 ( 1.62

a The affinities (K constants) of the biotinylated Fab fragments d for europium-labeled estradiol were calculated from the data obtained in the hot saturation experiment shown in Figure 6. b The Fab fragments were biotinylated either enzymatically (using the GST-BirA fusion protein as the biotin ligase) or chemically (using either 300 or 500 µM BITC reagent). c The binding data were initially fitted into models involving either one, two or three binding site subpopulations. The two site model was the statistically most plausible one in each case, even when the second sites were present at very low proportions. However, the binding parameters of the minor sites could not be accurately defined and were omitted from the table.

Figure 6. Affinity determination of the enzymatically and chemically biotinylated Fab fragments. Biotinylated Fab fragments were immobilized on streptavidin-coated wells, after which the binding sites were titrated with europium-labeled estradiol (C4 conjugate). (A) The bound concentration (pM) is plotted against the total added concentration of europiumlabeled estradiol. The replicates (two parallel wells) are plotted separately. (B) Scatchard plot presentation of the same data. As an exception to the conventional presentation (bound/free vs bound), the x values were normalized to the form B/Bmax. This way, all the curves cross the x-axis at the same point and can be more readily compared. (The absolute Bmax values are shown in Table 3). The curves drawn through the data points were fitted according to the model involving two binding sites.

ligand and either one, two, or three binders. Statistical comparison between the fits (performed by the RADLIG fitting program) supported the model involving two classes of binders in each case, although the exact nature of the minor classes could not be accurately defined. Realizing that the two-binder model only gave the statistically most plausible fit for the available binding data, we concentrate on the comparison of the major binder classes within that model (Table 3). While the major class always represented more than 90% of the total binding site population, it can be seen that the enzymatically biotinylated Fab fragments were the most uniform (98.6% major binder). The dissociation constants (Kd) of the chemically biotinylated Fab fragments were 2.6-3.6-fold higher than that of the enzymatically biotinylated Fab (Table 3), suggesting that the random attachment of biotin lead in most cases to the decrease of antigen-binding affinity. Competitive Immunoassay for Estradiol. To demonstrate the usage of the BioFab as an immunoassay reagent, we set up a competitive estradiol immunoassay in streptavidin-coated microtitration strips, using europium-labeled estradiol (C6 conjugate) as the tracer. The B0 signal (corresponding to zero estradiol concentration) was approximately 51 000 cps whereas the signals obtained with estradiol standards between 1 and 100 nM decreased from 44 800 down to 4300, covering practically the whole measurable concentration range (Figure 7).

Figure 7. Competitive immunoassay for estradiol. The assay was performed in streptavidin-coated microtitration plates to which purified, enzymatically biotinylated anti-estradiol Fab fragments (BioFab) were precaptured in a separate incubation. The assay incubations (60 min) contained a fixed concentration of tracer (0.2 nM europium-labeled C6 conjugate of estradiol) and a varying concentration (0-100 nM) of estradiol; the x-axis refers to the final estradiol concentrations in the wells. After washes, the bound tracer was measured with time-resolved fluorescence as described in materials and methods. Filled circles indicate the average signal (CPS, left y-axis) obtained from the measurement of three replicate wells for each estradiol concentration. The MultiCalc program was used for curve fitting as well as for the reverse calculation of estradiol concentrations from the measured signals for the individual wells. The coefficiency of variation (CV%, right y-axis) of the calculated estradiol concentrations for each set of triplicate wells is indicated by open triangles.

The europium signals were fitted with a sigmoidal doseresponse curve model (the four-parameter logistic equation) producing a standard curve with an ED50 of 6.7 nM and a slope factor of -0.94. When the estradiol concentrations of the standards were reverse calculated from the fitted curve using the measured signals, the coefficiency of variation (CV%) of the concentrations remained less than 10% throughout the range (Figure 7), indicating that the assay is technically reliable and could be applied to the measurement unknown estradiol concentrations. DISCUSSION

We describe the site-specific enzymatic biotinylation of recombinant anti-estradiol Fab fragments through a 13 amino acid acceptor peptide translationally fused at the C-terminus of the Fd chain. To our knowledge, this is the first report on the in vitro application of the biotin ligase to biotinylate antibodies. Moreover, we describe the usage of a novel fusion protein (GST-BirA) as the biotinylating enzyme.


The production of correctly folded recombinant Fab fragments in E. coli requires that they be secreted to the periplasm where the oxidative conditions allow for the formation of the necessary disulfide bridges (Plu¨ckthun and Skerra, 1989). Because efficient secretion is not ideally compatible with in vivo biotinylation (Reed and Cronan, 1991; Jander et al., 1996), we decided not to try to optimize the intracellular conditions but rather perform the biotinylation in vitro. Nevertheless, we observed a low (2%) basal level of biotinylation in the protein G purified apo-Fab fragments (Table 2). A similar figure was reported by Weiss et al. (1994) in a study of secreted recombinant Fab fragments to which a 101 amino acid C-terminal fragment of the E. coli BCCP was fused at the C-terminus of the Fd chain. Relying on the activity of the endogenous biotin ligase, they initially achieved an in vivo biotinylation level of 3%, although they reported a 5-fold enhancement of the basic level on the addition of biotin to the growth medium. The basal biotinylation level observed by us and by Weiss et al. (1994) may represent the fraction of fusion proteins that are biotinylated in the cytoplasm before they are secreted (Reed and Cronan, 1991); possibly some biotinylation occurs also when cytoplasmic biotin ligase enters the periplasm as a result of partial cell lysis typical of recombinant Fab expression. Furthermore, here we used a shortcut (sonication) instead of osmotic shock for the extraction of the periplasmic Fab fragments and, thus in effect, mixed the cytoplasm and periplasm for a while before the protein G purification. The biotinylation in the cell lysate could indeed be an effective alternative to in vivo biotinylation, as shown by Chapman-Smith et al. (1994). They achieved only limited biotinylation of the overexpressed BCCP biotin acceptor domain in vivo despite the coexpression of the biotin ligase. However, the biotinylation was taken to completion in the cell lysate on addition of ATP and biotin. The minimum size for the C-terminal fragment of BCCP that can be biotinylated as a fusion protein is about 84 residues (Li and Cronan, 1992). Compared to this, the 13 residue peptides described by Schatz (1993) seem to bring a definite advantage for recombinant fusion protein applications at least when the size of the tail is critical. Other parameters important for the general utility of these fusion tails include their efficiency as biotin ligase substrates, compatibility with secretion, and possible susceptibility to proteolysis. The biotinylated lysine of BCCP is located in a highly conserved hairpin β-turn (Athappilly and Hendrickson, 1995), and the cloned acceptor proteins from various bacterial as well as eucaryotic sources act as substrates for E. coli biotin ligase (Cronan, 1990). However, the consensus sequence of the 13 amino acid peptides differs considerably from the natural acceptors, indicating that different constraints and perhaps a different mechanism of recognition apply to the small acceptor peptides (Schatz, 1993). Despite this, the peptides have been shown to be quantitatively biotinylated in vivo, both as N- and C-terminal fusions, provided that biotin ligase is coexpressed (Tsao et al., 1996). The periplasmic secretion of the 13 amino acid biotin acceptor peptides as fusion proteins has not been attempted before. Our results indicate that the peptides fused at the C-terminus of the Fd chain are compatible with membrane translocation which is a prerequisite for the assembly of functional Fab fragments. We have constructed several similar Fab-acceptor peptide fusion proteins, and the production levels vary between 30 and 100% of that of the nonfusion versions, depending on the particular Fab fragment (M. Vehni-


a¨inen, unpublished data). There are large inherent differences in the folding and stability of different recombinant Fab fragments in the bacterial periplasm (Knappik and Plu¨ckthun, 1995), which may be reflected in the differing effects of the fusion tail on the production levels. The fact that more than half of the purified apoFab 57-2 could not be biotinylated suggests that the tail was not not always accessible for the biotin ligase or it was proteolytically degraded. The biotinylation degree did not improve with the use of more enzyme or prolonging the incubation time, nor in attempts to rebiotinylate the nonbiotinylated fraction (data not shown). Further studies will be needed to clarify whether the incomplete biotinylation was caused by proteolytic cleavage of the tail or by some conformation-dependent mechanism. To facilitate the purification of the biotin ligase, we cloned the birA gene into the GST fusion vector pGEX1λT (Smith and Johnson, 1988). The fusion of glutathione S-transferase to the amino terminus of BirA was considered a minor risk, especially because there was an option to remove the fusion partner by thrombin cleavage at a site in the joining linker (Figure 2). On the basis of genetic data as well as sequence comparisons, Buoncristiani et al. (1986) suggested that the N-terminal part of the BirA protein contains a helix-turn-helix motif responsible for DNA binding, while the central part contains the catalytic activities. In the crystal structure of BirA (Wilson et al., 1992), there is only a loose connection between the N-terminal domain (residues 1-60) and the central domain (residues 68-269), suggesting that the fusion of GST to the amino-terminus of BirA would not affect the catalytic activity. However, the catalytic and repressor functions are actually intimately connected. The biotin ligase activity consists of two steps: the formation of biotinyl-5′-AMP from biotin and ATP and the transfer of the activated biotin moeity to the acceptor protein (BCCP). If all acceptor proteins are already biotinylated, the biotinyl-5′-AMP remains bound to BirA and acts as the corepressor of biotin biosynthesis (Cronan, 1989). Not only is biotinyl-5′-AMP required for the binding of BirA to the bio operator but, in turn, also the presence of the DNA-binding domain is important for the binding of biotinyl-5′-AMP to the catalytic domain. In a study of a truncation mutant of BirA lacking the Nterminal DNA-binding domain, Xu and Beckett (1996) found that the affinities for biotin and biotinyl-5′-AMP were 100- and 1000-fold decreased, respectively, although the maximal catalytic rates for the formation of biotinyl5′-AMP and the transfer of biotin to BCCP were unchanged. These results implied that the N-terminal fusion of GST to BirA could, in an unfortunate case, affect the enzymatic activity. However, the fusion protein worked reasonably well for our purposes, so we did not attempt removing the GST part by taking advantage of the thrombin cleavage site. Biochemical studies are needed in the future to assess the possible effect of the GST fusion as well as to determine the catalytic parameters of the BirA biotin ligase for the peptide substrates. The enzymatically biotinylated Fab fragments were characterized for their interaction with surface-bound streptavidin in order to assess their potential usefulness as immunoassay reagents. First, the binding of BioFab to streptavidin-coated wells was found to be linear in a wide concentration range (almost 5 decades), indicating that the Fab fragments bind independently of each other. The binding was also specific as demonstrated by the blocking of binding by submicromolar concentrations of biotin. The stability of the complex between BioFab and the coated streptavidin was a concern as it is known that


some biotinylated macromolecules show considerably weaker binding to streptavidin than does biotin (Vincent and Samuel, 1993). We tested the binding stability by measuring the time-dependent dissociation of Bio-Fab from streptavidin in the presence of a vast excess of free biotin to block the rebinding. The dissociation rate was extremely low, less than 10% in 24 h, which should be adequate for most immunoassay applications. Moreover, typical assay conditions would allow the rebinding of dissociated BioFab and thus would show even higher apparent binding stability. In conclusion, the above results indicate that the fusion peptide tail presents the biotin moeity in a favorable position for streptavidin binding. The determination of the antigen-binding affinities of one enzymatically biotinylated and two chemically biotinylated Fab fragments indicated that the enzymatic biotinylation results in a more uniform binder population. Moreover, comparison of the major binders shows that the dissociation constants (Kd) of the chemically biotinylated Fabs are 2.6-3.6 times higher than that of the enzymatically biotinylated one (Table 3). These differences may be explained by the location of the biotin moiety on the surface of the Fab fragment. In the chemical procedure, in principle all surface lysines are targets for conjugation, although some may be preferred over the others. The outcome of the chemical biotinylation is therefore dependent on the content and distribution of surface lysines. The conjugation of a lysine residue at or near the antigen binding site can have an effect on the affinity either directly by interfering with the antibody-antigen interaction or indirectly by steering the immobilization of the Fab in an unfavorable orientation. The disadvantages of the chemical conjugation process can often be reduced by careful optimization of the conditions, aiming at one biotin per Fab. Even then, it depends on the primary structure of the Fab whether the single biotinylated lysine is preferentually found in the antigen binding site, elsewhere in the variable domains or in the constant domains. Ultimately, it is possible to engineer the primary structure of the binding site in order to decrease its sensitivity to chemical labeling, as shown in a recent study in which two lysine residues in the CDR loops of a recombinant immunotoxin were changed into arginines (Benhar et al., 1994). As this technique is by no means trivial or universally applicable, there will remain an increasing number of recombinant Fab (as well as single chain Fv) fragments that cannot be labeled with amine-reactive chemistries. Our results show that the enzymatic biotinylation through a peptide acceptor tail can produce a homogeneous, wellbehaving binder population when the chemical biotinylation through amino groups fails to do so. Finally, we demonstrated that the enzymatically biotinylated Fab fragments work as reagents in a competitive estradiol immunoassay. The standard curve (Figure 7) followed nicely the four-parameter logistic model and had a slope factor close to -1 which again indicated that the antigen and the tracer reacted with a single binder population. Although the assay is technically reliable (CV% was less than 10% throughout the measuring range), it is not yet useful for the measurement of clinical samples because the affinity of the antibody 57-2 for estradiol is not high enough. However, we are currently modifying its binding properties by random mutagenesis and phage display (Saviranta et al., 1998) and expect to use the mutated version for the assay of clinical samples. Furthermore, the site-specifically biotinylated Fab fragments should be ideal for the novel miniaturized assay

Saviranta et al.

formats, such as single microparticle-based assays (Lo¨vgren et al., 1997) where maximally efficient usage of the coated surface is required. ACKNOWLEDGMENT

The financial support from State Technology Development Centre of Finland (TEKES) is gratefully acknowledged. We thank H. Mikola and N. Meltola for delivering the europium-labeled estradiol conjugates. M. Pajunen, M. Vehnia¨inen, and P. Nurmikko are thanked for help in DNA cloning, protein purification, and chemical biotinylation, respectively. LITERATURE CITED Athappilly, F. K., and Hendrickson, W. A. (1995) Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing. Structure 3, 1407-1419. Barbas, C. F., III, Kang, A. S., Lerner, R. A., and Benkovic, S. J. (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. U.S.A. 88, 7978-82. Barker, D. F., and Campbell, A. M. (1981) Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli. J. Mol. Biol. 146, 469-492. Bayer, E. A., and Wilchek, M. (1990) Protein biotinylation. Methods Enzymol. 184, 138-166. Benhar, I., Brinkmann, U., Webber, K. O., and Pastan, I. (1994) Mutations of two lysine residues in the CDR loops of a recombinant immunotoxin that reduce its sensitivity to chemical derivatization. Bioconjugate Chem. 4, 321-326. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Buoncristiani, M. R., and Otsuka, A. J. (1988) Overproduction and rapid purification of the biotin operon repressor from Escherichia coli. J. Biol. Chem. 263, 1013-1016. Buoncristiani, M. R., Howard, P. K., and Otsuka, A. J. (1986) DNA-binding and enzymatic domains of the bifunctional biotin operon repressor (BirA) of Escherichia coli. Gene 44, 255-261. Casadaban, M. J., and Cohen, S. N. (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138, 179-207. Chapman-Smith, A., Turner, D. L., Cronan, J. E., Jr., Morris, T. W., and Wallace, J. C. (1994) Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase. Biochem. J. 302, 881-887. Cronan J. E., Jr. (1989) The E. coli bio operon: transcriptional repression by an essential protein modification enzyme. Cell 58, 427-429. Cronan, J. E., Jr. (1990) Biotination of proteins in vivo. A posttranslational modification to label, purify and study proteins. J. Biol. Chem. 265, 10327-10333. Diamandis, E. P., and Christopoulos, T. K. (1991) The Biotin(strept)avidin system: principles and applications in biotechnology. Clin. Chem. 37, 625-636. Hayden, M. S., Gilliland, L. K., and Ledbetter J. A. (1997) Antibody engineering. Curr. Opin. Immunol. 9, 201-212. Hermanson, G. T. (1996) Bioconjugate techniques, Academic Press, Inc., San Diego, CA. Jander, G., Cronan, J. E., and Beckwith, J. (1996) Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion. J. Bacteriol. 178, 3049-3058. Knappik, A., and Plu¨ckthun, A. (1995) Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng. 8, 81-89. Li, S. J., and Cronan, J. E., Jr. (1992) The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. J. Biol. Chem. 267, 855-863. Lo¨vgren, T., Heinonen, P., Lehtinen, P., Hakala, H., Heinola, J., Harju, R., Takalo, H., Mukkala, V. M., Schmid, R.,

In Vitro Enzymatic Biotinylation of Fab Fragments Lo¨nnberg, H., Pettersson, K., and Iitia¨, A. (1997) Sensitive bioaffinity assays with individual microparticles and timeresolved fluorometry. Clin. Chem. 43, 1937-1943. Mukkala, V.-M., Ha¨nninen, E., and Hemmila¨, I. (1993) Synthesis of two biotin isothiocyanate derivatives. Proceedings of 8th European Symposium on Organic Chemistry, p 422, Sitges, Spain. Pajunen, M., Saviranta, P., Jauria, P., Karp, M., Pettersson, K., Ma¨ntsala¨, P., and Lo¨vgren, T. (1997) Cloning, sequencing, expression, and characterization of three anti-estradiol-17β Fab fragments. Biochim. Biophys. Acta 1351, 192-202. Plu¨ckthun, A., and Skerra, A. (1989) Expression of functional antibody Fv and Fab fragments in Escherichia coli. Methods Enzymol. 178, 497-515. Rapley R. (1995) The biotechnology and applications of antibody engineering. Mol. Biotechnol. 3, 139-154. Reed, K. E., and Cronan, J. E., Jr. (1991) Escherichia coli exports previously folded and biotinated protein domains. J. Biol. Chem. 266, 11425-11428. Saviranta, P., Pajunen, M., Jauria, P., Karp, M., Pettersson, K., Ma¨ntsa¨la¨, P., and Lo¨vgren, T. (1998) Engineering the steroid-specificity of an anti-17β-estradiol Fab by random mutagenesis and competitive phage panning. Protein Eng. 11, 143-152. Schatz, P. J. (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: A 13 amino acids concensus peptide specifies biotinylation in Escherichia coli. BioTechnology 11, 1138-1143. Shenoy, B. C., and Wood, H. G. (1988) Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii. FASEB J. 12, 2396-2401.

Bioconjugate Chem., Vol. 9, No. 6, 1998 735 Smith, D. B., and Johnson, K. S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31-40. Tsao, K.-L., DeBarbieri, B., Michel, H., and Waugh, D. S. (1996) A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli. Gene 169, 59-64. Vincent, P., and Samuel, D. (1993) A comparison of the binding of biotin and biotinylated macromolecular ligands to an antibiotin monoclonal antibody and to streptavidin. J. Immunol. Methods 165, 177-182. Weiss, E., Chatellier, J., and Orfanoudakis, G. (1994) In vivo biotinylated recombinant antibodies: Construction, characterization, and application of a bifunctional Fab-BCCP fusion protein produced in Escherichia coli. Protein Expression Purif. 5, 509-517. Wilchek, M., and Bayer, E. A. (1990a) Introduction to avidinbiotin technology. Methods. Enzymol. 184, 5-13. Wilchek, M., and Bayer, E. A. (1990b) Biotin-containing reagents. Methods Enzymol. 184, 123-138. Wilson, K. P., Shewchuk, L. M., Brennan, R. G., Otsuka, A. J., and Matthews, B. W. (1992) Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains. Proc. Natl. Acad. Sci. U.S.A. 89, 9257-9261. Wood, H. G., Barden, R. E. (1977) Biotin enzymes. Annu. Rev. Biochem. 46, 385-413. Xu, Y. And Beckett, D. (1996) Evidence for interdomain interaction in the Escherichia coli repressor of the biotin biosynthesis from studies of an N-terminal domain deletion mutant. Biochemistry 35, 1783-1792.

BC9800217

In Vitro Enzymatic Biotinylation of Recombinant Fab Fragments

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