PEDS Advance Access originally published online on May 2, 2008
Protein Engineering Design and Selection 2008 21(7):443-451; doi:10.1093/protein/gzn021
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Rapid and easy development of versatile tools to study protein/ligand interactions
1Macromolécules biologiques, Centre d’Ingénierie des Protéines 2ProGenosis 3Laboratoire d’Enzymologie, Centre d’Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000 Liège, Belgium
4 To whom correspondence should be addressed. E-mail: pfilee{at}ulg.ac.be
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Abstract |
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The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the β-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid β-lactamases is achieved in the presence of β-lactams making further screening of correctly folded and secreted hybrid β-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the β-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the β-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.
Keywords: β-lactamase/antibodies/high through put screening/hybrid protein
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Introduction |
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Many Escherichia coli expression vectors are available to produce and to purify proteins fused to affinity tags, such as the Schistosoma japonicum glutathione S-transferase (GST), the E. coli maltose binding protein, the E. coli thioredoxin etc (LaVallie and McCoy, 1995
; Tsunoda et al., 2005). All these systems allow the subsequent proteolytic cleavage of the fusion protein to separate the protein of interest from the affinity tags (LaVallie and McCoy, 1995; Tsunoda et al., 2005). But a common feature of these systems is the limited utility of the affinity tag in the biochemical characterisation of the studied protein. This work describes new opportunities by using the Bacillus licheniformis β-lactamase BlaP as a protein carrier and a reporter enzyme.
BlaP is an active-serine enzyme belonging to class A of β-lactamases (Matagne et al., 1999). Class A β-lactamases share a similar three-dimensional structure characterised by all 
and /β domains (Moews et al., 1990; Fonzé et al., 1995, 2002). This enzyme is a natural proteases-insensitive protein adapted to the presence of various and numerous proteases that are co-expressed by B. licheniformis (Filée et al., 2002; Brans et al., 2004; Veith et al., 2004). Previously, we identified a common permissive insertion site in class A β-lactamases that is located in a solvent-exposed loop far away from the active site. This position limits interferences between the biological activities of the inserted polypeptide and the carrier protein (Hallet et al., 1997; Ruth et al., 2005; Chevigné et al., 2007). We demonstrated that small peptides (Ruth et al., 2005), random protein fragments (Chevigné et al., 2007) and protein-binding motifs (Vandevenne et al., 2007) can be accommodated in this loop without losing the β-lactamase activity of the carrier protein. Moreover, we showed that these hybrid proteins can be expressed at the surface of phage fdTet in fusion with the minor coat protein pIII and selected for their affinities towards specific targets (Chevigné et al., 2007). This new approach for displaying polypeptides is named β-lactamase hybrid protein technology (BHP) for bifunctional hybrid proteins.
In a recent report, we described the insertion of the chitin binding domain (CHBD) of the human macrophage chitotriosidase into BlaP (Vandevenne et al., 2007). CHBD is a protein domain of 73 residues that promotes binding to insoluble chitin. Three disulphide bridges have been reported as essential for the chitin binding activity (Tjoelker et al., 2000; Ujita et al., 2003). The BlaPCHBD construct behaved as a soluble chimeric protein that conserves both the chitin-binding and the β-lactamase activities of the related native proteins. The biochemical and biophysical properties of this protein have been studied and these results have contributed to a better understanding of the reciprocal effects between the carrier protein and the inserted protein fragment (Vandevenne et al., 2007).
In this work, we have engineered the permissive loop of the β-lactamase BlaP by the introduction of two thrombin (Thb)-cleavage sites surrounding the insertion site. We demonstrate here that the hybrid protein BlaP Thb/CHBD/Thb remains bifunctional and that the inserted polypeptide can be separated from its carrier protein without losing its biochemical properties. As a result, this approach has facilitated the high and soluble production of this small mammalian polypeptide, usually difficult to produce in E. coli. A relevant application has been reported where the β-lactamase activity of the hybrid protein was used to study the interaction between CHBD and chitin. Finally, it is also shown that bifunctional hybrid β-lactamases can be used in the screening of neutralizing agents.
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Construction of pNY02 and pNY03
The synthetic gene fragment encoding two contiguous thrombin-cleavage sites was obtained by annealing the complementary oligonucleotides Thb+ (5'-CTGGTTCCGCGTGGATCTCCCGGGTTAGTGCCACGTGGTAGC-3') and Thb- (5'-GCTACCACGTGGCACTAACCCGGGAGATCCACGCGGAACCAG-3'). In this construction, the dipeptide Pro-Glu separates the proteolytic clivage sites and permits to create a SmaI restriction site (CCC-GGG) in the nucleotidic sequence. The annealed oligonucleotides were successively phosphorylated using T4 polynucleotide kinase and purified on a QiaquickTM removal kit (Qiagen). The gene fragments were finally ligated into the SmaI-digested and dephosphorylated pNY01 (Chevigné et al., 2007) to yield pNY02.
The gene fragment coding for CHBD was PCR amplified using Pfu DNA polymerase with primers CHBD+ (5'-GGTAGCGGACCAGAGCTTGAAGTTCCTAAACCAGGACAGCCCTCT-3') and CHBD– (5'-GTGGGGTCCATTCCAGGTACAACATTTGCAGGAGTTGCTGAACACCAGACC-3'). The PCR products were successively purified on a GFXTM gel band purification kit (Amersham Biosciences, UK), phosphorylated using T4 polynucleotide kinase and purified again on a GFXTM gel band purification kit before to be cloned into the SmaI-digested and dephosphorylated pNY02 to yield pNY03.
Hybrid β-lactamase purification
To achieve production of the hybrid β-lactamases harbouring a polyhistidine tag, E. coli JM109 transformed with pNY02 or pNY03 were grown in Terrific Broth supplemented with 75 µg/ml spectinomycin and 10 µg/ml ampicillin at 37°C. Cells from an overnight culture (1 l) were harvested by centrifugation (9000g for 15 min) and resuspended in 40 ml of TES (20% sucrose, 30 mM Tris–HCl, 5 mM EDTA, pH 8) at 37°C. The bacterial suspension was placed under stirring at 37°C for 10 min. Cells were harvested by centrifugation (9000g for 15 min) and the pellet resuspended in 100 ml of 5 mM MgSO4 at 4°C. The bacterial suspension was stirred at 4°C for 10 min. The supernatant containing the periplasmic proteins was harvested by centrifugation (13 000g for 20 min) and diluted with three volumes of 50 mM phosphate (pH 7.4). The periplasmic proteins were loaded on a HisTrapTM Chelating HP column (GE healthcare) equilibrated in 50 mM phosphate (pH 7.4). The column was successively washed with 2 M NaCl and 50 mM phosphate (pH 7.4) supplemented with 10 mM Imidazole. The hybrid proteins were eluted by an imidazole linear gradient (10–500 mM) in 50 mM phosphate (pH 7.4). Fractions containing the purified hybrid proteins were pooled and dialysed against phosphate-buffered saline (PBS) (50 mM phosphate, 150 mM NaCl, pH 7.4).
Quantitation of the sulphhydryl groups with DTNB
Ellman’s reagent, 5,5'-Dithio-bis(2-nitrobenzoic acid), DTNB (Ellman, 1959), was used for the determination of free thiols with glutathione as a standard. BlaP Thb/SmaI/Thb was used as a negative control. All measurements were performed by incubating samples in urea 6 M during 2 h to expose non-accessible cysteines in the native structures. Proteins and DTNB (
= 13 600 M–1 cm–1) concentrations were, respectively, 5 µM and 10 mM. Hundred microlitres of protein or glutathione solutions were incubated during 5 min with 900 µl of DTNB solution. Absorbance of samples was measured at 420 nm.
Thrombin (Sigma, Washington DC, USA) cleavage was performed in PBS supplemented with 2.5 mM CaCl2 at 20°C under overnight agitation. Five units of protease were used to digest 1 mg of protein in a final volume of 1 ml. We avoided the use of 2-mercaptoethanol when working with BlaP Thb/CHBD/Thb to prevent reduction of the native disulphide bonds in CHBD. To eliminate the N- and C-terminal fragments of the β-lactamase BlaP which remain bound by non-covalent interactions, the sample was loaded on HisTrapTM Chelating HP column. Purified CHBD was collected in the flow through.
Identical amounts (70 pmol) of BlaP Thb/SmaI/Thb, BlaP Thb/CHBD/Thb and ChBD were incubated during 2 h with 25 mg of insoluble chitin (Sigma) in a final volume of 300 µl of 50 mM sodium phosphate buffer (pH 7.5) in the presence or in the absence of 50 mM 1,4-Dithio-DL-threitol (DTT). The solutions were centrifuged at 13 000 rpm during 10 min to separate the supernatants containing unbound proteins from the pellet containing chitin with the captured proteins. The free proteins in the supernatants were analysed by SDS–PAGE and compare with the supernatants before addition of chitin.
Binding assays were conducted as follows: various concentrations of the chimeric protein (25 nM to 3 µM) were incubated in the presence of 10 mg of chitin in final volume of 500 µl of 50 mM sodium phosphate buffer, 150 mM NaCl, pH 7.5 at 22°C with continual mixing. The mixtures were centrifuged at 4°C for 15 min at 13 000 rpm and the supernatant containing the free protein was collected. The free protein concentration was determined using the reporter β-lactamase activity. The amount of bound protein was calculated as the difference between the initial protein concentration and the free protein concentration after binding. The relative equilibrium association constants (Kr) was determined by the method described by Gilkes et al. (Gilkes et al., 1992) using the following equation:
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Where [B] is the concentration of bound ligand (moles. g chitin–1), [F] the concentration of free ligand (molar), [N0] the concentration of binding site in the absence of ligand, a the number of lattice units occupied by a single ligand molecule and Ka the equilibrium association constant (litres.mol–1). Ka value cannot be isolated from this equation but the relative equilibrium association constant, Kr (litres.g chitin–1) is defined as:
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Electrospray ionisation mass spectrometry
Purified CHBD was concentrated on a bed of PEG 20 000 to obtain a concentration of 0.25 mg/ml. Classical concentrating systems were avoided to prevent adsorption of the protein on membranes. Aggregation was observed at CHBD concentrations >0.25 mg/ml. Before analysing the protein by mass spectrometry, the protein was dialysed against 50 mM ammonium acetate (pH 7).
A polyclonal anti-CHBD antiserum was obtained by immunizing one New Zealand white rabbit with four injections of purified CHBD (Centre d’Economie Rurale et d’Hormonologie de Marloie).
The kinetic parameters of the purified bifunctional hybrid β-lactamases were determined using nitrocefin in 50 mM phosphate buffer at pH 7. The initial rate of hydrolysis was monitored at 482 nm. The Km and kcat values were calculated by fitting the data to the Henri-Michaelis equation and its linearised form according to the Hanes transformation (Frère, 1973).
Purified proteins (500 ng) were denatured at 100°C in the presence of denaturing buffer and separated by SDS–PAGE (15%). The proteins were electroblotted onto a polyvinylidene difluoride membrane. Immunoblot analyses using polyclonal preimmune or anti-CHBD rabbit sera were carried out and rabbit antibodies were detected with ECL kit (Amersham Biosciences) by using goat horseradish peroxidase-conjugated anti-rabbit antibodies.
The experiments were performed in PBS (50 mM sodium phosphate, 150 mM NaCl, pH 7.4). Identical quantities (20 ng) of proteins (BlaP Thb/SmaI/Thb and BlaP Thb/CHBD/Thb) were preincubated or not with various dilutions of preimmune or anti-CHBD sera for 1 h before to be incubated during 2 h with 10 mg of insoluble chitin (Sigma). All the incubations were performed at room temperature. The solutions were centrifuged at 13 000g during 10 min to separate the supernatant containing unbound proteins from the pellet containing chitin with the captured proteins. The pellets were washed three times with 1 ml of wash buffer (50 mM phosphate, 500 mM NaCl, pH 7.4) and then incubated in the presence of 1 ml of 100 µM nitrocefin a chromogenic β-lactam at 25°C for 30 min. Then the suspensions were rapidly filtered through a 0.22 µm cut-off filter and the absorbances of the hydrolysis products were measured at 482 nm to determine the chitin immobilised β-lactamase activity.
Interaction of phage particles with chitin
Production and purification of phage particles harbouring the bifunctional hybrid protein or the parental β-lactamase were performed as described in our previous work (Chevigné et al., 2007). Interaction with phage particles was performed as follow: 100 µl of the phage particles solution (DO260 nm = 5) in 500 µl final volume of 50 mM Tris buffer (pH 7.5) were incubated 2 h with 10 mg of insoluble chitin (Sigma). The solutions were centrifuged at 13 000 rpm during 10 min to separate the supernatant containing unbound protein from the pellet containing chitin with the captured protein. The pellets were washed three times with 50 mM Tris buffer, 500 mM NaCl and then incubated in the presence of 100 µM nitrocefin for 5 min at 25°C. Then the suspension was rapidly filtered, and the absorbance of the hydrolysis product was measured at 482 nm.
A colony of Candida albicans (ATCC 10231) was inoculated into 30 ml of YPD medium containing 2% dextrose and growth at 28°C for 20 h. To detect chitin, 4.8 ml of this culture (DO600 nm= 0.5) was centrifuged at 8000 rpm. The pellet washed twice with 50 mM sodium phosphate buffer (pH 7.5) and then resuspended in 500 µl of phosphate buffer containing 5 pmol of BlaP or BlaPChBD. The reaction mixture was incubated for 2 h at 4°C under continuous mixing. The cell suspensions were centrifuged at 8000 rpm during 10 min to separate the supernatant containing unbound protein from the pellet containing chitin with the captured protein. The pellets were washed three times with 50 mM sodium phosphate buffer, 0.2% Tween and then incubated in the presence of 100 µM nitrocefin for 35 min at 25°C. Then the suspension was rapidly filtered, and the absorbance of the hydrolysis product was measured at 482 nm.
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Results |
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PNY01 is an E. coli plasmid that allows a high constitutive periplasmic expression of the engineered class A BlaP/SmaI β-lactamase (Vandevenne et al., 2007
). The gene encoding BlaP/SmaI was obtained by insertion of two codons (CCC-GGG), corresponding to a SmaI restriction site and coding for the dipepetide Pro-Gly, between residues Asp211 and Lys212 of the BlaP β-lactamase. This plasmid was further modified by insertion into the SmaI restriction site of a nucleotidic sequence encoding two adjacent thrombin-cleavage sites characterised each by the consensus peptidic sequence ‘Leu-Val-Pro-Arg-Gly-Ser’ to give pNY02 (Fig. 1A). PNY02 leads to the production of the BlaP Thb/SmaI/Thb β-lactamase (Fig. 1B). In this construction, the thrombin-cleaveage sites are spaced by the Pro-Gly dipeptide, allowing a re-introduction of a SmaI restriction site. This site was subsequently used to clone the gene fragment encoding the CHBD of the human macrophage chitotriosidase Chit1 (residues Pro398 to Asn466) to give pNY03 (Fig. 1A). This plasmid allows the production of the BlaP Thb/CHBD/Thb β-lactamase. In these experiments, all the bacteria transformed with pNY01, pNY02 or pNY03 were selected and grown in the presence of 10 µg/ml ampicillin. In this case, the growth of cells results from the production in the periplasmic space of a correctly folded, functional and soluble β-lactamase. The BlaP Thb/SmaI/Thb and BlaP Thr/CHBD/Thr β-lactamases were overproduced in E. coli and the periplasmic extracts from overnight cultures were loaded on a HisTrapTM Chelating HP column. Thanks to the presence of a polyhistidine tag at the C-terminal end of BlaP, the two proteins were purified to homogeneity (Fig. 1C). After purification, 40 mg of BlaP Thb/SmaI/Thb and 18 mg of BlaP Thb/CHBD/Thb were obtained per litre of culture. In comparison, no cytoplasmic and periplasmic productions were obtained when we tried to express the isolated CHBD with the pET expression systems (pET 26b and pET 28a) in E. coli BL21 DE3 derivatives strains [BL21 (DE3) 69 450-3; BL21 (DE3) pLysS 69 451-3; OrigamiTM 2 (DE3) 71 345-3; RosettaTM (DE3) 70 954-3] at different temperatures (18, 28 and 37°C). In previous studies, CHBD was poorly expressed in E. coli as a fusion protein with the GST (Ujita et al., 2003).
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It has to be noticed that in our previous report (Vandevenne et al., 2007
), we pointed out a difference (183 Da) between the theoretical and experimental molecular weights of the chimeric protein. The nucleotide sequence was verified and we did not find any mutation in the sequence encoding the hybrid protein. To explain the observed difference we also controlled the sequence of the seven-histidine tag in which we found a substitution of the first histidine to a proline and a deletion of the second one resulting only on a five-histidine tag. But fortunately, this probable recombination event did not perturb the purification process.
As shown by Table I, no free thiol was detected with BlaP Thb/CHBD/Thb by using Ellman’s reagent in denaturing condition (6 M urea). This suggests that the six CHBD cysteines of BlaP Thb/CHBD/Thb are involved in the formation of three disulphide bounds. The same result was obtained with purified CHBD. The literature indicates that these disulphide bounds are of crucial importance for the correct folding and thus the chitin binding activity of CHBD (Tjoelker et al., 2000). The SDS–PAGE presented in Fig. 2 are in agreement with these results because the chitin binding activity of BlaP Thb/CHBD/Thb is completely inactivated in the presence of the reducing agent DTT. BlaP Thb/SmaI/Thb was used as a negative control to demonstrate the specificity of the interaction between BlaP Thb/CHBD/Thb and chitin.
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The Km and kcat values of the two purified hybrid β-lactamases were compared with those of the native and BlaP/SmaI enzymes (Table II). In regard to BlaP WT, it can be concluded that the main impacts of the loop engineering are 8- and 9-fold decreases of the kcat for BlaP Thb/CHBD/Thb and BlaP Thb/SmaI/Thb, respectively. But these effects can be considered as minor since they do not indicate significant structural perturbations at the level of the BlaP active site. This suggests also that the β-lactamase is correctly folded and that the CHBD inserted polypeptide is well displayed at the surface of the carrier protein. We have observed that the thrombin cleavage of BlaP Thb/SmaI/Thb (Fig. 1C) does not yield an inactive β-lactamase. This observation is in agreement with the data reported by Wehrman et al. (Wehrman et al., 2002
). The comparison of the kinetic parameters between the cleaved and non-cleaved forms of BlaP Thb/SmaI/Thb (Table II) shows no significant change in the catalytic efficiency (kcat/Km) suggesting that the N-terminal and C-terminal fragments of BlaP stay in close interaction even after cleavage and that this complex is stabilised by strong non-covalent interactions.
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To explain this feature, we have analysed the crystallographic three-dimensional structure of BlaP WT [PDB entry ID 4BLM (Moews et al., 1990
)], and tried to identify the interactions which bind the N-terminal to the C-terminal parts of the protein. The Fig. 3A shows the structures of BlaP wt and its two related fragments when the protein is cleaved by thrombin. The side chains of the residues involved in the enzymatic catalysis (Ser70, Lys73, Ser130, Glu166, Asn 170 and Lys234) have been also represented. Except for Lys234, they are all included in the N-terminal fragment of BlaP. According to the literature, Lys234 could be involved in the stabilisation of β-lactam in the catalytic site during its hydrolysis (Jacob et al., 1990). Moreover, it appears that the C-terminal fragment of BlaP also participates in the formation of the cavity harbouring the catalytic site. If we refer to the structural organisation of class A β-lactamases, the C-terminal fragment of BlaP participates in the formation of the /β domain of BlaP (Fig. 3B). This domain is composed of an anti-parallel β-sheet and two anti-parallel -helices coming from the N- and C-extremities of BlaP. The C-terminal fragment of BlaP bears three β-strands of the β-sheet and one helix of the two anti-parallel -helices. Figure 3C highlights two features involving the apolar side chains of the residues that compose the /β domain. The first is that hydrophobic interactions appear to stabilised the anti-parallel -helices. The second is that the two anti-parallel -helices are bound to the β-sheet by a hydrophobic interface. The hydrogen bonds in the /β domain and the β-sheet alone are shown in Fig. 3D and E, respectively. No hydrogen bond is established between the two anti-parallel -helices and between the anti-parallel -helices and the β-sheet. As expected, the five β-strands of the β-sheet establish an important number of stabilising hydrogen bonds. As mentioned early, three of the five β-strands come from the C-terminal fragment of BlaP. Thus, it is proposed that the two driving forces that stabilise the protein complex after thrombin cleavage are due to both hydrogen bonds and hydrophobic interactions.
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The mutual affinity of the N-terminal and C-terminal fragments of BlaP after thrombin cleavage and the presence of a polyhistidine tag at the C-terminal end of BlaP constitute an ideal opportunity to prepare pure CHBD polypeptides. A two-step procedure has thus been validated (Fig. 4A) consisting in a cleavage of BlaP Thb/CHBD/Thb with thrombin in solution and followed by a depletion of the sample of BlaP fragments by loading on a HisTrapTM Chelating HP column. This step can be repeated until the desired level of purity is obtained (Fig. 4B). Mass spectrometry analysis of the depleted sample (Fig. 5) demonstrates the efficacy of the system. Only CHBD is detected as monomeric and aggregated forms. The monomeric form displays a molecular mass of 9072 Da which corresponds exactly to the expected mass after formation of the three disulphide bridges. The presence of the aggregates is in agreement with the fact that solubility problems arise upon concentration of CHBD domain when devoid of its carrier. In comparison, purified CHBD could not concentrated >0.25 mg/ml against >20 mg/ml of BlaP Thb/ChBD/Thb (corresponding to 4 mg/ml of ChBD). This shows that the β-lactamase carrier can increase the solubility of protein fragments. The chitin binding assay presented on Fig. 2 shows that the purified CHBD is still active. We also determined the relative equilibrium association constant (Kr) of CHBD to insoluble chitin, the calculated value is 8.5 ± 1 l g–1 which is similar to that calculated for the hybrid protein BlaPChBD namely 5.4 ± 0.5 l g–1.
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Previously, we used the β-lactamase activity carried by the hybrid protein BlaP CHBD to calculate the relative equilibrium association constant (Kr) of CHBD to insoluble chitin and to quantify the amount of bound protein by measuring the free and immobilised β-lactamase activities. These β-lactamase assays were performed with nitrocefin in a colorimetric assay. In this work, we took advantage of this feature to demonstrate that the BHP offers the opportunity to screen therapeutic molecules. To validate this system, we immunised a rabbit with the purified CHBD polypeptide described above. The sera were collected after four injections. No seroneutralisation of the β-lactamase activity was observed when BlaP Thb/CHBD/Thb was preincubated with rabbit polyclonal preimmune and anti-CHBD sera. The western blot presented on Fig. 6 indicates that anti-CHBD antibodies have been induced after immunisation with the purified CHBD and that these antibodies recognise free CHBD, BlaP Thb/CHBD/Thb and the native human macrophage chitotriosidase. Figure 7 shows a seroneutralisation titration curve of the chitin binding activity of BlaP Thr/CHBD/Thr. The curve was obtained by plotting the percentage of chitin-immobilised β-lactamase activity versus the dilution of the tested sera. One hundred percent corresponds to the chitin-immobilised β-lactamase activity in the absence of the serum. These data show that immunisation with purified CHBD has induced anti-CHBD antibodies which can prevent the interaction of CHBD with chitin.
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Another application of our hybrid protein system is the possibility to transpose it to phage display technology. The hybrid β-lactamase BlaPChBD can be successfully expressed at the surface of phages so that phage particles retain a β-lactamase activity which is used to monitor phage/ligand interactions. Figure 8 illustrates the β-lactamase activity immobilised on chitin powder and shows that this interaction is specific.
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Finally, Fig. 9 shows the interaction of purified BlaPChBD with a pathogenic fungi such as C. albicans. The monitoring of the immobilised β-lactamase activity on this micro-organism demonstrates that our hybrid protein is able to interact with chitin present in fungal cell wall and thus can be used for diagnosis fungal infections.
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During the last decade, the class A β-lactamases have emerged as promising reporter enzymes to study protein interactions. These proteins are attractive enzymes because they are monomeric and of small size (Philippon et al., 1998
). Their three-dimensional structures are well-defined and they are classified as very efficient enzymes (Matagne et al., 1999). β-lactamase activity is also highly specific with no equivalent in eukaryotic cells and production of β-lactamase by these cells does not result in toxic effects (Matagne et al., 1998; Philippon et al., 1998; Galarneau et al., 2002). These proteins have been expressed successfully in prokaryotic and eukaryotic cells allowing their utilisations as well for in vitro as in vivo applications (Galarneau et al., 2002). A massive expression of these enzymes can be obtained with prokaryotic expression systems allowing to drastically reduce their production times and costs (Frate et al., 2000). A large variety of β-lactam substrates has been developed including chromogenic β-lactams for immunoassay, fluorescein membrane-permeant β-lactams which allow cell imagery, flow cytometry and gene expression assay, and non-toxic β-lactam prodrugs for antibody-directed enzyme prodrug therapy (Bebrone et al., 2001; Vrudhula et al., 2003; Lee et al., 2005; Naqvi and Singh, 2007). For this latter application, a mutated β-lactamase with reduced immunogenicity has been developed (Harding et al., 2005).
Many applications have been reported in the literature. For example, genetic insertion of small peptides into loops bordering the active site of β-lactamase was also used to create allosteric regulatable enzyme for homogeneous immunoassays (Legendre et al., 1999). A common permissive-insertion site in class A β-lactamase has been used to insert random gene fragments. When combined with phage display, this modified enzyme has been used for epitope mapping (Chevigné et al., 2007), immunoassay development and vaccine development (Ruth et al., 2005). Assays based on the complementation of β-lactamase fragments fused to interacting polypeptides that regenerate enzymatic activity were used to monitor inducible protein–protein interactions in eukaryotic cells (Galarneau et al., 2002; Wehrman et al., 2002; Qureshi, 2007). The hydrolysis of β-lactams generates protons so that the β-lactamases are employed for the development of potentiometric signal transduction in biosensors (article submitted for publication). These examples show that β-lactamases have been successfully utilised in a wide range of applications.
This work contributes to enlarge the application fields of β-lactamase by proposing an original tool that elicits the production of difficult to express protein fragments and facilitates their purifications and studies. In a first step, this approach delivers purified hybrid proteins in which the protein fragments of interest are each displayed into a reporter enzyme, BlaP. Thanks to the presence of the carrier protein, the binding properties of the protein fragments can be studied by using the β-lactamase activity. To allow the study of the properties of the insert independently of the presence of the carrier, two specific protease-recognition sites surrounding the insert have been introduced into the hybrid protein. By combining specific proteolytic cleavage and affinity chromatography, a purified insert was isolated. No residual trace of the carrier was detected by mass spectrometry after purification. We have demonstrated that the purified insert deleted of its carrier protein retained its biological properties. Moreover we showed that the use of the β-lactamase carrier system can be successfully used to enhance peptide expression in E. coli but also to increase protein solubility during sample concentration.
In a second step, we have showed that the hybrid β-lactamase is an efficient tool in the screening of molecules which can neutralise the biological properties of the studied protein fragment. In this work, the purified insert was used to immunise a rabbit and the collected polyclonal serum was shown to inhibit the binding properties of the insert by monitoring the binding of the hybrid protein to its specific ligand. This was done by measuring the immobilised β-lactamase activity. This approach can be extended to drug discovery of molecule enable to prevent or to elicit specific interactions.
We also showed that this system can be used to detect fungal infections and also easily transposed to phage display technology. This approach can be applied to the screening of enzyme-coupled protein binding domains which can also be overexpressed as recombinant proteins and directly implemented in immunoassays. This method is advantageous for affinity maturation of protein domains because the β-lactamase activity of phage particles allows the monitoring of the affinity maturation during the enrichments steps or the comparison of affinities of individual mutants without the necessity to produce a purified recombinant protein.
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This work was supported by funding from the Walloon region (grant initiative no. 215123) and the FRS-FNRS (FRFC grants 2.4511.06 and 2.4561.07). M.V. is a recipient of a FRS-FNRS (Brussels, Belgium) doctoral fellowship. G.G. beneficiates from a grant from the Walloon region (project reseau I no. 415701). P.F. (project initiative no. 215123) is post-doctoral fellows from the Walloon region.
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Edited by Bauke Dijkstra
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References |
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Received December 19, 2007; revised April 2, 2008; accepted April 2, 2008.
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