INTRODUCTION

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-Lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobial agents. Because of their widespread use, bacterial resistance to these antibiotics has become an increasing problem (7). The most common mechanism of resistance is the production of -lactamases. -Lactamases are secreted to the periplasm of gram-negative bacteria, or extracellularly in gram-positive bacteria, where they hydrolyze -lactam antibiotics to create ineffective antimicrobials. There are a large number of -lactamases which are found to be encoded either on plasmids or on the bacterial chromosome (3). In gram-negative bacteria, the most common plasmid-mediated -lactamase is the TEM-1 -lactamase (30). This enzyme efficiently hydrolyzes penicillins and many cephalosporins and is therefore a widespread source of -lactam resistance (18).

An effective means of combating TEM-1 -lactamase-mediated resistance has been the clinical use of small-molecule -lactamase inhibitors such as sulbactam and clavulanic acid (21). These molecules do not possess significant antimicrobial activity themselves but are used in conjunction with other -lactam antibiotics, such as ampicillin. The inhibitor protects the antibiotic from the action of -lactamase and thereby restores the therapeutic value of the antibiotic. However, in recent years, there have been many reports of resistance to -lactam--lactamase inhibitor combinations (12). This resistance is due to mutations in -lactamase that enable the enzyme to avoid inactivation by the inhibitor while retaining the ability to hydrolyze -lactam antibiotics (15).

The small-molecule inhibitor clavulanic acid is a natural product from Streptomyces clavuligerus (19). In addition to clavulanic acid, S. clavuligerus also produces a protein inhibitor of -lactamase, called -lactamase inhibitory protein (BLIP) (8). BLIP is a 165-amino-acid protein encoded by the bli gene that binds and inhibits TEM-1 -lactamase, with a reported K_i of 0.6 nM (28). BLIP also binds to other -lactamases from both gram-negative and gram-positive bacteria, albeit with reduced affinity. In addition, BLIP has been reported to inhibit the Enterococcus faecalis PBP 5 with a K_i of 12 µM (28). The X-ray structure of BLIP has been solved both alone and in complex with TEM-1 -lactamase and has revealed the residues making up the binding surface of BLIP (27, 28). Using this information, it may be possible to use site-directed mutagenesis techniques as a means of increasing BLIP binding and inhibition of -lactamases and penicillin-binding proteins. If BLIP can be exploited as a molecular scaffolding to engineer binding interactions, it may be possible to create new BLIP-based antibiotics and inhibitors.

Phage display has proven to be an effective methodology for the selection of binding partners of high affinity or altered specificity (26). Monovalent display of a protein of interest can often be achieved by fusing the gene encoding the protein to the N terminus of the M13 gene III coat protein (17). High-affinity variants can then be obtained by creating phage libraries containing mutants of the protein of interest and sequencing the corresponding DNA packaged in the phagemid particles after several rounds of binding selection (20). Here we describe the cloning of the BLIP gene into a new phagemid vector. The vector encodes chloramphenicol resistance and contains an amber codon at the 5' end of gene III. BLIP is fused at its N terminus to the -lactamase signal sequence and at its C terminus to the protein encoded by gene III of the phage (g3p). Transcription of the fusion is controlled by the constitutive -lactamase promoter (4). Specific binding of phage containing the BLIP-g3p fusion to immobilized -lactamase is demonstrated to occur with nanomolar affinity. This system can now be used to engineer new BLIP binding specificities.

	MATERIALS AND METHODS

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Bacterial strains. Escherichia coli XL1-Blue (2) [F'::Tn10 proA⁺B⁺ lacI^q (lacZ)M15/recA1 endA1 gyr96(Nal^r) thi hsdR17 (r m⁺) supE44 relA1 lac] was used for transformations of ligations, and E. coli TG1 (10) [F' traD36⁺ lacI^q (lacZ)M15 proA⁺ B⁺/supE (hsdM mcrB) (r m McrB) thi (lac proAB) was used for production, amplification, and determination of the titer of bacteriophage.

PCR and cloning. The phagemid encoding the BLIP-g3p fusion was constructed by inserting a 1,365-bp XbaI-BamHI fragment containing gene III from phagemid phGHamg3 (16) into XbaI-BamHI-digested pBCKS+ (Stratagene) to create plasmid pG3-CMP. The pG3-CMP plasmid was digested with SalI, the 5' overhangs were filled-in with Klenow polymerase and deoxynucleoside triphosphates, and the ends were religated to destroy the SalI site and create plasmid pG3-C2 (Fig. 1). A construct containing the coding sequence of TEM-1 -lactamase fused to gene III was then created by PCR amplification of the bla_TEM-1 gene from plasmid pBG66-N78 (14). This plasmid was constructed previously and contains a bla_TEM-1 gene with a SalI site inserted at nucleotide position 284 of the published sequence (29), which is the codon for the third amino acid position beyond the cleavage site of the -lactamase signal sequence (14). Therefore, the SalI site can be used to fuse other genes behind the promoter and signal sequence of -lactamase. The bla_TEM-1 gene was amplified with the following primers: PD-bla1, 5'-CGGGGAGCTCGTTTCTTAGACGTCAGGTGGC-3'; and PD-bla2, 5'-CCCCGTCTAGACCCAATGCTTAATCAGTGAG-3'. Enzyme restriction sites are underlined. The primer PD-bla1 contains a SacI site; the primer PD-bla2 contains an XbaI site. The PCR was performed with the Advantage cDNA PCR kit from Clontech, Inc., using the buffer conditions recommended by the manufacturer. The reaction mixtures were cycled 30 times at 94°C for 1 min, followed by 64°C for 4 min. After cycle 30, the reaction mixtures were incubated at 64°C for 10 min. The PCR product was digested with SacI and XbaI and inserted into SacI-XbaI-digested pG3-C2 plasmid to create pG3-C3. The SacI-XbaI fragment of pG3-C3 contains approximately 150 bp of sequence upstream of the bla_TEM-1 gene and therefore contains the constitutive -lactamase promoter. The XbaI site is the point of fusion between -lactamase and gene III. There is an amber codon at the fusion site, and so the fusion protein will only be made in an amber suppressor strain of E. coli.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Maps of the plasmids used in this study. Restriction endonuclease sites used for the construction of the vectors (Materials and Methods) are also shown.

Phage preparation and panning. After overnight growth, E. coli cells were removed by centrifugation, and the phage were precipitated from the supernatant with a 1/5 volume of 20% polyethylene glycol-2.5 M NaCl. The phage were pelleted by centrifugation and resuspended in 1/100 of the original culture volume of STE (0.1 M NaCl, 10 mM Tris-Cl [pH 8.0], 1 mM EDTA [pH 8.0]). The phage titer was determined by making serial dilutions of a 0.1-ml total volume and adding 0.2 ml of E. coli TG1 cells. Aliquots of 0.15 ml were plated on Luria-Bertani agar supplemented with 12.5 µg of chloramphenicol per ml. After overnight growth at 37°C, the number of colonies was determined and the titer was calculated.

Phage ELISA. A two-step phage enzyme-linked immunosorbent assay (ELISA) (6) was performed to measure BLIP phage affinity for TEM-1 -lactamase. Microtiter plates (Nunc; Maxisorp, 96 wells each) were coated with purified TEM-1 -lactamase (at 10 µg/ml) in 50 mM sodium carbonate (pH 9.6) at 4°C overnight. The plates were then blocked with SuperBlock (Pierce) for 2 h at room temperature. Serial dilutions of the BLIP phage stock were added to the wells and incubated for 2 h at room temperature in buffer A at a final volume of 0.15 ml. The plates were then washed several times with buffer A, and the bound phage were probed with a sheep anti-M13 polyclonal antibody conjugated to horseradish peroxidase (HRP). To determine phage affinity, serial dilutions of -lactamase and a subsaturating concentration of 1.3 × 10¹¹ BLIP phage were added to wells in 0.1 ml of buffer A. After 2 h at room temperature, the wells were washed multiple times with buffer A, and bound phage were probed as described above. Affinities (IC₅₀) were calculated as the concentration of competing -lactamase that resulted in half-maximal BLIP phage binding. The half-maximal concentration was calculated by converting the data in Fig. 3 from log to linear values and fitting the binding curve to the equation for a hyperbola.

	RESULTS

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Construction of a BLIP phage display vector. A new phagemid vector, pG3-C3, was constructed for the display of BLIP (Fig. 1). The pG3-C3 phagemid encodes chloramphenicol resistance and is designed to express heterologous proteins as fusions at their N terminus to the signal sequence of TEM-1 -lactamase. In addition, the heterologous proteins are fused at their C terminus to the g3p of M13 for display on the surface of the bacteriophage. The fusion protein is expressed under the control of the constitutive -lactamase promoter. Because there is an amber codon at the fusion junction between the heterologous protein and g3p, it is necessary to express the fusion in an amber suppressor-containing strain of E. coli. The pG3-C3 plasmid was constructed by insertion of a SacI-XbaI DNA fragment containing the promoter and gene encoding TEM-1 -lactamase into the pG3-C3 plasmid, as shown in Fig. 1. The bla_TEM-1 gene that was inserted was previously engineered to contain a SalI linker insertion at the codon for the third amino acid position beyond the cleavage site of the -lactamase signal sequence (14). The SalI linker provides a site to fuse a heterologous gene encoding a protein that will be secreted under the control of the -lactamase signal sequence. pG3-BLIP was constructed by PCR amplification of the bli gene by using primers containing XhoI and XbaI sites. The bli fragment was then inserted into the SalI-XbaI-digested pG3-C3 vector (Fig. 1). The resulting phagemid is designed to express the BLIP-g3p fusion, which, after cleavage of the -lactamase signal sequence, contains the entire amino acid sequence of the mature form of BLIP with an additional seven N-terminal amino acids (Fig. 2). DNA sequence analysis of the entire BLIP-encoding fragment as well as the fusion junctions with the -lactamase signal and gene III was performed with clones that were shown to contain the BLIP insertion by DNA restriction enzyme analysis. All four of the clones sequenced were found to contain missense or frameshift mutations and therefore were not suitable for further testing.

View larger version (6K): [in this window] [in a new window]   FIG. 2.   Amino acid sequence of junctions between the -lactamase signal sequence (bla signal) and the N terminus of BLIP and the C terminus of BLIP and the N terminus of g3p. The site of the cleavage site for signal peptidase is shown. The glutamate residue three positions downstream from the signal cleavage site is the last amino acid encoded by the blaTEM-1 gene. It is followed by four-amino-acid segment encoded by the SalI linker DNA that was inserted in the blaTEM-1 gene. The alanine marked as "start BLIP" is the first amino acid in the construct encoded by the bli gene. The amber codon at the junction of BLIP-g3p is shown as a glutamine, which is incorporated in the amber suppressor strain used in this study. The BLIP protein is fused to amino acid position 267 of g3p.

Phage ELISA to demonstrate -lactamase-specific binding. A two-step phage ELISA method was used to demonstrate that BLIP is functionally displayed on the surface of the bacteriophage and that the phage bind specifically to -lactamase. For this purpose, ELISA plates were coated with purified TEM-1 -lactamase, and serial dilutions of phage displaying wild-type BLIP were allowed to bind to the immobilized protein in the presence of a large excess of BSA. After washing, bound phage were stained with HRP-conjugated anti-M13 antibody. The binding curve in Fig. 3A demonstrates that the pG3-BLIP phage bind -lactamase. To quantitate binding, ELISA plates were coated with -lactamase and a constant subsaturating concentration of pG3-BLIP phage was added with serial dilutions of purified -lactamase. As seen in Fig. 3B, the pG3-BLIP phage were competed off of the immobilized -lactamase with soluble -lactamase at an IC₅₀ of 1 nM. This affinity compares favorably to the published K_i value of 0.6 nM for the BLIP--lactamase interaction (28). These results show that BLIP is expressed on the surface of the bacteriophage in a form that binds tightly and specifically to -lactamase.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Two-step phage ELISA results. (A) Binding curve to determine the subsaturation concentration of phage to use in step 2. A total of 1.3 × 1011 phage were used in step 2. (B) Plot showing ELISA measurements of pG3-BLIP phage binding to immobilized -lactamase. The IC50 given shows the concentration of competing -lactamase that results in half-maximal binding to the phagemid.

Specific enrichment of BLIP phage by panning on -lactamase. In order to use the pG3-BLIP phagemid to engineer BLIP for altered binding properties, it is necessary to be able to select binding phage by panning on an immobilized substrate. This was tested by attaching purified -lactamase to oxirane-acrylic beads and incubating the beads with 10¹¹ phage from the pG3-BLIP phage stock in the presence of a large excess of BSA. Two control experiments were performed to demonstrate that phage binding to the beads was dependent on the BLIP--lactamase interaction. First, an internal control experiment was performed by adding 10¹¹ phage that did not display BLIP along with 10¹¹ pG3-BLIP phage to the oxirane beads conjugated to -lactamase. The phagemid pG3-SPT, which produced the nondisplaying phage, was constructed by inserting a gene cassette encoding spectinomycin resistance (23) into the chloramphenicol resistance gene of pG3-C2 (Fig. 1). The advantage of this system is that the extent of enrichment of nondisplaying phage versus BLIP-displaying phage can be determined simply by determining the titer of the phage recovered from the oxirane beads on spectinomycin-containing agar plates as well as chloramphenicol-containing agar plates. The results in Table 1 illustrate that 27-fold more pG3-BLIP phage were recovered from the -lactamase beads than nondisplaying phage when no soluble -lactamase was added to complete for binding to the pG3-BLIP phage (compare pG3-BLIP versus pG3-SPT in the column with 0 µM -lactamase added). As increasing amounts of soluble -lactamase were added, the enrichment of pG3-BLIP phage over nondisplaying phage decreased to threefold when 10 µM soluble -lactamase was added. Therefore, soluble -lactamase is able to compete off the binding of pG3-BLIP phage to the -lactamase-coated oxirane beads. These data show that BLIP phage can be specifically enriched by a round of panning against immobilized -lactamase.

View this table: [in this window] [in a new window]   TABLE 1.   Phage bound to -lactamase- and BSA-coated beads

	DISCUSSION

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Phage display is a powerful method for engineering the binding affinity and specificity of many different molecules, including antibody fragments, enzymes, hormones, protease inhibitors, and DNA binding proteins (reviewed in references 5 and 20). The ability to use phage display to alter the binding properties of a protein depends on whether a polypeptide can be expressed and displayed on the surface of the bacteriophage in a folded, functional form. Here we have displayed BLIP as a fusion to g3p and have shown that bacteriophage containing the fusion bind specifically and with high affinity to -lactamase.

Display of functional BLIP was accomplished by constructing a new phage display vector that allowed BLIP to be expressed under the control of the constitutive TEM-1 -lactamase promoter and secreted under the direction of the -lactamase signal sequence. Many phage display vectors use the lac promoter for expression of the heterologous protein-g3p fusion (1, 13). Exposure of E. coli to IPTG can induce the lac promoter to high levels of transcription. However, in the absence of IPTG, there is still significant transcription because the bacterial cell usually does not contain enough lac repressor to bind all of the lac promoters from a multicopy plasmid (9). For phage production, IPTG is not used because of toxicity of the g3p fusion proteins and the resultant low phage titer (13). The -lactamase promoter is weak and is not inducible (4). The low levels of transcription of the BLIP-g3p fusion are not toxic to E. coli, and titers of 10¹² to 10¹³ phage/ml were routinely obtained in the experiments reported here.

Because of the high frequency of frameshift mutations in the BLIP gene found among transformants after insertion of the BLIP gene into the pG3-C3 vector, it was necessary to obtain a nonmutant sequence by functional selection for BLIP phage that bound to immobilized -lactamase. It is unclear why such a high frequency of clones contained frameshift mutations in BLIP in the nonselected population. Presumably it is due to a high frequency of polymerase errors during PCR. A possible reason for this is the very high G-C content of the BLIP gene. The fragment of BLIP cloned into the pG3-C3 vector is 69% G-C. G-C DNA templates are often difficult to amplify by PCR (11). The difficulty may be due to the higher melting temperature of G-C-rich template DNA or to extensive secondary structure in the template (24). The finding of a high frequency of frameshift mutations in the amplified BLIP DNA may indicate that PCR of G-C-rich DNA has reduced fidelity.

The development of the BLIP phage system will now be exploited to determine which residues on BLIP are critical for TEM-1 -lactamase binding and to select for variants that bind tightly to other -lactamases or penicillin-binding proteins. To achieve this, libraries of random mutants of BLIP will be created in the pG3-BLIP vector. The phage libraries will then be panned on purified TEM-1 -lactamases and other -lactamases as described here.