1 Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland and 2 Dipartimento di Biologia Molecolare, Sez. di Chimica Biologica, Università di Siena, Via Fiorentina 1, 53100 Siena, Italy
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Abstract |
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Keywords: biotin ligase/Bir A/calmodulin/phage display/trypsin
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Introduction |
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A number of selection strategies have been proposed, which aim at anchoring the reaction product on the surface of catalytically active phageenzyme, followed by the physical isolation of the desired phageenzymes by means of anti-product affinity reagents (Pedersen et al., 1998; Atwell and Wells, 1999
; Demartis et al., 1999
; Jestin et al., 1999
). One of these strategies, recently described by our laboratory, features the stable (but non-covalent) anchoring of reaction substrates on calmodulin-tagged phageenzymes by means of calmodulin-binding peptide derivatives (Demartis et al., 1999
). In our selection scheme, the phageenzyme catalyses the intramolecular conversion of substrate into product, which cannot dissociate from the phage, since it is anchored to the minor coat protein pIII by means of the stable calmodulin/peptide complex (Kd = 2 pM). Phage displaying the product of reaction can be physically isolated from non-catalytically active phage using anti-product affinity reagents. The calmodulinpeptide complex can be dissociated in a number of ways, e.g. by addition of calcium chelators, liberating the phageenzymes which can be amplified by bacterial infection.
The selection scheme of Demartis et al. (Demartis et al., 1999) was tested in a number of model selection experiments, including those with a calmodulin-tagged subtilisin mutant displayed on phage by means of a phagemid vector, which showed enrichment factors up to 54-fold, relative to negative controls. The paper demonstrated that, among other parameters, good levels of functional enzyme display on phage were essential in order to achieve selection for catalysis. In this work, we aimed at characterizing and improving the parameters which determine the efficiency of selection for catalysis by phage display. We chose a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57
Ala mutant) as representative model enzymes. BirA is a biotin ligase which catalyses the formation of biotinyl-5'-adenylate from biotin and ATP followed by a covalent attachment of biotin on to a specific lysine residue of the biotin carboxyl carrier protein (BCCP). The trypsin His57
Ala mutant (H57A) is an endopeptidase, which cleaves polypeptides containing the HisLys or HisArg substrate motif (Corey et al., 1995
) by a mechanism of substrate-assisted catalysis (Carter and Wells, 1987
). While the trypsin H57A mutant bears an exquisite substrate specificity, its turnover number kcat is reduced more than 10 000-fold compared with wild-type trypsin. The two enzymes were displayed on phage as fusion protein with calmodulin and the minor coat protein pIII, using novel phage vectors.
Phage displaying the calmodulin-tagged active enzyme were captured with anti-product reagents according to the selection scheme depicted in Figure 1. For BirA selections, we used the peptide GAAARWKKAFIAVSAANRFKKISTSGGGPGGLVSIFEAQKIEWH as reaction substrate, which contains a sequence capable of high-affinity binding to calmodulin (underlined) fused to a biotinylatable peptidic tag (bold) (Schatz, 1993
). The lysine residue of the peptide moiety LVSIFEAQKIEWH is biotinylated. Selections with trypsin mutants were performed with peptide GGHRDYKDEGGGAAARWKKAFIAVSAANRFKKIS, which contains a cal-modulin binding sequence (underlined), fused to a peptidic appendix (bold), which can be cleaved by the trypsin H57A mutant. Upon cleavage, the resulting calmodulin-binding peptide displays an N-terminal DYKDE sequence (italics), which is bound specifically by the M1 antibody (Demartis et al., 1999
). The DYKDE sequence is not bound by the M1 antibody when it is located in internal peptide positions (Hopp et al., 1988
; Huber et al., 1999
). We demonstrated in model selection experiments that phageenzymes could be enriched on the basis of their catalytic activity and we investigated the parameters which dictate the efficiency of selection. Finally, a phage library of trypsin mutants was constructed, characterized and selected, with the aim of isolating improved trypsin mutants, which were able to cleave the HisLys or HisArg substrate motifs.
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Materials and methods |
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The E.coli strain TG1 (K12, (lac-pro), supE, thi, hsdD5/F'traD36, proA+B+, lacIq, lacZ
M15) was used as a host for phage production and gene expression. Peptides were synthesized and purified by HPLC as described (Montigiani et al., 1996
). Peptide identity was confirmed by amino acid analysis and by MALDI-TOF mass spectrometry (Protein Service Labor, ETH Zürich).
The fluorogenic substrates benzyloxycarbonyl-Gly-Gly-Arg-4-methyl-7-aminocoumarin and benzyloxycarbonyl-Gly-His-Arg-4-methyl-7-aminocoumarin were purchased from Bachem Bioscience (Bubendorf, Switzerland).
Ampicillin was purchased from Applichem (Darmstadt, Germany) and tetracycline from Sigma (Buchs, Switzerland). All the restriction enzymes and polymerases were purchased from Appligene Oncor (Basel, Switzerland), Axon Lab (Baden, Switzerland) and Boehringer Mannheim (Rotkreuz, Switzerland). T4 DNA ligase was obtained from Eurogentec (Seraing, Belgium). DNA primers were purchased from Eurogentec (Seraing, Belgium) and Microsynth (Balgach, Switzerland).
Construction of vectors
DNA manipulations were performed according to standard protocols (Sambrook et al., 1989).
pCHH1 is a derivative of fd-tet-DOG1 (Clackson et al., 1991) in which the multiple cloning site was altered. An insert containing a unique restriction site SfiI was prepared by polymerase chain reaction (PCR) using the primers Bsrfdba and LeaderSfifo and fd-tet-DOG1 as template. The PCR product was digested with the restriction enzymes BsrGI and SfiI and ligated into fd-tet-DOG1.
pCHH2 was constructed by cloning the genes of rat anionic trypsin II, a 13-amino acid linker and calmodulin sequentially into the vector pCHH1. The cDNA of rat anionic trypsin II was obtained from rat pancreas using a reverse transcriptase (Pharmacia, Dübendorf, Switzerland), followed by PCR amplification using the primers Tyba and Tyfo. The gene coding for the 13-amino acid linker and calmodulin was PCR amplified from the vector pSD4 (Demartis et al., 1999) with the primers Linkba and CaMfoNot. An SfiI restriction site and an overlapping sequence were appended to the trypsin gene in a PCR reaction using the primers SfincobaTsn and Linkfo. The two PCR products were assembled in a PCR reaction with the primers SfincobaTsn and CaMfoNot and ligated into the restriction sites SfiI and NotI of the vector pCHH1.
pCHH3 is a derivative of pCHH1 which harbors the genes of trypsin H57A, a 13-amino acid linker and calmodulin. The vector was cloned with the same strategy as for pCHH2. The mutation H57A was inserted into the trypsin gene as follows. Two gene fragments were PCR amplified with the primer pairs SfincobaTsn/Ty57Afo and Ty57Aba/Linkfo, respectively. The two PCR fragments were assembled and amplified with the primers SfincobaTsn and Linkfo.
pCHH4 is a derivative of pCHH1 in which a dummy DNA fragment of 930 base pairs was inserted into the restriction sites SfiI and NotI.
pSD17 is a phagemid vector based on pSD4 (Demartis et al., 1999). The biotin ligase gene of E.coli was PCR amplified from vector pGEX-BirA (a kind gift from Prof. Petri Saviranta, University of Turku, Finland) (Saviranta et al., 1998
) with the primers BirbaNco1 and BirfoNot and cloned into the NcoI and NotI sites of pSD4.
pSD22 is an fd-tet-DOG1 (Clackson et al., 1991) based vector in which the genes of BirA, a 13-amino acid linker and calmodulin were inserted. The fused genes of BirA, the 13-amino acid linker and calmodulin were PCR amplified from pSD17 with the primers BirbaApa1 and PhVecCamfoXho and subcloned into the ApalI and XhoI sites of fd-tet-DOG1.
pCHH5 is a vector for the expression of hexahistidine-tagged wild-type trypsin. The gene of rat anionic trypsin II was inserted into the SfiI and NotI sites of the expression vector pDN321 which is a derivative of pUC119SNpolymyc (Neri et al., 1996). The gene was PCR amplified from pCHH2 with the primers SfincobaTsn containing the SfiI restriction site and TsnHisGlyfo containing the NotI restriction site and a hexahistidine tag.
pCHH6 was constructed following the same strategy as for the vector pCHH5. It harbors the trypsin mutant H57A instead of the wild-type trypsin. The gene for the mutant trypsin was PCR amplified from pCHH3 with the primers SfincobaTsn and TsnHisGlyfo.
pCHH7 is a vector for the expression of the hexahistidine-tagged trypsin mutant H57K. The mutation H57K was introduced into wild-type trypsin as follows. Two gene fragments were PCR amplified with the primer pairs Sfincobatsn/H57Kfo and TsnHisGlyfo/tsnlib-mut55-57ba, respectively. These two PCR fragments were assembled and amplified with the primers Sfincobatsn and TsnHisGlyfo. The resulting gene was cloned with SfiI/NotI sites into pDN321, a derivative of pUC119SN polymyc (Neri et al., 1996).
DNA primers: BirbaApa1, 5'-GGCGGCCACAGTGCA-CAGGTCCAAGATAACACCGTGCCACTGAAATTG-3';BirbaNco1, 5'-AATCGACCCATGGCCCAGGTCCAGGATAACACCGTGCCACTGAAATTG-3'; BirfoNot, 5'-GAGTCA-TTCTGCGCCGCGTTTTCTGCACTACGCAGGGATAT-TTC-3'; Bspbafd, 5'-CCGTTTAATGGAAACTTCCTCAT-G3'; Bsrfdba, 5'-TACGTTGATTTGGGTAATGAATATC-3'; CaMfonot, 5'-CCGATTGCGGCCGCCTTTGCTGTCATCATTTGTACA-3'; H57Kfo, 5'-CTTGGATGCGGGACTTATAGCATTTAGCTGCAGACACCACCCA-3'; LeaderSfifo, 5'-TGATGCGGCCGCGGCTATGGCCGGCTGGGCCGCATA-GAAAGGAACAACTAAAGGAAT-3' Linkba, 5'-ACCGCC-ACTTCCACCGCCACCAGAACCACCGTTGGCAG-3';Linkfo, 5'-CCACCAGAACCACCGTTGGCAGCAA TTGTGTCCTGA-3'; PhVecCamfoXho, 5'-GGCCCGCTCGAGCTTTGCTGTCATCATTTGTACAAAC-3'; SFINCOBATSN, 5'-ATGCGGCCCAGCCGGCCATGGCCATCGTTGGAGGATA-CACCTGC-3'; TsnHisGlyfo, 5'-CCATTGCGGCCGCTATC-AGTGATGGTGATGGTGATGGCCACCGTTGGCAGCAA-TTGTGTCCTG-3'; Ty57Aba, 5'-GTGTCTGCAGCTGCTTGCTATAAGTCCCGCATCCAAG3'; Ty57Afo, 5'-CTTATAGCAAGCAGCTGCAGACACCACCCACTGG-3'
Phage preparation and characterization
Phages were produced in E.coli TG1 cells at 20°C for 20 h in 2YT medium supplied with tetracycline (10 µg/ml final concentration), isolated and purified using polyethylene glycol (PEG) according to standard procedures (Nissim et al., 1994).
The number of infective phage particles was determined by incubating phage with an excess of exponentially growing E.coli TG1 cells for 30 min at 37°C and plating them immediately. The concentration of physical phage particles was measured by absorption spectroscopy (Day et al., 1978).
The display of protein on phage was analysed by Western blotting. A total of 5x108 infective phage were denatured by heating at 90°C for 5 min in the presence of 20 µl of reducing SDS-sample buffer. The samples were loaded on a 10% (w/v) polyacrylamide gel (Novex, Frankfurt, Germany), separated by electrophoresis and transferred on to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The membrane was blocked with PBS buffer [phosphate-buffered saline; 50 mM phosphate (pH 7.4), 100 mM NaCl] containing 1% (w/v) milk powder and proteins were detected with an anti-pIII murine monoclonal antibody (Tesar et al., 1995) followed by a secondary anti-mouse HRP-goat IgG antibody (Sigma, St. Louis, MO). The peroxidase activity was detected by electrochemiluminescence using an ECL kit from Amersham (Dübendorf, Switzerland).
Calmodulin binding activity was assayed by phage ELISA. Phages were incubated with the biotinylated peptide CAAARWKKAFIAVSAANRFKKIS, washed, blocked and immobilized on streptavidin-coated plates (Boehringer Mannheim, Rotkreuz, Switzerland) as described (Demartis et al., 1999).
Fluorogenic activity assays of wild-type trypsin displayed on phage (pCHH2) were performed in 50 mM TrisHCl pH 8.0, 20 mM CaCl2, 100 mM NaCl at 25°C using Z-Gly-Gly-Arg-AMC (AMC = aminomethylcoumarin) as substrate. Fluorescence measurements were carried out with an excitation wavelength of 381 nm and an emission wavelength of 455 nm. Typically, 50 µl of phage [5x1011 transducing units (t.u.)/ml] were added to 950 µl of buffer containing 50 mM Z-Gly-Gly-Arg-AMC substrate.
Model selection experiments with trypsin H57A
Trypsin H57Acalmodulinphage were blocked in TBSC (50 mM TrisHCl pH 7.4, 100 mM NaCl, 1 mM CaCl2) containing 3% (w/v) BSA for 30 min at room temperature. These phages (1010 t.u.) were incubated with the peptides DYKDEGGGAAARWKKAFIAVSAANRFKKIS, GGHRDYKDEGGGAAARWKKAFIAVSAANRFKKIS, GAAHYDYKDEGGGAAARWKKAFIAVSAANRFKKIS and GDDDDKDYKDEGGGAAARWKKAFIAVSAANRFKKIS in individual reactions. The final concentration of peptide was 1 µM in a 400 µl volume (5 mM CaCl2, 20 mM TrisHCl pH 8, 100 mM NaCl). Reactions were carried out for 30 min at 37°C, then the unbound peptide was removed by precipitating the phage with 100 µl of ice-cold PEG solution [20% (w/v) polyethylene glycol, 2.5 M NaCl) followed by centrifugation at 13 000 r.p.m. for 1 min. The phage pellet was dissolved in 100 µl of TBSC containing 1% (w/v) milk powder and incubated with 1 µg/ml biotinylated M1 anti-FLAG antibody (Kodak, New Haven, CT) for 20 min at room temperature. A 50 µl volume of pre-blocked streptavidin-coated M280 Dynabeads (10 mg beads/ml; Dynal, Oslo, Norway) were added and mixed for 20 min. The magnetic beads were captured on a magnet (Dynal) and washed seven times with TBSCTween-20 (0.1%) and four times with TBSC. Phages were eluted in 300 µl of TBS (50 mM TrisHCl pH 7.4, 100 mM NaCl) containing EDTA (10 mM), saturated with calcium chloride and used to infect exponentially growing TG1 cells (OD = 0.4). The cells were plated on 2YT agar plates containing tetracycline (10 µg/ml).
Model selection experiments with BirA
BirAcalmodulinphage (pSD22, 5x108 t.u.) pre-blocked in TBSCBSA (3%, w/v) were incubated with the peptide GAAARWKKAFIAVSAANRFKKISTSGGGPGGLVSIFE-AQKIEWH (10-7 M), 0.1 M biotin, 10 mM ATP, 12 mM MgCl2, 1 mg/ml inorganic pyrophosphatase (Sigma) and 0.06% Tween-20 in a total volume of 200 µl of TBSC. In negative control reactions either biotin or peptide was omitted, and in positive control reactions the phage were incubated with the biotinylated CAAARWKKAFIAVSAANRFKKIS peptide (Demartis et al., 1999). All individual reactions were incubated for 20 min at room temperature. Phage were then PEG precipitated twice (as described above) and resuspended in 200 µl of TBSC containing milk powder (3%, w/v) and BSA (1 mg/ml). Phage were captured, washed and eluted from streptavidin-coated beads as described (Demartis et al., 1999
).
In competition experiments, phage of two different species were mixed and captured as described above. After selective elution, phage was used to infect E.coli bacteria. The primers LMB3 (5'-CAGGAAACAGCTATGAC-3') and fdseq (5'-GAATTTTCTGTATGAGG-3') were used for PCR screening of individual colonies.
Construction of the trypsin library
The trypsin library was constructed using partially degenerate primers in a PCR-based method, which introduced random mutations at positions 55, 57, 102 and 214 of rat anionic trypsin II. Primers containing the codon 5'-NNK-3' were used. They allow the integration of all 20 amino acids and only one stop codon. The stop codon UAG is suppressed in E-suppressor strains, such as TG1. For residue 57, the codon 5'-DNK-3' was used to knock out histidine in this position. This codon also prevented the amino acids Pro and Gln at this position. Four overlapping PCR fragments were produced with the primer pairs Bspbafd/Tsnlin-mut5557fo, Tsnlib-mut5557ba/Tsnlib-mut102fo, Tsnlib-mut102ba/Tsnlib-mut214fo and Tsnlib-mut214ba/fdseq1 (15 s at 94°C, 45 s at 56°C, 1 min at 72°C for 10 cycles and 15 s at 94°C, 1 min at 58°C, 2 min at 72°C for 15 cycles). Gel-purified PCR fragments were assembled in a single reaction with the primers Bspbafd and fdseq1, double-digested with SfiI/NotI and ligated into 5 µg of SfiI/NotI-digested pCHH4 phage vector. A 930 bp dummy fragment between the restriction sites SfiI and NotI of pCHH4 allowed a good separation of doubly digested vector from an agarose gel (0.8%, w/v), from which the DNA was recovered with QIAEX II gel extraction kit (Qiagen, Hilden, Germany). The ligation product was purified in a phenol extraction followed by ethanolsodium acetate precipitation and washed twice with 70% ethanol. The vector was transformed into electrocompetent TG1 E.coli cells, which were then plated on agar plates supplemented with 10 µg/ml tetracycline at 30°C overnight. The colonies were pooled by washing the large plates (20 cm diameter) with 7.5 ml of 2YT + 2.5 ml of glycerol. Cells were frozen in liquid nitrogen and stored at -80°C. Phage were produced and purified as described above.
DNA primers: Bspbafd, 5'-CCGTTTAATGGAAACTTCCTCATG-3'; fdseq1, 5'-GAATTTTCTGTATGAGG-3'; Tsnlib-mut102ba, 5'-ATCATGCTGATCAAGCTCTCTTC-3'; Tsnlib-mut102fo, 5'-GAAGAGAGCTTGATCAGCATGATMNNATTGTTCAGGGTCTTCCTATCG-3'; Tsnlib-mut214ba, 5'-TGGGGCTATGGCTGTGCCCTGC-3'; Tsnlib-mut214fo, 5'-GCAGGGCACAGCCATAGCCCCAMNNGACAATGCCC-TGCAGCTCTCC-3'; Tsnlib-mut55-57ba, 5'-TGCTATAAGTCCCGCATCCAAG-3'; Tsnlib-mut55-57fo, 5'-TTGGATGC-GGGACTTATAGCAMNHAGCMNNAGACACCACCCA-CTGGTCAT-3'.
Trypsin library selection
Biopanning experiments on the trypsin library were performed essentially as described in the section Model selection experiments with trypsin H57A. Between 109 and 1011 phage were subjected to selection in each round of panning. The washing conditions were less stringent in the first round of selection [three times with TBSCTween-20 (0.1%) and three times with TBSC]. The reaction time was reduced to 1 min in the third and fourth rounds of panning; however, the reaction could proceed for some time during the PEG precipitation step. Eluted phages were used to infect an excess of exponentially growing TG1 cells, which were then plated on 2YT agar plates containing tetracycline (10 µg/ml). The colonies were pooled and used to produce new phages.
Expression and purification of trypsin
Trypsin mutants were PCR-amplified from single colonies using the primers Sfincobatsn and TsnHisGlyfo. The primer TsnHisGlyfo adds a hexahistidine tag C-terminally to the trypsin mutant. PCR products were SfiI/NotI digested and ligated into the expression vector pDN321, a derivative of pUC119SNpolymyc (Neri et al., 1996).
The vector contains a PelB leader sequence, which directs the recombinant proteins into the periplasm. A 5 ml overnight culture of E.coli strain TG1 containing the mutant plasmids was used to inoculate 400 ml of 2YT medium containing 100 µg/ml ampicillin and 0.1% glucose. Cells were grown at 37°C with vigorous shaking until an OD600 of 0.6 was reached. Protein expression was induced by adding IPTG to a final concentration of 1 mM. Cells were incubated for an additional 12 h at 20°C and then harvested. The periplasmic fraction was obtained as described (Hennecke et al., 1997). A modified extraction buffer (50 mM TrisHCl pH 8, 300 mM NaCl, 1 mM EDTA and 1 mg/ml polymyxin) was used which is compatible with nickel affinity chromatography purification.
The native fraction of the expressed protein was purified by nickel affinity chromatography. An 8 ml volume of periplasmic extract (from a 400 ml culture) was supplied with imidazole (15 mM final concentration) and MgCl2 (final concentration 1 mM), incubated with 200 µl of Ni-NTA agarose (Qiagen) and mixed for 60 min on a rotary shaker (200 r.p.m.) at 4°C. The lysateNi-NTA mixture was loaded into a column (Bio-Rad, Glattbrugg, Switzerland) and washed twice with 6 ml of a buffer containing 50 mM TrisHCl pH 8, 300 mM NaCl and 15 mM imidazole by gravity flow. The enzyme was eluted in 200 µl of buffer containing 50 mM TrisHCl pH 8, 300 mM NaCl and 250 mM imidazole. Alternatively, the column was first washed with 6 ml of a denaturing buffer (50 mM TrisHCl pH 8, 300 mM NaCl, 4 M urea and 15 mM imidazole) in order to remove ecotin, which is bound to some of the trypsin mutants.
Activity measurement of mutant trypsin
Fluorescence assays were performed by following the release of free aminomethylcoumarin (AMC) from the substrates Z-Gly-Gly-Arg-AMC and Z-Gly-His-Arg-AMC. Time-dependent fluorescence intensity was measured with a VersaMAX Gemini plate spectrofluorimeter (Molecular Devices, Sunnyvale, CA). A typical assay consisted of a 100 µl volume of 50 mM TrisHCl pH 8.0, containing 10 mM CaCl2, 100 mM NaCl, 5 µM2 mM Z-Gly-Gly-Arg-AMC or Z-Gly-His-Arg-AMC and 0.11 µM of a trypsin mutant at 25°C for 2 h. Fluorescence measurements were carried out with an excitation wavelength of 381 nm and an emission wavelength of 455 nm.
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Results |
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The efficient display of functional enzymecalmodulin fusion on phage is an absolute prerequisite for the selection of enzymatic activities according to the strategy outlined in Figure 1 and described in the Introduction. In this study, we displayed the two enzymes trypsin H57A and BirA as calmodulinpIII fusion on filamentous phage.
The gene of trypsin H57A was fused to the calmodulin gene and cloned into the phage vector pCHH1, a derivative of fd-tet-DOG1 (Clackson et al., 1991). The resulting vector pCHH3 is shown in Figure 2a
. Bioactive rat anionic trypsin II was displayed on phage without the pro-peptide, in analogy to wild-type trypsin which had previously been functionally displayed on phage as a fusion to the C-terminal portion (residues 198406) of pIII (Corey et al., 1993
). Here we linked the calmodulin-tagged enzyme to the entire pIII protein (residues 1406). Fluorogenic activity assays, monitoring the release of free AMC from the substrate Z-Gly-Gly-Arg-AMC, performed on phage displaying wild-type trypsincalmodulin fusion (pCHH2), showed that the phageenzyme is catalytically active (data not shown). The activity of trypsin H57A on phage (pCHH3) could not be measured, as the turnover number of this mutant is too low (Corey et al., 1995
).
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The display of calmodulin-tagged enzyme on phage was further analysed by Western blotting using an anti-pIII monoclonal antibody (Tesar et al., 1995). The immunoblot confirmed that trypsin H57Acalmodulin fusion is efficiently displayed on phage (Figure 3
). Additionally, some degradation products of the fusion protein can be observed. The same pattern of bands is observed in the immunoblot of phage displaying the wild-type trypsincalmodulin fusion (pCHH2). By comparison, trypsincalmodulinpIII fusions can hardly be detected in immunoblot experiments, whenever phage particles are produced using phagemid vectors (Demartis et al., 1999
). The fusion protein BirAcalmodulinpIII (pSD22) could be detected by immunoblot experiments, which were dominated by a band corresponding to calmodulinpIII fusion, indicating extensive proteolysis at the level of the linker between BirA and calmodulin (Figure 3
). This low level of functional display of BirA on phage has consequences on the results of the selection experiments (see below).
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We systematically investigated the performance of selection for catalysis using BirAcalmodulin phage, according to the strategy described in the Introduction (Figure 1). The selection scheme relies on the efficient capture of catalytically active phage displaying biotinylated peptides, by means of streptavidin-coated magnetic beads and on the selective separation of phage particles from unreacted biotin, by means of PEG precipitation.
In order to monitor biopanning efficiency, control reactions were performed as follows. A biotinylated calmodulin-binding peptide was used as positive control, simulating the reaction product of the biotinylation reaction of Figure 1a. In negative controls, either biotin or the substrate peptide was not added to the reaction mixture. The phageenzyme (
5x108 phage particles in each of the four separate reactions) was allowed to catalyse the reaction for 20 min at room temperature, before PEG precipitation and streptavidin capture. Nine independent sets of experiments were performed, in order to assess the reproducibility of the biopanning results. The phage titres rescued after each individual selection experiment are presented in Figure 4
. On average,
110% of the input phage was recovered in positive control selections, performed with the biotinylated peptide, indicating a good level of functional display of calmodulin on phage and an efficient phage capture scheme. Selection experiments performed with peptide GAAARWKKAFIAVSAANRFKKISTSGGGPGGLVSIFEA-QKIEWH, biotin and ATP gave consistently higher titres (range: between 4- and 800-fold) compared with negative control experiments, in which either the peptide or biotin had been omitted (Figure 4
). Notably, the phage titres rescued in the selection experiments were consistently lower (range: between 3- and 100-fold) than positive control selections, indicating a lower level of BirA display on phage, compared with the level of calmodulin display (Figure 3
), with a variability among different batches of phage preparation.
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It is tempting to speculate that BirA mutants, with improved folding properties and higher levels of display on phage, should allow an even more efficient recovery of catalytically active phageenzymes. Under the experimental conditions used, the kcat of BirA towards the biotin carboxyl carrier protein is 0.6 s-1 (Xu and Beckett, 1994). Furthermore, it can be excluded that the substrate peptide is biotinylated in an uncatalysed reaction, as kuncat is negligible for this reaction.
Model selection experiments with the trypsin mutant H57A
In model selection experiments, we tested whether phage displaying trypsin H57A can be enriched for catalysis over appropriate negative controls. Selection experiments were performed as depicted schematically in Figure 1b, using the substrate peptide GGHRDYKDEGGGAAARWKKAFIAVSAANRFKKIS and allowing for a 30 min. reaction time (kcat = 0.002 s-1 for peptides containing the HisArg substrate) (Corey et al., 1995
). The trypsin mutant H57A displayed on phage recognizes the HisArg sequence and cleaves the peptide C-terminally to this site. The monoclonal antibody M1 is used as capture reagent (Hopp et al., 1988
), binding to peptides carrying the sequence DYKDE (underlined) at the N-terminal extremity, but not peptides with the DYKDE sequence at internal positions (Huber et al., 1999
). A PEG precipitation step was performed before phage capture by means of streptavidin-coated magnetic beads, pre-incubated with the biotinylated M1 antibody. In positive control experiments, the peptide DYKDEGGGAAARWKKAFIAVSAANRFKKIS was used, which represents the product of the cleavage reaction. As a negative control, the peptides GAAHYDYKDEGGGAAARWKKAFIAVSAANRFKKIS and GDDDDKDYKDEGGGAAARWKKAFIAVSAANRFKKIS were used, which contain only either the His or the Lys residue located N-terminally relative to the DYKDE sequence and are therefore not cleaved by trypsin H57A. In another negative control experiment, no peptide was added to the phage.
Three independent sets of experiments were performed in which phage were incubated with different peptides in five individual reactions. Elution titres of phageenzyme anchoring the substrate peptide were reproducibly higher (range: between 15- and 2000-fold) compared with the titres of negative control selections (Figure 5). While these results demonstrate that phage displaying trypsin H57A can be enriched for catalysis, phage captured with the peptide DYKDEGGGAAARWKKAFIAVSAANRFKKIS (positive control) gave consistently higher titres (range: between 1.4- and 17-fold) compared with selection for catalysis experiments (Figure 5
). The more efficient phage rescue in the positive control experiments can be explained by the fact that not all phage display an enzyme (Figure 3
), stressing once again the importance of efficient functional enzyme display on phage. Additionally, the endogenous E.coli protein ecotin, a nanomolar inhibitor of trypsin, might partially inhibit the enzymatic reaction on phage (Chung et al., 1983
). However, enrichment factors did not improve when phageenzymes were dialysed prior to selections (data not shown).
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In substrate-assisted catalysis (Carter and Wells, 1987), the His residue in the catalytic triad of a serine protease is mutated into a different residue. At the same time, a His residue is inserted in the P2 position of the peptidic substrate. To a certain extent, the His residue in the substrate complements the missing histidine in the catalytic triad, leading to an active protease with a two-amino acid substrate specificity (e.g. HisLys or HisArg for the trypsin H57A mutant (Corey et al., 1995
). Structural studies of substrate-assisted catalysis of the trypsin mutant H57A revealed that the catalytic histidine residue provided by the substrate does not optimally restore the stereoelectronic arrangement of the catalytic triad (Corey et al., 1995
). In particular, it is expected that Asp102 of the catalytic triad may not assist catalysis. We therefore aimed at creating a new trypsin mutant, which maintains the substrate specificity of trypsin H57A and which has an improved catalytic activity.
A trypsin library was constructed which bears mutations in the proximity of the trypsin's active site. We hoped that the residues selected for mutation would complement the catalytic unit formed by Ser195 and the histidine residue provided by the substrate. We combinatorially randomized Ala55, Asp102 and Ser214 (Figure 6). Additionally, His57 was combinatorially mutated with codon DNK, which does not allow for a histidine residue at this position. Ala55 points into the catalytic cavity and interacts with His57, which together with Asp102 and Ser195 constitutes the catalytic triad of the enzyme. Ser214 points towards the catalytic triad and forms a hydrogen bond with Asp102. It has been shown that single mutations of these residues do not disrupt the overall structure of trypsin (Corey and Craik, 1992
; McGrath et al., 1992
; Takeda-Shitaka and Umeyama, 1998
). Using partially degenerate oligonucleotidic primers in a PCR assembly protocol and the phage vector pCHH4 (see Materials and methods), we constructed a library containing >5x105 transformants, which was significantly larger than the calculated theoretical complexity of 1.36x105 possible amino acid combinations. Sequencing of randomly picked clones indicated that the library had been properly constructed and that the expected diversity was present at the desired positions (Table I
). Phage were produced from cells representing the trypsin library, obtained by electroporation. However, we found that this phage population was less infective (0.01%) than the monoclonal phage used in model selections, as determined by measuring transforming units by bacterial infection and number of phage particles by UV absorption methods (Day and Wisemann, 1978
).
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Four rounds of panning were performed with the phage library of trypsin mutants described in the previous section, using GGHRDYKDEGGGAAARWKKAFIAVSAANRFKKIS as peptide substrate and following the selection scheme described in Figure 1b. In parallel, positive control selections were performed using the peptide DYKDEGGGAAARWKKAFIAVSAANRFKKIS, and also negative control selections, in which no peptide was added. In the first round of selection, 109 infective phage were used, representing each trypsin variant
10 000 times on average. Enzymes were allowed to react for 20 min at 37°C in the first two rounds of panning, while the reaction time was reduced in the third and fourth rounds of selection. Phage titres rescued at the end of each round of panning are listed in Table II
. Surprisingly, elution titres of the first round of selection were enriched more than 60-fold, compared with the negative control. The number of phage captured in this selection was nearly as high as in the positive control. Similar enrichment factors were observed in the three successive rounds of panning.
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Discussion |
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In this work, we implemented the selection scheme of Demartis et al. (Demartis et al., 1999) for two different classes of enzymes: an endoprotease and a ligase. As expected, the use of phage vectors (rather than phagemids) appears to improve enzyme display on phage dramatically. However, proteolytic degradation (particularly prominent in the case of biotin ligase) may also limit the number of enzyme molecules displayed on each phage particle. It is tempting to speculate that the selection scheme of Figure 1
may yield enzyme mutants with improved folding properties and superior display on phage, from libraries obtained, for example, by DNA shuffling (Stemmer, 1994
).
In model selection experiments, phage displaying BirA were consistently enriched (between 4- and 800-fold) per round of panning, relative to negative controls. In competition experiments we showed that the enzymes act on the substrate displayed on the same phage rather than on the substrate of other phage. Phage displaying BirA mixed with phage displaying an irrelevant enzyme were enriched by a factor of 10 per round of selection.
Phage displaying the trypsin His57Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15- and 2000-fold), relative to negative controls. These promising results suggest that the selection scheme of Figure 1
may be useful for the isolation of novel and/or improved catalysts. This task remains a significant challenge in the field of protein engineering, particularly when large protein repertoires are selected with phage display methodologies.
In order to improve the catalytic properties of the trypsin His57Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phageenzymes was shown by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57
Ala mutant could not be isolated. These results indicate that further work is needed to demonstrate the usefulness of phageenzyme selection schemes at the level of large combinatorial libraries. The catalytically active His57
Ala mutant was contained in the library, but was not recovered after four rounds of biopanning (Table I
). This result suggests that more stringent selection schemes will have to be implemented, e.g. by blocking the reaction with suitable inhibitors after addition of the substrate to the phage and before the capture step (Figure 1
). A potential problem with selections performed with peptidic substrates is represented that proteolytic cleavage by the His57
Ala mutant is extremely slow (kcat = 0.002 s-1) and approaches the level of background proteolysis in mixtures of unpurified biological material. In the future, we will direct our attention to trypsin mutants with an intact catalytic triad in order to start the selection with more active trypsin variants. The substrate specificity could be altered at the P1 position of the peptide by changing the binding pocket of trypsin. A human enzyme with novel substrate specificity, which maintains its catalytic activity in serum, could be used for effective tumor targeting. The antibody-mediated delivery of such enzymes to the tumor environment (Halin and Neri, 2001
) will allow the selective generation of drugs from non-toxic precursors (`pro-drugs') in situ, therefore sparing normal tissues and improving the therapeutic index of available chemotherapeutic agents (Niculescu-Duvaz et al., 1999
).
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Notes |
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Acknowledgments |
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Received March 7, 2001; revised August 10, 2001; accepted September 5, 2001.