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Substrate Phage
C H A P T E R
Substrate Phage David J. Matthews
Substrate phage is a powerful adaptation of phage display which makes it possible to determine substrate specificity by screening peptide phage libraries. The method has been successfully applied to determining the substrate specificity of several different proteases (Table 1) but could also be applied to determining peptide sequence specificity for a wide variety of peptide post-translational modifications. The requirements for a substrate phage experiment are described below, illustrated by reference to protease substrate phage.
A S U I T A B L E LIBRARY OF R A N D O M PE P TIDE S D I S P L A Y E D O N T H E SURFACE OF P H A G E PARTICLES In the case of protease substrate phage, a randomized peptide coding sequence is inserted between an "affinity domain" (in Fig. 1, a variant of human growth hormone [hGH]) and a truncated form of M 13 gene III. Other forms of affinity domain could also be used for protein immobilization, such as "epitope tag" peptides which bind to anti-peptide antibodies (for example, the Flag-tag (Hopp et al., 1988; Brizzard et al., 1994) and the Myc-tag (Munro and Pelham, 1984; Ward et al., 1989). The phage library is ideally expressed in a phagemid system (Wells and Lowman, 1992), so that only a small percentage of phage particles display the Phage Display of Peptides and Proteins Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
255
256
David J. Matthews
TABLE I ProteasesScreened Using Substrate Phage Display
FIGURE
I
Protease
Reference
Subtilisin BPN' mutant S24C/H64A/ E 156S/G166A/G169A/Y217L Human factor Xa HIV protease (preliminary data only) Human furin Human matrilysin Human stromelysin Human tissue-type plasminogen activator Subtilisin BPN' mutant N62D/G 166D
Matthews and Wells (1993) Matthews and Wells (1993) Matthews and Wells (1993) Matthews et al. (1994) Smith et al. (1995) Smith et al. (1995) Ding et al. (1995) Ballinger et al. (1996)
Scheme for protease substrate phage selection. Reprinted with permission from Matthews, D. J., and Wells, J. A. (1993) Substrate phage: Selection of Protease substrates by monovalent phage display. Science 260, 1113-7, 9 1993 AAAS.
growth h o r m o n e - p e p t i d e - g e n e III fusion and the majority of these contain only one copy per phage particle. However, it should be noted that Smith e t al. ( 1 9 9 5 ) have reported the use of a polyvalent phage display library for discovery of substrates of the metalloproteinases matrilysin and stromelysin.
Chapter 14 Substrate Phage
257
A M E T H O D FOR IN VITRO M O D I F I C A T I O N OF PEPTIDE SUBSTRATES For protease substrates, in vitro modification involves simply cleaving the peptide substrates by incubation with protease under appropriate conditions. This selection may be employed to generate information on both protease-sensitive and proteaseresistant sequences. The amount of protease used will depend on its specificity and catalytic efficiency, and usually must be determined empirically. If possible, it is helpful to initially make two phage constructs, one bearing a known substrate and the other a known nonsubstrate sequence. Phage produced from these constructs may then be used to determine appropriate selection conditions as follows: i. Immobilize equal numbers of substrate and nonsubstrate phage particles to separate wells of a microtiter plate via the affinity domain. Set up several wells for each phage construct. ii. Wash extensively to remove unbound phage particles. iii. Incubate with varying concentrations of protease for a fixed period of time (based on an estimate of the enzyme's turnover number if known). It is important to include a control incubation with buffer alone. iv. Remove the eluted phage from the well and measure the phage titer. This should allow determination of the protease concentration which gives the best enrichment for substrate over nonsubstrate and for protease incubation over control incubation. v. Proceed with phage selection using the protease concentration determined in the above steps.
A M E T H O D FOR S E P A R A T I N G M O D I F I E D PEPTIDES FROM U N M O D I F I E D PEPTIDES Substrate phage are immobilized on a solid support via the affinity domain (in this case, binding of the hGH variant to immobilized hGH binding protein). Techniques for protein immobilization, phage binding, and washing are essentially as discussed elsewhere in this volume; however, phage elution is performed by incubation with protease. On proteolytic cleavage, phage particles bearing substrates are released into solution whereas those containing nonsubstrates remain bound to the solid support. If desired, the protease-resistant phage may be subsequently eluted using a low-pH buffer (50 mM glycine, pH 2.0). The pools of protease-sensitive and protease-resistant phage may be propagated by infection of Escherichia coli and the process repeated to further enrich for protease-sensitive and protease-resistant sequences. DNA sequencing of individual clones is performed to obtain detailed sequence information about substrate specificity. Phage titers may be measured after each round of selection, although this may be of limited benefit; it has been found that consensus sequences may emerge even when little or no enrichment is observed by phage titer (D. J. Matthews and J. A. Wells, unpublished observations).
258
DavidJ. Matthews Following phage sorting, it is useful to perform a rapid assay to confirm that sequences isolated from the phage library are indeed good substrates. Matthews and Wells (1993) have described such an assay for protease substrates. Sequences from phage sorting are assembled by PCR into a vector which directs expression of a tripartite fusion protein. The fusion protein comprises the substrate sequence fused between hGH at the N-terminus and alkaline phosphatase (AP) at the C-terminus. This protein is expressed, partially purified, and bound via hGH to immobilized hGH binding protein. On incubation with protease the AP moiety is released, thus allowing substrate cleavage to be monitored by measurement of AP activity in the supernatant. It is also possible to determine the N-terminal sequence of the cleaved AP component, thus allowing the scissile bond to be identified (Matthews and Wells, 1993; Matthews et al., 1994; Ballinger et al. (1996).
CAVEATS,TROUBLESHOOTING,
AND
HINTS
9 In the case of protease substrate phage, it is important that cleavage occurs preferentially in the linker between the affinity domain and gene III. Flanking the substrate region with flexible linker peptides may help to ensure that cleavage occurs preferentially within the library cassette. Fortunately, filamentous phage are remarkably resistant to proteolysis; among commonly used proteases, only subtilisin is known to degrade phage coat proteins (Schwind et al., 1992) 9 E. coli periplasmic proteases may cleave some substrates before they even leave the cell. Nevertheless, substrates with trypsin-like motifs (which one might expect to be degraded in the periplasm) have been successfully screened using substrate phage (Matthews et al., 1994). 9 If experiments with a test system involving known substrate/nonsubstrate sequences are successful but no selection from a library is observed, it may be that the enzyme has broad specificity and thus the sorting process will not converge on any one family of substrate sequences. This is a limitation of any technique used to determine substrate specificity. There are some 300 amino acid modifications which occur in vivo (Yan et al., 1989), many of which could be studied using the substrate phage concept. For example, to study protein tyrosine phosphorylation one may construct a random peptide library with a fixed tyrosine residue surrounded by random codons. The selection procedure would involve incubating the phage library with a tyrosine kinase, and phosphorylated peptides could be separated from unphosphorylated peptides using an anti-phosphotyrosine antibody. In this case, care must be taken that the antibody recognizes phosphorylated tyrosine alone and is not sequencespecific; otherwise, this step introduces another selective pressure on the substrate phage screening process. Other post-translational modifications which could be studied with this technique include dephosphorylation, glycosylation, isoprenylation, methylation, acylation, and carboxylation. Such uses of phage display have not been reported to date. However, Westendorf et al. (1994) have recently reported the
Chapter 14 Substrate Phage
259
use of peptide phage libraries to determine the epitope of an anti-phosphopeptide antibody which binds to M-phase phosphoproteins, and Schatz (1993) has used a related techniquemthe so-called "peptides on plasmids" method--to determine the substrate specificity of E. coli biotin holoenzyme synthetase. A variation of the protease substrate phage technique has also been used to determine the nucleophile specificity of "subtiligase," a mutant of subtilisin which acts as a peptide ligase (Chang et al., 1994).
References Chang, T. K., Jackson, D. Y., Burnier, J. E and Wells, J. A. (1994). Subtiligase: A tool for semisynthesis of proteins. Proc. Natl. Acad. Sci. U.S.A. 91, 12544-12548. Ballinger, M. D., Tom, J., and Wells, J. A. (1996). Designing Subtilisin BPN' to cleave substrates containing dibasic residues. Biochemistry 34, 13312-13319. Ding, L., Coombs, G. S., Strandberg, L., Navre, M., Corey D. R., and Madison E. L. (1995). Origins of the substrate specificity of tissue-type plasminogen activator. Proc. Natl. Acad. Sci. U.S.A. 92, 7627-7631. Brizzard, B. L., Chubet, R. G., and Vizard, D. L. (1994). Immunoaffinity purification of Flag epitopetagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques 16, 730-735. Hopp, T. P., Prickett, K. S., Price, V. L., Libby, R. T., March, C. J., Ceretti, D. P., Urdal, D. L. and ConIon, P. J. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204-1210. Matthews, D. J., Goodman, L. J., Gorman, C. M., and Wells, J. A. (1994). A survey of furin substrate specificity using substrate phage display. Prot. Sci. 3, 1197-1205. Matthews, D. J., and Wells, J. A. (1993). Substrate phage: Selection of protease substrates by monovalent phage display. Science 260, 1113-1117. Munro, S., and Pelham, H. R. B. (1984). Use of peptide tagging to detect proteins expressed from cloned genes: Deletion mapping functional domains of Drosophila hsp 70. EMBO J. 3, 3087-3093. Schatz, P. (1993). Use of peptide library to map the substrate specificity of a peptide-modifying enzyme: A 13 residue consensus peptide specified biotinylation in Escherichia coli. Bio/Technology 11, 1138-1142. Schwind, P., Kramer, H., Kremser, A., Ramsberger, U., and Rasched, I. (1992). Subtilisin removes the surface layer of the phage fd coat. Eur J Biochem 210, 431-436. Smith, M., Shi, L., and Navre, M. (1995). Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries. J. Biol. Chem. 270, 6440-6449. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989). Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544-546. Wells, J. A., and Lowman, H. B. (1992). Rapid evolution of peptide and protein binding properties in vitro. Curr. Op. Struct. Biol. 597-604. Westendorf, J. M., Rao, P. N., and Gerace, L. (1994). Cloning of cDNAs for M-phase phosphoproteins recognized by the MPM2 monoclonal antibody and determination of the phosphorylated epitope. Proc. Natl. Acad. Sci. U.S.A. 91,714-718. Yan, S. C. B., Grinnell, B. W., and Wold, F. (1989). Post-translational modifications of proteins: Some problems left to solve. Trends Biochem. Sci. 14, 264-268.
Substrate Phage David J. Matthews
Substrate phage is a powerful adaptation of phage display which makes it possible to determine substrate specificity by screening peptide phage libraries. The method has been successfully applied to determining the substrate specificity of several different proteases (Table 1) but could also be applied to determining peptide sequence specificity for a wide variety of peptide post-translational modifications. The requirements for a substrate phage experiment are described below, illustrated by reference to protease substrate phage.
A S U I T A B L E LIBRARY OF R A N D O M PE P TIDE S D I S P L A Y E D O N T H E SURFACE OF P H A G E PARTICLES In the case of protease substrate phage, a randomized peptide coding sequence is inserted between an "affinity domain" (in Fig. 1, a variant of human growth hormone [hGH]) and a truncated form of M 13 gene III. Other forms of affinity domain could also be used for protein immobilization, such as "epitope tag" peptides which bind to anti-peptide antibodies (for example, the Flag-tag (Hopp et al., 1988; Brizzard et al., 1994) and the Myc-tag (Munro and Pelham, 1984; Ward et al., 1989). The phage library is ideally expressed in a phagemid system (Wells and Lowman, 1992), so that only a small percentage of phage particles display the Phage Display of Peptides and Proteins Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
255
256
David J. Matthews
TABLE I ProteasesScreened Using Substrate Phage Display
FIGURE
I
Protease
Reference
Subtilisin BPN' mutant S24C/H64A/ E 156S/G166A/G169A/Y217L Human factor Xa HIV protease (preliminary data only) Human furin Human matrilysin Human stromelysin Human tissue-type plasminogen activator Subtilisin BPN' mutant N62D/G 166D
Matthews and Wells (1993) Matthews and Wells (1993) Matthews and Wells (1993) Matthews et al. (1994) Smith et al. (1995) Smith et al. (1995) Ding et al. (1995) Ballinger et al. (1996)
Scheme for protease substrate phage selection. Reprinted with permission from Matthews, D. J., and Wells, J. A. (1993) Substrate phage: Selection of Protease substrates by monovalent phage display. Science 260, 1113-7, 9 1993 AAAS.
growth h o r m o n e - p e p t i d e - g e n e III fusion and the majority of these contain only one copy per phage particle. However, it should be noted that Smith e t al. ( 1 9 9 5 ) have reported the use of a polyvalent phage display library for discovery of substrates of the metalloproteinases matrilysin and stromelysin.
Chapter 14 Substrate Phage
257
A M E T H O D FOR IN VITRO M O D I F I C A T I O N OF PEPTIDE SUBSTRATES For protease substrates, in vitro modification involves simply cleaving the peptide substrates by incubation with protease under appropriate conditions. This selection may be employed to generate information on both protease-sensitive and proteaseresistant sequences. The amount of protease used will depend on its specificity and catalytic efficiency, and usually must be determined empirically. If possible, it is helpful to initially make two phage constructs, one bearing a known substrate and the other a known nonsubstrate sequence. Phage produced from these constructs may then be used to determine appropriate selection conditions as follows: i. Immobilize equal numbers of substrate and nonsubstrate phage particles to separate wells of a microtiter plate via the affinity domain. Set up several wells for each phage construct. ii. Wash extensively to remove unbound phage particles. iii. Incubate with varying concentrations of protease for a fixed period of time (based on an estimate of the enzyme's turnover number if known). It is important to include a control incubation with buffer alone. iv. Remove the eluted phage from the well and measure the phage titer. This should allow determination of the protease concentration which gives the best enrichment for substrate over nonsubstrate and for protease incubation over control incubation. v. Proceed with phage selection using the protease concentration determined in the above steps.
A M E T H O D FOR S E P A R A T I N G M O D I F I E D PEPTIDES FROM U N M O D I F I E D PEPTIDES Substrate phage are immobilized on a solid support via the affinity domain (in this case, binding of the hGH variant to immobilized hGH binding protein). Techniques for protein immobilization, phage binding, and washing are essentially as discussed elsewhere in this volume; however, phage elution is performed by incubation with protease. On proteolytic cleavage, phage particles bearing substrates are released into solution whereas those containing nonsubstrates remain bound to the solid support. If desired, the protease-resistant phage may be subsequently eluted using a low-pH buffer (50 mM glycine, pH 2.0). The pools of protease-sensitive and protease-resistant phage may be propagated by infection of Escherichia coli and the process repeated to further enrich for protease-sensitive and protease-resistant sequences. DNA sequencing of individual clones is performed to obtain detailed sequence information about substrate specificity. Phage titers may be measured after each round of selection, although this may be of limited benefit; it has been found that consensus sequences may emerge even when little or no enrichment is observed by phage titer (D. J. Matthews and J. A. Wells, unpublished observations).
258
DavidJ. Matthews Following phage sorting, it is useful to perform a rapid assay to confirm that sequences isolated from the phage library are indeed good substrates. Matthews and Wells (1993) have described such an assay for protease substrates. Sequences from phage sorting are assembled by PCR into a vector which directs expression of a tripartite fusion protein. The fusion protein comprises the substrate sequence fused between hGH at the N-terminus and alkaline phosphatase (AP) at the C-terminus. This protein is expressed, partially purified, and bound via hGH to immobilized hGH binding protein. On incubation with protease the AP moiety is released, thus allowing substrate cleavage to be monitored by measurement of AP activity in the supernatant. It is also possible to determine the N-terminal sequence of the cleaved AP component, thus allowing the scissile bond to be identified (Matthews and Wells, 1993; Matthews et al., 1994; Ballinger et al. (1996).
CAVEATS,TROUBLESHOOTING,
AND
HINTS
9 In the case of protease substrate phage, it is important that cleavage occurs preferentially in the linker between the affinity domain and gene III. Flanking the substrate region with flexible linker peptides may help to ensure that cleavage occurs preferentially within the library cassette. Fortunately, filamentous phage are remarkably resistant to proteolysis; among commonly used proteases, only subtilisin is known to degrade phage coat proteins (Schwind et al., 1992) 9 E. coli periplasmic proteases may cleave some substrates before they even leave the cell. Nevertheless, substrates with trypsin-like motifs (which one might expect to be degraded in the periplasm) have been successfully screened using substrate phage (Matthews et al., 1994). 9 If experiments with a test system involving known substrate/nonsubstrate sequences are successful but no selection from a library is observed, it may be that the enzyme has broad specificity and thus the sorting process will not converge on any one family of substrate sequences. This is a limitation of any technique used to determine substrate specificity. There are some 300 amino acid modifications which occur in vivo (Yan et al., 1989), many of which could be studied using the substrate phage concept. For example, to study protein tyrosine phosphorylation one may construct a random peptide library with a fixed tyrosine residue surrounded by random codons. The selection procedure would involve incubating the phage library with a tyrosine kinase, and phosphorylated peptides could be separated from unphosphorylated peptides using an anti-phosphotyrosine antibody. In this case, care must be taken that the antibody recognizes phosphorylated tyrosine alone and is not sequencespecific; otherwise, this step introduces another selective pressure on the substrate phage screening process. Other post-translational modifications which could be studied with this technique include dephosphorylation, glycosylation, isoprenylation, methylation, acylation, and carboxylation. Such uses of phage display have not been reported to date. However, Westendorf et al. (1994) have recently reported the
Chapter 14 Substrate Phage
259
use of peptide phage libraries to determine the epitope of an anti-phosphopeptide antibody which binds to M-phase phosphoproteins, and Schatz (1993) has used a related techniquemthe so-called "peptides on plasmids" method--to determine the substrate specificity of E. coli biotin holoenzyme synthetase. A variation of the protease substrate phage technique has also been used to determine the nucleophile specificity of "subtiligase," a mutant of subtilisin which acts as a peptide ligase (Chang et al., 1994).
References Chang, T. K., Jackson, D. Y., Burnier, J. E and Wells, J. A. (1994). Subtiligase: A tool for semisynthesis of proteins. Proc. Natl. Acad. Sci. U.S.A. 91, 12544-12548. Ballinger, M. D., Tom, J., and Wells, J. A. (1996). Designing Subtilisin BPN' to cleave substrates containing dibasic residues. Biochemistry 34, 13312-13319. Ding, L., Coombs, G. S., Strandberg, L., Navre, M., Corey D. R., and Madison E. L. (1995). Origins of the substrate specificity of tissue-type plasminogen activator. Proc. Natl. Acad. Sci. U.S.A. 92, 7627-7631. Brizzard, B. L., Chubet, R. G., and Vizard, D. L. (1994). Immunoaffinity purification of Flag epitopetagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques 16, 730-735. Hopp, T. P., Prickett, K. S., Price, V. L., Libby, R. T., March, C. J., Ceretti, D. P., Urdal, D. L. and ConIon, P. J. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204-1210. Matthews, D. J., Goodman, L. J., Gorman, C. M., and Wells, J. A. (1994). A survey of furin substrate specificity using substrate phage display. Prot. Sci. 3, 1197-1205. Matthews, D. J., and Wells, J. A. (1993). Substrate phage: Selection of protease substrates by monovalent phage display. Science 260, 1113-1117. Munro, S., and Pelham, H. R. B. (1984). Use of peptide tagging to detect proteins expressed from cloned genes: Deletion mapping functional domains of Drosophila hsp 70. EMBO J. 3, 3087-3093. Schatz, P. (1993). Use of peptide library to map the substrate specificity of a peptide-modifying enzyme: A 13 residue consensus peptide specified biotinylation in Escherichia coli. Bio/Technology 11, 1138-1142. Schwind, P., Kramer, H., Kremser, A., Ramsberger, U., and Rasched, I. (1992). Subtilisin removes the surface layer of the phage fd coat. Eur J Biochem 210, 431-436. Smith, M., Shi, L., and Navre, M. (1995). Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries. J. Biol. Chem. 270, 6440-6449. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989). Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544-546. Wells, J. A., and Lowman, H. B. (1992). Rapid evolution of peptide and protein binding properties in vitro. Curr. Op. Struct. Biol. 597-604. Westendorf, J. M., Rao, P. N., and Gerace, L. (1994). Cloning of cDNAs for M-phase phosphoproteins recognized by the MPM2 monoclonal antibody and determination of the phosphorylated epitope. Proc. Natl. Acad. Sci. U.S.A. 91,714-718. Yan, S. C. B., Grinnell, B. W., and Wold, F. (1989). Post-translational modifications of proteins: Some problems left to solve. Trends Biochem. Sci. 14, 264-268.