Abstract
Regulatory proteases modulate proteomic dynamics with a spectrum of specificities against substrate proteins. Substrate phage display is one of the key methodologies in producing substrate sequence information in vitro. Factor Xa, a key regulatory protease in the blood coagulation system, is used as a model system to demonstrate a high-throughput procedure to quantitatively characterize substrate sequences and their susceptibilities for enzymatic cleavage. This methodology can be generalized to proteases for which the active forms (not necessarily purified forms) are available for the in vitro experiments.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gosalia DN, Salisbury CM, Maly DJ, Ellman JA, Diamond SL (2005) Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays. Proteomics 5:1292–1298
Salisbury CM, Maly DJ, Ellman JA (2002) Peptide microarrays for the determination of protease substrate specificity. J Am Chem Soc 124:14868–14870
Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik CS (2000) Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci U S A 97:7754–7759
Marnett AB, Craik CS (2005) Papa’s got a brand new tag: advances in identification of proteases and their substrates. Trends Biotechnol 23:59–64
Hsu HJ, Tsai KC, Sun YK, Chang HJ, Huang YJ, Yu HM, Lin CH, Mao SS, Yang AS (2008) Factor Xa active site substrate specificity with substrate phage display and computational molecular modeling. J Biol Chem 283:12343–12353
Ding X, Rasmussen BF, Petsko GA, Ringe D (2006) Direct crystallographic observation of an acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of a peptidyl ester substrate: Exploiting the “glass transition” in protein dynamics. Bioorg Chem 34:410–423
Laskowski M, Qasim MA (2000) What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim Biophys Acta 1477:324–337
Coombs GS, Rao MS, Olson AJ, Dawson PE, Madison EL (1999) Revisiting catalysis by chymotrypsin family serine proteases using peptide substrates and inhibitors with unnatural main chains. J Biol Chem 274:24074–24079
Brandstetter H, Kuhne A, Bode W, Huber R, von der Saal W, Wirthensohn K, Engh RA (1996) X-ray structure of active site-inhibited clotting factor Xa implications for drug design and substrate recognition. J Biol Chem 271:29988–29992
Deperthes D (2002) Phage display substrate: a blind method for determining protease specificity. Biol Chem 383:1107–1112
Ohkubo S, Miyadera K, Sugimoto Y, Matsuo K, Wierzba K, Yamada Y (2001) Substrate phage as a tool to identify novel substrate sequences of proteases. Comb Chem High Throughput Screening 4:573–583
Smith MM, Shi L, 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
Matthews DJ, Wells JA (1993) Substrate phage: selection of protease substrates by monovalent phage display. Science 260:1113–1117
Chen CT, Yang EW, Hsu HJ, Sun YK, Hsu WL, Yang AS (2008) Protease substrate site predictors derived from machine learning on multilevel substrate phage display data. Bioinformatics 24:2691–2697
Sharkov NA, Davis RM, Reidhaar-Olson JF, Navre M, Cai D (2001) Reaction kinetics of protease with substrate phage. Kinetic model developed using stromelysin. J Biol Chem 276:10788–10793
Sidhu SS, Weiss GA (2004) Constructing phage display libraries by oligonucleotide-directed mutagenesis: Phage display (Clackson T, Lowman HB eds.), Oxford University Press, NY, pp 27–41
Bianchini EP, Louvain VB, Marque PE, Juliano MA, Juliano L, Le Bonniec BF (2002) Mapping of the catalytic groove preferences of factor Xa reveals an inadequate selectivity for its macromolecule substrates. J Biol Chem 277:20527–20534
Acknowledgment
This work is supported by grants from NHRI and NSC of Taiwan.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hsu, HJ., Yang, AS. (2014). Substrate Phage Display for Protease Substrate Sequence Characterization: Bovine Factor Xa as a Model System. In: Nixon, A. (eds) Therapeutic Peptides. Methods in Molecular Biology, vol 1088. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-673-3_8
Download citation
DOI: https://doi.org/10.1007/978-1-62703-673-3_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-672-6
Online ISBN: 978-1-62703-673-3
eBook Packages: Springer Protocols