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Surface Expression and Ligand-Based Selection of cDNAs Fused to Filamentous Phage Gene VI

Bio/Technology volume 13, pages 378–382 (1995)Cite this article

Abstract

We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3′-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.

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References

  1. Helfman, D.M., Fiddes, J.R., Thomas, G.P. and Hughes, S. 1983. Identification of clones that encode chicken tropomyosin. Proc. Natl. Acad. Sci. USA 80: 31–35.

    Article  CAS  Google Scholar 

  2. Young, R.A. and Davis, R.W. 1983. Efficient isolation of genes using antibody probes. Proc. Natl. Acad. Sci. USA 80: 1194–1198.

    Article  CAS  Google Scholar 

  3. Sikela, J.M. and Hahn, W. 1987. Screening an expression library with a ligand probe: Isolation and sequence of a cDNA corresponding to a brain calmodulin-binding protein. Proc. Natl. Acad. Sci. USA 84: 3038–3042.

    Article  CAS  Google Scholar 

  4. Singh, S.J.H., LeBowitz, A.S., Baldwin, Jr. and Sharp, P.A. 1988. Molecular cloning of an enhancer binding protein: Isolation by screening of an expression library with a recognition site DNA. Cell 52: 415–423.

    Article  CAS  Google Scholar 

  5. Fields, S. and Song, O.K. 1989. A novel genetic system to detect protein-protein interactions. Nature 340: 245–246.

    Article  CAS  Google Scholar 

  6. Seed, B. and Aruffo, A. 1987. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. USA 84: 3365–3369.

    Article  CAS  Google Scholar 

  7. Germino, F.J., Wang, Z.X. and Weissman, S.M. 1993. Screening for in vivo protein-protein interactions. Proc. Natl. Acad. Sci. USA 90: 933–937.

    Article  CAS  Google Scholar 

  8. Crameri, R. and Suter, M. 1993. Display of biologically active proteins on the surface of filamentous phages: A cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production. Gene 137: 69–75.

    Article  CAS  Google Scholar 

  9. Parmley, S.F. and Smith, G.P. 1988. Antibody-selectable filamentous fd phage vectors: Affinity purification of target genes. Gene 73: 305–318.

    Article  CAS  Google Scholar 

  10. De Maeyer, M., Lambeir, A.M., Laroche, Y., Lasters, I., Lauwereys, M., Mattyssens, G., Stanssens, P., Ripka, W.C. and Vlasuk, G.P. 1993. Recombinant Kunitz inhibitors of coagulation proteases II: Random mutagenesis. Thromb. Haemostas. 69: 888.

    Google Scholar 

  11. Vieira, J. and Messing, J. 1987. Production of single-stranded plasmid DNA. Meth. Enzymol. 153: 3–11.

    Article  CAS  Google Scholar 

  12. Winter, G., Griffiths, A.D., Hawkins, R.E. and Hoogenboom, H.R. 1994. Making antibodies by phage display technology. Annu. Rev. Immunol. 12: 433–455.

    Article  CAS  Google Scholar 

  13. Clackson, T. and Wells, J.A. 1994. In vitro selection from protein and peptide libraries. TIBTECH 12: 173–184.

    Article  CAS  Google Scholar 

  14. Roberts, B.L., Markland, W., Ley, A.C., Kent, R.B., White, D.W., Guterman, S.K., and Ladner, R.C. 1992. Directed evolution of a protein: Selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage. Proc. Natl. Acad. Sci. USA 89: 2429–2433.

    Article  CAS  Google Scholar 

  15. Altman, J.D., Henner, D., Nilsson, B., Anderson, S. and Kuntz, I.D. 1991. Intracellular expression of BPTI fusion proteins and single column cleavage/ affinity purification by chymotrypsin. Protein Eng. 4: 593–600.

    Article  CAS  Google Scholar 

  16. McCafferty, J., Griffiths, A.D., Winter, G. and Chiswell, D.J. 1990. Phage antibodies: Filamentous phage displaying antibody variable domains. Nature 348: 552–554.

    Article  CAS  Google Scholar 

  17. Zacher, A.N., Stock, C.A., Golden, J.W. and Smith, G.P. 1980. A new filamentous phage cloning vector: fd-tet. Gene 9: 127–140.

    Article  CAS  Google Scholar 

  18. Cunningham, B.C., Lowe, D.G., Li, B., Bennett, B.D. and Wells, J.A. 1994. Production of an atrial natriuretic peptide variant that is specific for type A receptor. EMBO J. 13: 2508–2515.

    Article  CAS  Google Scholar 

  19. Creighton, T.E. and Charles, I.G. 1987. Biosynthesis, processing, and evolution of bovine pancreatic trypsin inhibitor. Cold Spring Harbor Symp. Quant. Biol. 52: 511–519.

    Article  CAS  Google Scholar 

  20. Von Heijne, G. 1986. A new method for predicting signal sequence cleavage site. Nucl. Acids Res. 14: 4683–4690.

    Article  CAS  Google Scholar 

  21. Goodman, R.B. and Peanasky, R.J. 1982. Isolation of the trypsin inhibitors in Ascaris lumbricoides var suum using affinity chromotography. Anal. Biochem. 120: 387–393.

    Article  CAS  Google Scholar 

  22. Babin, D.R., Peanasky, R.J. and Goss, S.M. 1984. The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: The primary structure. Arch. Biochem. Biophys. 232: 143–161.

    Article  CAS  Google Scholar 

  23. Huang, K., Strynadka, N.C.J., Bernard, V.D., Peanasky, R.J. and James, M.N.G. 1994. The molecular structure of the complex of Ascaris chy-motrypsin/elastase inhibitor with porcine elastase. Stucture. 2: 679–689.

    Article  CAS  Google Scholar 

  24. Simons, G.F.M., Konings, R.N.H. and Schoenmakers, J.G.G. 1981. Genes VI, VII and IX of phage M13 code for minor capsid proteins of the virion. Proc. Natl. Acad. Sci. USA 78: 4194–4198.

    Article  CAS  Google Scholar 

  25. Makowski, L. 1992. Terminating a macromolecular helix: structural model for the minor proteins of bacteriophage M13. J. Mol. Biol. 228: 885–892.

    Article  CAS  Google Scholar 

  26. Eiff, J. A. 1966. Nature of an anticoagulant from the cephalic glands of Ancylostoma caninum. J. Parasit. 52: 833–843.

    Article  CAS  Google Scholar 

  27. Capello, M., Clyne, L.P., McPhedran, P. and Hotez, P.J. 1993. Ancylostoma Factor Xa inhibitor: Partial purification and its identification as a major hookworm-derived anticoagulant in vitro.. J. Infect. Dis. 167: 1474–1477.

    Article  Google Scholar 

  28. Short, J.M., Femandez, J.M., Sorge, J.A. and Huse, W.D. 1988. λ ZAP: A bacteriophage λ expression vector with in vivo excision properties. Nucl. Acids Res. 16: 7583–7600.

    Article  CAS  Google Scholar 

  29. Crameri, R., Menz, G. and Blaser, K. 1994. Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction. Eur. J. Biochem. 226: 53–58.

    Article  CAS  Google Scholar 

  30. Maruyama, I.N., Maruyama, H.I. and Brenner, S. 1994. λfoo: A λ phage vector for the expression of foreign proteins. Proc, Natl. Acad. Sci. USA 91: 8273–8277.

    Article  CAS  Google Scholar 

  31. Bode, W., Papamokos, E., Musil, D., Seemueller, U. and FritZ, H. 1986. Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin. EMBO J. 5: 813–818.

    Article  CAS  Google Scholar 

  32. Hoogenboom, H.R., Griffiths, A.D., Johnson, K.S., Chiswell, D.J., Hudson, P., and Winter, G. 1991. Multi-subunit proteins on the surface of filamentous phage: Methodologies for displaying antibody (Fab) heavy and light chains. Nucl. Acids Res. 19: 4133–4137.

    Article  CAS  Google Scholar 

  33. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68.

    Article  CAS  Google Scholar 

  34. Ward, E.S., Güssow, 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.

    Article  CAS  Google Scholar 

  35. Bock, P.E., Craig, P.A., Olson, S.T. and Singh, P. 1989. Isolation of human blood coagulation α-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active site titration with fluorescein mono-P-guanidinobenzoate. Arch. Biochem. Biophys. 273: 375–388.

    Article  CAS  Google Scholar 

  36. Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D. and Winter, G. 1989. By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222: 581–597.

    Article  Google Scholar 

  37. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.

  38. Dower, W.J., Miller, J.F. and Ragsdale, C.W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res. 16: 6127–6145.

    Article  CAS  Google Scholar 

  39. Güssow, D. and Clackson, T.P. 1989. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucl. Acids Res. 17: 4000.

    Article  Google Scholar 

  40. Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.

    Article  CAS  Google Scholar 

  41. Ishii, S.-I., Yokosawa, H., Kumazaki, T. and Nakamura, I. 1983. Immobilized anhydrotrypsin as a specific affinity adsorbent for tryptic peptides. Meth. Enzymol. 91: 378–383.

    Article  CAS  Google Scholar 

  42. Williams, G.W. and Morrison, J.F. 1979. The kinetics of reversible tight-binding inhibition. Meth. Enzymol. 63: 437–467.

    Article  CAS  Google Scholar 

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Author notes

  1. Laurent S. Jespers: Center for Molecular and Vascular Biology, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.

Authors and Affiliations

  1. Corvas International N.V., Jozef Plateaustraat 22, B-9000, Gent, Belgium

    Laurent S. Jespers, Joris H. Messens, Annick De Keyser, Dominique Eeckhout, Ilse Van Den Brande, Yannick G. Gansemans, Marc J. Lauwereys & Patrick E. Stanssens

  2. Corvas International Inc., Science Park Road 3030, San Diego, California, 92121

    George P. Vlasuk

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Jespers, L., Messens, J., Keyser, A. et al. Surface Expression and Ligand-Based Selection of cDNAs Fused to Filamentous Phage Gene VI. Nat Biotechnol 13, 378–382 (1995). https://doi.org/10.1038/nbt0495-378

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