Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 May 11;23(9):1531–1535. doi: 10.1093/nar/23.9.1531

Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes.

M R Dyson 1, V Germaschewski 1, K Murray 1
PMCID: PMC306893  PMID: 7784206

Abstract

Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance with recent successes of screening techniques based upon random phage display libraries (biopanning). Since a number of different phage are usually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative dissociation constants (KdRel) between filamentous phage carrying peptide fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage selected from a random fusion phage library for binding to a monoclonal antibody or a viral structural protein exhibited KdRel values in the nanomolar and micromolar ranges for their respective substrates, thus validating the method over a wide range of binding affinities.

Full text

PDF
1531

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balass M., Heldman Y., Cabilly S., Givol D., Katchalski-Katzir E., Fuchs S. Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10638–10642. doi: 10.1073/pnas.90.22.10638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blond-Elguindi S., Cwirla S. E., Dower W. J., Lipshutz R. J., Sprang S. R., Sambrook J. F., Gething M. J. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell. 1993 Nov 19;75(4):717–728. doi: 10.1016/0092-8674(93)90492-9. [DOI] [PubMed] [Google Scholar]
  3. Choo Y., Sánchez-García I., Klug A. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature. 1994 Dec 15;372(6507):642–645. doi: 10.1038/372642a0. [DOI] [PubMed] [Google Scholar]
  4. Clackson T., Wells J. A. In vitro selection from protein and peptide libraries. Trends Biotechnol. 1994 May;12(5):173–184. doi: 10.1016/0167-7799(94)90079-5. [DOI] [PubMed] [Google Scholar]
  5. Dedman J. R., Kaetzel M. A., Chan H. C., Nelson D. J., Jamieson G. A., Jr Selection of targeted biological modifiers from a bacteriophage library of random peptides. The identification of novel calmodulin regulatory peptides. J Biol Chem. 1993 Nov 5;268(31):23025–23030. [PubMed] [Google Scholar]
  6. Dyson M. R., Murray K. Selection of peptide inhibitors of interactions involved in complex protein assemblies: association of the core and surface antigens of hepatitis B virus. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2194–2198. doi: 10.1073/pnas.92.6.2194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Felici F., Luzzago A., Folgori A., Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides, II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries. Gene. 1993 Jun 15;128(1):21–27. doi: 10.1016/0378-1119(93)90148-v. [DOI] [PubMed] [Google Scholar]
  8. Friguet B., Chaffotte A. F., Djavadi-Ohaniance L., Goldberg M. E. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods. 1985 Mar 18;77(2):305–319. doi: 10.1016/0022-1759(85)90044-4. [DOI] [PubMed] [Google Scholar]
  9. Friguet B., Djavadi-Ohaniance L., Goldberg M. E. Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen. Mol Immunol. 1984 Jul;21(7):673–677. doi: 10.1016/0161-5890(84)90053-1. [DOI] [PubMed] [Google Scholar]
  10. Germaschewski V., Murray K. Screening a monoclonal antibody with a fusion-phage display library shows a discontinuity in a linear epitope within PreS1 of hepatitis B virus. J Med Virol. 1995 Mar;45(3):300–305. doi: 10.1002/jmv.1890450311. [DOI] [PubMed] [Google Scholar]
  11. Hammer J., Valsasnini P., Tolba K., Bolin D., Higelin J., Takacs B., Sinigaglia F. Promiscuous and allele-specific anchors in HLA-DR-binding peptides. Cell. 1993 Jul 16;74(1):197–203. doi: 10.1016/0092-8674(93)90306-b. [DOI] [PubMed] [Google Scholar]
  12. Heermann K. H., Goldmann U., Schwartz W., Seyffarth T., Baumgarten H., Gerlich W. H. Large surface proteins of hepatitis B virus containing the pre-s sequence. J Virol. 1984 Nov;52(2):396–402. doi: 10.1128/jvi.52.2.396-402.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Koivunen E., Gay D. A., Ruoslahti E. Selection of peptides binding to the alpha 5 beta 1 integrin from phage display library. J Biol Chem. 1993 Sep 25;268(27):20205–20210. [PubMed] [Google Scholar]
  14. Lane D. P., Stephen C. W. Epitope mapping using bacteriophage peptide libraries. Curr Opin Immunol. 1993 Apr;5(2):268–271. doi: 10.1016/0952-7915(93)90016-l. [DOI] [PubMed] [Google Scholar]
  15. Lasonder E., Schellekens G. A., Welling G. W. A fast and sensitive method for the evaluation of binding of phage clones selected from a surface displayed library. Nucleic Acids Res. 1994 Feb 11;22(3):545–546. doi: 10.1093/nar/22.3.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lowman H. B., Bass S. H., Simpson N., Wells J. A. Selecting high-affinity binding proteins by monovalent phage display. Biochemistry. 1991 Nov 12;30(45):10832–10838. doi: 10.1021/bi00109a004. [DOI] [PubMed] [Google Scholar]
  17. McCafferty J., Griffiths A. D., Winter G., Chiswell D. J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990 Dec 6;348(6301):552–554. doi: 10.1038/348552a0. [DOI] [PubMed] [Google Scholar]
  18. Rebar E. J., Pabo C. O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science. 1994 Feb 4;263(5147):671–673. doi: 10.1126/science.8303274. [DOI] [PubMed] [Google Scholar]
  19. Scott J. K., Smith G. P. Searching for peptide ligands with an epitope library. Science. 1990 Jul 27;249(4967):386–390. doi: 10.1126/science.1696028. [DOI] [PubMed] [Google Scholar]
  20. Smith G. P., Scott J. K. Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 1993;217:228–257. doi: 10.1016/0076-6879(93)17065-d. [DOI] [PubMed] [Google Scholar]
  21. Stahl S. J., Murray K. Immunogenicity of peptide fusions to hepatitis B virus core antigen. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6283–6287. doi: 10.1073/pnas.86.16.6283. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES