PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of narLink to Publisher's site
 
Nucleic Acids Res. 1995 April 11; 23(7): 1152–1156.
PMCID: PMC306824

Protein-peptide interactions analyzed with the yeast two-hybrid system.

Abstract

The yeast two-hybrid system was used to screen a library of random peptides fused to a transcriptional activation domain in order to identify peptides capable of binding to the retinoblastoma protein (Rb). Seven peptides were identified, all of which contain the Leu-X-Cys-X-Glu motif found in Rb-binding proteins, although their activity in the yeast assay varied over a 40-fold range. Mutagenesis of the DNA encoding two of these peptides followed by screening in the two-hybrid system allowed the delineation of residues apart from the invariant Leu, Cys and Glu that affect binding to Rb. Binding affinities of a peptide and one of its variants to Rb, determined by surface plasmon resonance, correlated with results from the two-hybrid assay. This method offers several advantageous features compared to existing technology for screening peptide libraries: in vivo detection of protein-peptide interactions, high sensitivity, the capacity for rapid genetic screening to identify stronger and weaker binding peptide variants, and the use of a simple assay (transcriptional activity) as a means to assess binding affinity.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (887K), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science. 1990 Jul 27;249(4967):386–390. [PubMed]
  • Cwirla SE, Peters EA, Barrett RW, Dower WJ. Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6378–6382. [PubMed]
  • Devlin JJ, Panganiban LC, Devlin PE. Random peptide libraries: a source of specific protein binding molecules. Science. 1990 Jul 27;249(4967):404–406. [PubMed]
  • Cull MG, Miller JF, Schatz PJ. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1865–1869. [PubMed]
  • Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmierski WM, Knapp RJ. A new type of synthetic peptide library for identifying ligand-binding activity. Nature. 1991 Nov 7;354(6348):82–84. [PubMed]
  • Needels MC, Jones DG, Tate EH, Heinkel GL, Kochersperger LM, Dower WJ, Barrett RW, Gallop MA. Generation and screening of an oligonucleotide-encoded synthetic peptide library. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10700–10704. [PubMed]
  • Ohlmeyer MH, Swanson RN, Dillard LW, Reader JC, Asouline G, Kobayashi R, Wigler M, Still WC. Complex synthetic chemical libraries indexed with molecular tags. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10922–10926. [PubMed]
  • Mattheakis LC, Bhatt RR, Dower WJ. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9022–9026. [PubMed]
  • Fields S, Sternglanz R. The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 1994 Aug;10(8):286–292. [PubMed]
  • Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. [PubMed]
  • Li B, Fields S. Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system. FASEB J. 1993 Jul;7(10):957–963. [PubMed]
  • Hannon GJ, Demetrick D, Beach D. Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev. 1993 Dec;7(12A):2378–2391. [PubMed]
  • Durfee T, Becherer K, Chen PL, Yeh SH, Yang Y, Kilburn AE, Lee WH, Elledge SJ. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. [PubMed]
  • Iwabuchi K, Li B, Bartel P, Fields S. Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene. 1993 Jun;8(6):1693–1696. [PubMed]
  • Iwabuchi K, Bartel PL, Li B, Marraccino R, Fields S. Two cellular proteins that bind to wild-type but not mutant p53. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6098–6102. [PubMed]
  • Wiman KG. The retinoblastoma gene: role in cell cycle control and cell differentiation. FASEB J. 1993 Jul;7(10):841–845. [PubMed]
  • DeCaprio JA, Ludlow JW, Lynch D, Furukawa Y, Griffin J, Piwnica-Worms H, Huang CM, Livingston DM. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell. 1989 Sep 22;58(6):1085–1095. [PubMed]
  • Dyson N, Howley PM, Münger K, Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. [PubMed]
  • Figge J, Breese K, Vajda S, Zhu QL, Eisele L, Andersen TT, MacColl R, Friedrich T, Smith TF. The binding domain structure of retinoblastoma-binding proteins. Protein Sci. 1993 Feb;2(2):155–164. [PubMed]
  • Moran E. A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products. Nature. 1988 Jul 14;334(6178):168–170. [PubMed]
  • Fisher RJ, Fivash M. Surface plasmon resonance based methods for measuring the kinetics and binding affinities of biomolecular interactions. Curr Opin Biotechnol. 1994 Aug;5(4):389–395. [PubMed]
  • Gordon EM, Barrett RW, Dower WJ, Fodor SP, Gallop MA. Applications of combinatorial technologies to drug discovery. 2. Combinatorial organic synthesis, library screening strategies, and future directions. J Med Chem. 1994 May 13;37(10):1385–1401. [PubMed]

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