1. ¹Pharmexa A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark
2. ²Cancer Research UK Cell Transformation Group, University of Dundee, UK

Correspondence: Dr GB Karlsson, Current address: Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. E-mail: Nilla.Karlsson@mtc.ki.se

³(Jensen's) current address: Symphogen A/S, Elektrovej, Bldg. 375, DK-2800 Lyngby, Denmark

⁴(Sørensen's) current address: Zealand Pharma A/S, Smedeland 26B, Dk-2600 Glostrup, Denmark

Received 11 February 2004; Revised 23 June 2004; Accepted 16 July 2004; Published online 21 September 2004.

Abstract

Small peptides that perturb intracellular signalling pathways are useful tools in the identification and validation of new drug targets. To facilitate the analysis of biologically active peptides, we have developed retroviral vectors expressing an intracellular scaffold protein that significantly enhances the stability of small peptides in mammalian cells. This approach was chosen because retroviral transduction results in efficient and controlled delivery of the gene encoding the effector peptide, while the scaffold protein not only stabilises the peptide but also facilitates the analysis and potential isolation of the target protein. Here, we have adapted a p53-responsive reporter assay to flow cytometry to demonstrate the versatility of this approach by using peptides with known Mdm2-binding activities inserted into a stable scaffold protein that is suitable for intracellular expression in multiple compartments of mammalian cells. This strategy should be generally applicable to the study of small biologically active peptides in diverse functional assays.