Optimization of a PDGFRβ binding peptide through peptide array analysis

Objectives Peptide arrays are a powerful method to identify peptide motifs that mediate protein-peptide interactions. Hundreds of peptides can be synthesized on the chip surface and therefore checked in one assay. This work focuses on the linear peptide PDGFR-P1 binding to the extracellular domain of PDGFRβ. Randomizing the complete dodecapeptide on an array should give information about the binding region of the peptide and improve the affinity to its target.

Methods A peptide array was constructed, randomizing the complete original peptide sequence by replacing every amino acid of the original peptide by all 20 naturally occurring amino acids. The results of the array were validated in cell assays. The peptides were radiolabelled with 125I and characterized further in kinetic and competition experiments in comparison to the original peptide on human pancreatic BxPC-3 cells, known to overexpress the target. Binding to the extracellular domain of PDGFRβ was examined using Biacore X100.

Results The pattern of the array showed that amino acids 1-4 are not important for binding to PDGFRβ, amino acid positions 5, 6 and 12 are tentatively crucial and amino acids 7 to 11 are indispensible. The spot G2 with a change from serine to arginine in position 7 of the original peptide was found to be the most prominent one and therefore used for further studies. A Fragment of G2 was also evaluated. All results of the peptide array could be confirmed in cell assays as well as on Biacore X100. In vitro, the two peptides showed a binding of 45 % binding/ [applied dose/ 1 mio. cells] to BxPC-3 cells compared to 12 % of the original peptide. Competition assays demonstrated an inhibition of radiolabelled peptides with cold peptide up to 96 %.

Conclusions Using a peptide array the original peptide could be improved by amino acid exchange in position 7 from serine to arginine. Both peptides, the fragment and G2, show a significantly higher affinity to the target protein compared to the original peptide PDGFR-P1