Figure 3

Assembly of multi-domain fusion protein devices. (A) Basic parts containing protein-protein interaction ligands were built using three independent, degenerate sequences encoding an N-terminal glycine-serine linker and an SH3 interaction peptide. Composite parts were created by standard assembly of these basic parts. (B) Experimental design for testing interactions between bait and prey parts. The number of prey molecules pulled-down by bait should be dependent on the number of SH3 peptide ligands. (C) A GST-pulldown experiment was conducted and the proteins separated via SDS-PAGE and then imaged by Coomassie staining. No visible interaction was observed when either the bait lacked any SH3 peptide ligand or the prey lacked an SH3 domain. Bait containing composite part J62008 with four SH3 peptide ligands, despite visible proteolytic degradation, was sufficient to pull down increased amounts of prey compared to bait containing the single SH3 ligand part J62002.