Difference between revisions of "Part:BBa K5302023"

Revision as of 01:51, 2 October 2024

pBBR-OmpA-V107

This work is derived from pBBRMCS-OmpA-mCherry and PUC19-ZVEGF-V114-V107-peptide, and it has undergone codon optimization. This composite part combines OmpA(21.4kda) and V107(approximately 2kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express V107. The plasmid uses lac promotor and has kanamycin resistence.

V107 is a small potent VEGF-binding peptide, which has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two V107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. It was shown that V107 is strikingly amphipathic, and in the complex with the VEGF, it adopts a mixed conformation with the disordered N-tail (residues 1–4), type-I β-turn (residues 6–9), extended region (residues 9–12), and C-terminal α-helix (residues 13–19) Hydrophobic Ile7, Ala8, Met10, Trp11, Trp13, Phe16, and Leu19 are situated at the interface with the VEGF molecule As for binding site, the hydrophobic side-chains of V107-Trp13, Phe16, and Leu19 contact VEGF residues Phe17, Met18, Tyr21, Lys48, Met81 and Ile83. The same cluster of residues on VEGF interacts with Flt-1D2 residues Pro143, Ile145, Leu204, and Leu221. This peptide is composed of 19 amino acids, that is GGNECDAIRMWEWECFERL, and it shows great affinity with VEGF(Kd=0.53 μM).We used pBBR1MCS-2 plasmid as a backbone and transfered V107 into Escherichia coli Nissle 1917, and finally succeeded in expressing V107.

Figure 1. VEGF-binding site of V107

Figure 2. VEGF-binding site of V107

Figure 3. Colony PCR results of pBBR-INP-V107

Figure 4. Structure of v107 in the VEGF-bound state

Sequence and Features