Part:BBa_K5302023

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.

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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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3979
  Illegal XbaI site found at 3140
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3979
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  Illegal NotI site found at 1008
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3979
  Illegal BglII site found at 1754
  Illegal BamHI site found at 3146
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3979
  Illegal XbaI site found at 3140
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3979
  Illegal XbaI site found at 3140
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  Illegal NgoMIV site found at 2418
  Illegal NgoMIV site found at 2701
  Illegal NgoMIV site found at 4637
  Illegal AgeI site found at 4477
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1330
  Illegal SapI.rc site found at 2267
  Illegal SapI.rc site found at 2477