Part:BBa_K5302031

pBBR-INP-V107

This work is derived from pBBR1MCS-mCherry and PUC19-ZVEGF-V114-V107-peptide, and it has undergone codon optimization. This composite part combines INP(about 30kda) 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.

Figure 1. Colony PCR results of pBBR-INP-V107

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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 2. VEGF-binding site of V107

Figure 3. VEGF-binding site of 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 4338
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4338
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal NotI site found at 1057
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4338
  Illegal BglII site found at 1803
  Illegal BamHI site found at 3195
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4338
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4338
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal NgoMIV site found at 2467
  Illegal NgoMIV site found at 2750
  Illegal NgoMIV site found at 3616
  Illegal NgoMIV site found at 5003
  Illegal AgeI site found at 4843
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1379
  Illegal SapI.rc site found at 2316
  Illegal SapI.rc site found at 2526