Part:BBa_K5302030

pBBR-INP-V114

This work is derived from pBBR1MCS-INP-mCherry and PUC19-ZVEGF-V114-V107-peptide, and it has undergone codon optimization. This composite part combines INP(about 30kda) and V114(approximately 2.3kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express V114. The plasmid uses lac promotor and has kanamycin resistence.

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V114 has been identified as a small potent VEGF-binding peptide, which is composed of 8 amino acids--- that is VEPNc[CDIHVnLWEWEC]FERL, and there is a disulfide bond between N and F, making the stucture more stable. This peptide has a similiar structure and VEGF-binding site as V107, and it shows great affinity with VEGF(Kd=0.11 μM). We used pBBR1MCS-2 plasmid as a backbone and transfered V114 into Escherichia coli Nissle 1917, and finally succeeded in expressing V114.

Figure 1. This picture shows its VEGF-binding site and its affinity with VEGF

Figure 2. This picture shows the dependence of the amount of V114, expressed in normalized fluorescence values, on the peptide concentration

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