Part:BBa_K5302041

pBBR-INP-VEGFR1D2-l2masking#29

This work is derived from pBBR-INP-VEGFR1D2 and pUC19-VEGFR-masking#29, and it has undergone codon optimization. This composite part combines INP(about 30kda) and VEGFR1D2(approximately 12kda), and it adds a fragment as a mask. We succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express VEGFR1D2. The plasmid uses lac promotor and has kanamycin resistence.

Figure 1. Colony PCR results of pBBR-INP-VEGFR1D2-l2masking#29

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This linear 13-mer peptide is derived from the N-terminal amino acids of Vascular Endothelial Growth Factor (VEGF) and forms a helix structure. It exhibits high affinity for VEGFR-like receptors, with an IC50 value of 0.05 μM, allowing it to effectively compete with VEGF for binding to VEGFR. Consequently, this peptide has been utilized as a masking agent. Upon administration into the human body, it preemptively binds to VEGFR, preventing VEGF from engaging with the receptor. Given that matrix metalloproteinases (MMPs) are present at high concentrations in the tumor microenvironment (TME), they can degrade this VEGFR-masking peptide (designated as #29). This degradation allows VEGF to subsequently bind to the VEGFR-like receptors, triggering their activation. Therefore, this peptide functions as a biological switch, becoming active when exposed to the TME upon the entry of engineered chimeric nanoparticles ( Escherichia coli Nissle 1917).

Figure 2. Dose–response curve for compound 29

Figure 3. VEGFR-mask-#29, from α-fold

Figure 4. The binding site of D2 and the mask can be identified as being identical to the binding site of D2 with VEGF, indicating that they can compete for binding. The linker employed consists of five repeats of the GGGGS sequence, from α-fold

Figure 5. The linker has been reduced to three repeats of the GGGGS sequence, from α-fold

Figure 6. The linker has been modified by replacing the middle GGGGS sequence with a matrix metalloproteinase (MMP) recognition sequence, while maintaining the binding site unchanged, from α-fold

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4605
  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 4605
  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 4605
  Illegal BglII site found at 1803
  Illegal BamHI site found at 3195
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4605
  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 4605
  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 5270
  Illegal AgeI site found at 4193
  Illegal AgeI site found at 5110
  
• 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