Part:BBa_K5302024

pBBR-INP-VEGFR1D2

This work is derived from pBBR1MCS-INP-mCherry and pUC19-VEGFR1D2, and it has undergone codon optimization. This composite part combines INP(about 30kda) and VEGFR1D2(approximately 12kda), 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 （1）

Figure 2. Colony PCR results of pBBR-INP-VEGFR1D2 （2）

Figure 3. Colony PCR results of pBBR-INP-VEGFR1D2 （3）

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VEGF binds to two tyrosine kinase receptors, VEGF receptor 1 (VEGFR1/Flt-1) and receptor 2 (VEGFR2/KDR), which are localized on the cell surface of various cell types. The receptors are organized in two intracellular kinase domains, a short transmembrane region and an extracellular portion constituted of seven immunoglobulin-like domains. The binding of VEGF to the extracellular domain induces receptor dimerization, transphosphorylation of the kinase domains, and activation of the intracellular signaling. Deletion studies have shown that the ligand binding function resides within the first three domains of VEGFR14 and in domains 2 and 3 of VEGFR2. Deletion of the second extracellular domain of VEGFR1 (VEGFR1D2) completely abolishes the binding to VEGF, whereas VEGFR1D2 binds VEGF with an affinity that is only 60 times lower than the entire extracellular portion of the receptor. The equilibrium constant for the binding of VEGF to one VEGFR1D2 molecule was determined by surface plasmon resonance. The obtained value, 2.9 ± 0.2 nM, is consistent with the constant reported for the binding of VEGF to monomeric VEGFR1D2. We used pBBR plasmid as a backbone and transfered VEGFR1D2 into Escherichia coli Nissle 1917, and finally succeeded in VEGFR1D2-expressing.

Figure 4. Predictions of VEGFR1D2 Secondary Structure

Figure 5. VEGFR1D2 Trypsin Digestion Analysis(Numbers refer to the human VEGFR1 protein sequence, underlined residues are involved in a disulfide bridge, the proline in bold indicates that the Arg-Pro bond is not cleaved by trypsin22. exp = experimental, calc = calculated.)

Figure 6. structure of VEGFR1D2

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4584
  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 4584
  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 4584
  Illegal BglII site found at 1803
  Illegal BamHI site found at 3195
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4584
  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 4584
  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 5249
  Illegal AgeI site found at 4193
  Illegal AgeI site found at 5089
  
• 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