Part:BBa_K5302019

pBBR-OmpA-ZVEGF

This work is derived from pBBR1MCS-OmpA-mCherry and PUC19-ZVEGF-V114-V107-peptide, and it has undergone codon optimization. This composite part combines OmpA(21.4kda) and ZVEGF(approximately 6kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express ZVEGF. The plasmid uses lac promotor and has kanamycin resistence.

Figure 1. Colony PCR results of pBBR-OmpA-ZVEGF

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ZVEGF is a 59-residue three-helix peptide that was developed by Fedorova et al. by randomizing 9 residues on helices 1 and 2 of the Z-domain scaffold via phage display, followed by selection for binding to the VEGF8-109 dimer. A co-crystal structure of ZVEGF with VEGF8-109 shows that the engineered Z-domain adopts the expected three-helix bundle tertiary structure and engages the receptor-recognition sites on the VEGF dimer through a surface formed by helices 1 and 2 of ZVEGF. It shows great affinity with VEGF(Ki=0.41 μM).We used pBBR1MCS-2 plasmid as a backbone and transfered ZVEGF into Escherichia coli Nissle 1917, and finally succeeded in expressing ZVEGF.

Figure 2. ZVEGF sequence

Figure 3. VEGF-binding site of ZVEGF

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4100
  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 4100
  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 4100
  Illegal BglII site found at 1754
  Illegal BamHI site found at 3146
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4100
  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 4100
  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 4758
  Illegal AgeI site found at 3789
  Illegal AgeI site found at 4598
  
• 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