Part:BBa_K5302005

miniZ

This year, the USTC iGEM team has utilized the competitive binding of vascular endothelial growth factor (VEGF) to develop a targeted bacterial therapy for solid tumors. Our quest for the optimal VEGF-binding protein(or peptide) led us to an in-depth exploration of proteins structurally akin to the vascular endothelial growth factor receptor (VEGFR), which we have named VEGFR-like. This part is derived from three helix 58-residue Z-domain of staphylococcal protein A. And through stabilizing mutations and the addition of a disulfide constraint the Z-domain is reengineered into a two-helix 34-residue “mini-Z” version that retains the parent's affinity. This is supposed to be more potent binders against VEGF. We used pBBR1MCS-2 plasmid as a backbone and transfered miniZ into Escherichia coli Nissle 1917, and finally succeeded in expressing miniZ.

Figure 1. sequence of miniZ and its KD with VEGF

Figure 2. Cartoon representation of the crystal structure of the mini-Z highlighting randomized residues shown as sticks and coloring the helices

Figure 3. Colony PCR results of pBBR-INP-miniZ

Figure 4. SDS-PAGE analysis of pBBR1MCS-INP-miniZ expression in Escherichia coli Nissle 1917

Figure 5. Colony PCR results of pBBR-OmpA-miniZ

Figure 6. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 (1)

Figure 7. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 (2)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]