Part:BBa_K1639007

DAMP-Pexiganan

Synthetic 22-amino-acid antimicrobial peptide(AMP) pexiganan, which is a magainin AMP analog isolated from the skin of the African clawed frog.

Usage and Biology

Pexiganan, a 22-amino-acid antimicrobial peptide, is an analog of the antibiotic magainin peptides isolated from the skin of the African clawed frog has been shown to exhibit broad-spectrum microbicidal activity and acts with a bactericidal mechanism against which the likelihood of the development of resistance may be low. Furthermore Pexiganan molecules’ dose for destroying erythrocytes is 250 μg/ml, the other dose for destroying H.pylori is 16 μg/ml.(Figure 1)

Figure 1: MICs of pexiganan and all-D-amino-acid pexiganan (MSI-214) for ATCC reference strain

Figure 2: Structure of DAMP4-Pexiganan

A significant challenge in production of this antimicrobial peptide(AMP) in E.coli is it's intrinsic antimicrobial activity and proteolytic degradation of peptide during expression. To overcome this AMP is fused to carrier protein. DAMP4 is a protein formed by connecting, at the DNA level, four surface-active AM1 peptides. DAMP4 expresses at high levels of solubility in recombinant bacteria, and forms a four helix bundle structure that is thermostable to 90 C and remains soluble at relatively high salt conditions(Figure 2)

Research conducted by Zhang, X. L. et al (2015) proves bactericidal activity of Pexiganan against H.pylori.(Figure 3) In the same research In vitro development of resistance studies were conducted.(Figure 4) In the 15th generation of H. pylori that had developed resistance to antibiotics, the H. pylori were still susceptible to pexiganan. The results showed that the pexiganan could be used to treat H. pylori infection that is resistant to some antibiotics.

Figure 3: Bactericidal kinetics study. The bactericidal activity of pexiganan against H. pylori were monitored for the first 1 h. After 0, 10, 20, and 60 min of exposure time at 37 °C, aliquots were diluted (serial 10-fold dilutions) and plated for CFU counts after 72 h incubation at 37 °C

Figure 4:In vitro development-of-resistance studies Evolution of MICs after successive exposures of H. pylori to subinhibitory concentrations of the Pexiganan. After 15 serial passages, the relative MIC was the normalized ratio of the MIC obtained for a given subculture to the MIC that obtained for first-time exposure.

In our project we aim produce enough pexiganan molecules to eradicate all of the H.pylori population in the stomach. We inserted TEV protease cleavage site between DAMP4-Pexiganan to control releasing of Pexiganan. Due to the presence of H. pylori in the environment, extremely high concentrations of NH3 and Al2 molecules induce AND GATE the system (BBa_K1639000). As a result of this activation, TEV protease molecules will be produced. The produced TEV Protease break the large amount of connections accumulated between DAMP and Pexiganan in the cells and releases pexiganan molecules (Figure 5)

Figure 5: Schematic diagram of AND gate system

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 108
  Illegal XhoI site found at 508
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]