Part:BBa_K4739004

Hcp-Minihepcidin fusion protein

The fusion protein was constructed based on BBa_K4739000(AAAGGG linker), BBa_K4739001(Minihepcidin), BBa_K4739002(Hcp), and a FLAG tag for antigen binding in western blot analysis. By replacing the sequence of Minihepcidin and FLAG tag with the sequence of required protein <25kD, the fusion protein should enable the T6SS of the engineered bacteria to deliver the protein into target or in its environment.

The T6SS needle's inner tube, vital for the functionality of T6SS, is constructed from hexamers of the Hcp protein, which are donut-shaped and stacked in a head-to-tail orientation to form a hollow tube. Its inner diameter is ∼40 Å and has an external diameter of ∼110 Å, limiting the size of effector <25kD (Cherrak et al., 2019). The Hcp hexameric rings and associated structures form the tube through which toxins or effectors can be transported during the secretion process. In our project, we utilize this structure to carry the drugs we aim to carry. For example, minihepcidin, and we witnessed a successful secretion through western blot. Results can be viewed from the result section of our wiki.

Minihepcidin is a peptide drug treating β-thalassemia and polycythemia vera (PV), two disorders related to erythropoiesis (red blood cell production). β-Thalassemia is characterized by ineffective erythropoiesis, anemia, and iron overload, while PV is associated with an overproduction of erythrocytes and an increased risk of blood clots. Minihepcidins are agonists of hepcidin, a key regulator of iron homeostasis, which has been shown to prevent iron overload and induce iron-restricted erythropoiesis in certain contexts. In mouse models of β-thalassemia, minihepcidin alleviated ineffective erythropoiesis, anemia, and iron overload. Furthermore, in PV mouse models that mimic the human condition through the expression of an orthologous JAK2 mutation, minihepcidin administration reduced spleen size and normalized hematocrit levels. Thus, minihepcidins showcase therapeutic potential for managing β-thalassemia and PV by mitigating various symptomatic aspects of these disorders. In our project, we link this drug to Hcp component of T6SS to utilize T6SS to deliver this drug. Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 487
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 142
  Illegal NgoMIV site found at 265
  
• 1000
  COMPATIBLE WITH RFC[1000]

Engineering and Results

Testing results can be viewed in BBa_K4739002.

Reference

Cherrak, Y., Flaugnatti, N., Durand, E., Journet, L., & Cascales, E. (2019). Structure and Activity of the Type VI Secretion System. Microbiology Spectrum, 7(4). https://doi.org/10.1128/microbiolspec.psib-0031-2019