Part:BBa_K2609010

mcp-1 with HlyA under T7 expression system

This part generates MCP-1 (mouse equivalent of the monocyte chemokine) fused to a HlyA secretion tag (through an OmpT cleavable linker) when transformed into a T7 expression system like BL21 (DE3). The mcp-1 protein is a monocyte chemoattractant belonging to the CC family of chemokines. The HlyA secretion tag fused to its C terminus transports the protein to extracellular space using the Type 1 Hemolysin system, where it performs its function as a chemoattractant.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 665
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

Monocyte Chemoattractant protein (MCP-1/CCL2) is a key member of the C-C chemokine family, involved in the regulation of the recruitment and migration of monocytes/macrophages to the site of infection^[1]. This protein has been tagged with the HlyA secretion signal peptide (from the iGEM registry) to allow for the transport of protein from the cytoplasm to the extracellular medium via the Type I secretion system in Gram negative bacteria. The secretion system requires the presence of HlyB and HlyD proteins, which form a channel across the cell membrane and cell wall, only accessible to proteins with the HlyA signal peptide attached at the C - terminus.^[2] The signal peptide ends up being attached to the protein even after its transport to the extracellular medium, so a linker with evidence^[4] for getting cleaved by the OmpT protease, present in the periplasmic space in E coli, was used.

Usage

This part can be used to characterize the chemotaxis of bacteria produced MCP-1 using the HlyA signal peptide, to try and quantify the amount of chemokine that we can expect to be released in its properly folded and functional state using the HlyA signal peptide.

To produce MCP-1 tagged with HlyA

This part can be transformed into E.coli BL21(DE3) and overexpression of MCP-1 tagged with HlyA can be induced with IPTG.

Utility as T7 expression backbone

The T7 expression adapter, obtained by the digestion of this part with HindIII and NheI, can be used to express any protein of interest (the coding sequence of the required protein, with the mentioned restriction sites added using overhang PCR can be inserted into the backbone).

The 2018 IISc-Bangalore iGEM Team used this part to asses whether HlyA tagged MCP-1, when delivered to a bacterial pathogen using a phage, will allow quick removal of the pathogen by the circulating monocytes in the region.

References

[1] Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29(6):313–326.

[2] GRAY, LINDSAY, et al. "A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium." J Cell Sci 1989.Supplement 11 (1989): 45-57.

[3] Zhang, Fang, et al. "Secretion and circular dichroism analysis of the C-terminal signal peptides of HlyA and LktA." Biochemistry 34.13 (1995): 4193-4201.

[4] Hanke, Christian, et al. "Processing by OmpT of fusion proteins carrying the HIyA transport signal during secretion by the Escherichia coli hemolysin transport system." Mol Gen Genet (1992) 233:42-48