Part:BBa_K2609008

mcp-1 with HlyA signal peptide under a T4 phage recombination cassette

This part consists of MCP-1 (mouse equivalent of the monocyte chemokine) attached to the HlyA signal peptide through an OmpT cleavable linker.The sequence is flanked by the downstream and upstream sequences of the e-gene from the T4 phage. This part is intended for recombination into the phage using the Lambda red recombination system.

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]

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 with 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

IISc-Bangalore iGEM 2018:

This part was used for recombination into T4 phage genome, by means of the homologous sites (the 50 bp sequences upstream and downstream of the e-gene in the T4 genome), to replace the existing e-gene with the MCP-1 gene. The HlyA signal on the C-term ensures carriage of the protein to the extracellular space. The linker helps in cleavage of the HlyA peptide and ensures that the protein outside the cell is unattached to the signal peptide^[4] (OmpT dependent).

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