Part:BBa_K2609011

mcp-1 with PelB under T7 expression system

This part generates MCP-1 (a monocyte chemokine: this is the mouse equivalent - JE) fused to a PelB secretion tag when transformed into a T7 expression system like BL21 (DE3). The mcp-1 protein is a monocyte chemoattractant belonging to the C-C family of chemokines. The PelB secretion tag fused to its N terminus transports the protein to the periplasmic space and is subsequently cleaved off.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 488
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 65
  Illegal NgoMIV site found at 91
  
• 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 PelB secretion signal peptide to allow the transport of protein from the cytoplasm to the periplasmic space, via the Sec-dependent pathway^[4]), where it is cleaved off by a protease before further transport of the fused protein. We believe that the small size of the protein compared to the average pore size in the cell wall will translate to a significant enough diffusion of the chemokine to the extracellular medium for an observable chemotaxis to occur.

Usage

IISc- Bangalore iGEM 2018:

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

Additional uses

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).

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.