Part:BBa_K5382120

InaK-linker-Im7_Cell membrane anchoring protein and immune protein complex

The InaK is a kind of ice nucleoprotein of Pseudomonas syringae KCTC1832. Ice nucleoprotein is a kind of protein that can promote the formation of ice crystals in organisms. Its N-terminal domain can be tightly anchored to the surface of E. coli cells as an anchor moda, so as to enable surface display.
The linker here is a short peptide sequence used to link InaK and Im7.
The Im7 is an immune protein that has a high affinity with CL7(an engineered variant of colicin CE7).Through their high affinity, CL7-linker-sfGFP (another composite part we registered) can be tightly bound to InaK-linker-Im7 and the InaK in CL7-linker-sfGFP can be tightly anchored to the cell membrane, thus anchoring the entire system to the membrane surface. Finally, the InaK-linker-Im7-CL7-linker-sfGFP system is displayed on the surface of the membrane(Figure 1).Additionally, the sfGFP is a green fluorescent protein. In this system, sfGFP can be used to verify the anchoring of the system through fluorescence confocal experiments (for details, please refer to the engineering section).
After validation is completed, replacing sfGFP with targeting elements such as single-chain antibodies can achieve targeting functions. It should be noted that our designed system utilizes the high affinity of CL7 and Im7, so some components can be flexibly replaced according to needs, suitable for different usage scenarios. For example, we can replace the ice nucleation protein with different ones for different cells, and replace sfGFP with different single-chain antibodies, nanobodies, etc., according to different targeting targets.

Figure 1. Schematic diagram of the targeting plug-in pairing system for Im7 protein to CL7 fusion pair displayed on the EcN surface.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1800
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 722
  Illegal NgoMIV site found at 962
  Illegal NgoMIV site found at 1130
  Illegal AgeI site found at 517
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 692
  Illegal BsaI.rc site found at 1182
  Illegal SapI.rc site found at 326