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 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, 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, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1800
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 692
Illegal BsaI.rc site found at 1182
Illegal SapI.rc site found at 326
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