Part:BBa_K4444003
PhoCl Shed GFP
PhoCl Shed mRuby is modified from the RELEASE system. It contains a GFP as a protein of interest, PhoCl2c, and a tri-transmembrane domain; the ER retention motif, furin cut site, and HCV cut site were all removed. PhoCl Shed will keep the protein of interest on the cell membrane until violet light is shined on it, which then breaks PhoCl and releases all the protein from the surface of the cell membrane.
Usage and Biology
Our project builds on the protein circuit RELEASE (Vlahos et al.) by introducing light-inducible proteins or photoswitches. Photocleavable protein (PhoCl) is a recently developed optogenetic technology that self-cleaves when stimulated by 405 nm ultraviolet light. PhoCl was engineered from a green-to-red photoconvertible fluorescent protein. When stimulated with ultraviolet light, the protein undergoes a β-elimination reaction, producing a small peptide fragment and a large barrel fragment that will spontaneously dissociate (Zhang et al.).
Mechanism: POI has no retention tag and continues along the secretion pathway to the plasma membrane where it remain bound. Upon light-activation Phocl breaks and the POI is shed from the membrane.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 52
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 775
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 946
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 60
Illegal BsaI.rc site found at 1468
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