Difference between revisions of "Part:BBa K4160011"
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Revision as of 22:51, 11 October 2022
GEMS receptor construct containing PR3 as affinity domain with 31 amino acid linker
This composite part encodes for a Generalized Extracellular Molecule Sensor (GEMS) receptor construct. This part was developed by replacing the RR120 VHH affinity domain of BBa_K4160008 with a PR3 affinity domain (BBa_K4160004) containing a linker of 31 amino acids (Figure 1).
This PR3 domain is fused to the erythropoietin receptor (EpoR) (BBa_K4160001), a transmembrane receptor that forms the foundation of the GEMS receptor. At the intracellular side of the EpoR, the intracellular signal transduction domain IL-6RB (BBa_K4160002) is attached. Sensing and binding of ligand anti-PR3 to the affinity domain should induce dimerization of the EpoR. As a result, the IL-6RB domain should activate downstream signaling of the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. In this part, an Igκ secretion signal (BBa_K4160000) is incorporated. This signal localizes the GEMS receptor to the membrane of mammalian cells. Furthermore, at the C-terminus of the part, a bovine growth Hormone polyadenylation (bGH poly A) signal is located which medicates efficient transcription termination and polyadenylation.1
Figure 1 | |GEMS receptor construct containing PR3 as affinity domain. PR3 was fused to the GEMS receptor via a linker of 31 amino acids. This receptor should sense the ligand anti-PR3.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 733
Illegal XbaI site found at 2414
Illegal PstI site found at 191
Illegal PstI site found at 1102
Illegal PstI site found at 2097
Illegal PstI site found at 2258 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 733
Illegal NheI site found at 1471
Illegal PstI site found at 191
Illegal PstI site found at 1102
Illegal PstI site found at 2097
Illegal PstI site found at 2258
Illegal NotI site found at 2401 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 733
Illegal BglII site found at 1575
Illegal BglII site found at 1761
Illegal BglII site found at 2025
Illegal BamHI site found at 64
Illegal XhoI site found at 985
Illegal XhoI site found at 2408 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 733
Illegal XbaI site found at 2414
Illegal PstI site found at 191
Illegal PstI site found at 1102
Illegal PstI site found at 2097
Illegal PstI site found at 2258 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 733
Illegal XbaI site found at 2414
Illegal PstI site found at 191
Illegal PstI site found at 1102
Illegal PstI site found at 2097
Illegal PstI site found at 2258 - 1000COMPATIBLE WITH RFC[1000]
Usage & biology
This GEMS receptor construct is based on the GEMS system that is developed by Scheller et al., 2018.2 The authors developed this highly modular synthetic receptor construct that allows for the coupling of an extracellular input to an intracellular signaling pathway.2 The modularity of this receptor allows the designing of GEMS platforms that sense and respond to a wide variety of extracellular molecules.2
The TU-Eindhoven team 2022 developed this part to investigate whether the GEMS receptor could be activated using autoantibodies, specifically anti-PR3, as a ligand. This part is a member of a library that was created. Additional parts of this library are the GEMS receptor constructs containing the PR3 affinity domain fused to EpoR with no (BBa_K4160009) and an 8 amino acid (BBa_K4160010) linker.
This composite part was used in combination with the transcription factor Signal Transducer and Activator of transcription 3 (STAT3) (BBa_K4160005) and the part that encodes STAT-induced SEAP (BBa_K4160016). This part was expressed using a pLeo619-Psv40 mammalian expression vector (GenBank accession no. MG437012).3
Characterization
Unfortunately, cloning of PR3 containing a 31 amino acid linker into the pLeo619-Psv40 expression vector was unsuccessful. Therefore, no colonies were grown after transfection and no characterization experiments could be performed.
References