Part:BBa_K4160014
GEMS receptor construct containing PR3 fused to HA-tag 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 (BBa_K4160004) fused to HA-tag (BBa_K1150016) as affinity domain (Figure 1) containing a linker of 31 amino acids.
This PR3 domain with HA-tag 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 fused to HA-tag as affinity domain. PR3 with HA-tag was fused to the GEMS receptor via a linker of 31 amino acids. In addition to ligand anti-PR3, this receptor should sense the ligand anti-HA as illustrated.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 760
Illegal XbaI site found at 2441
Illegal PstI site found at 191
Illegal PstI site found at 1129
Illegal PstI site found at 2124
Illegal PstI site found at 2285 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 760
Illegal NheI site found at 1498
Illegal PstI site found at 191
Illegal PstI site found at 1129
Illegal PstI site found at 2124
Illegal PstI site found at 2285
Illegal NotI site found at 2428 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 760
Illegal BglII site found at 1602
Illegal BglII site found at 1788
Illegal BglII site found at 2052
Illegal BamHI site found at 64
Illegal XhoI site found at 1012
Illegal XhoI site found at 2435 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 760
Illegal XbaI site found at 2441
Illegal PstI site found at 191
Illegal PstI site found at 1129
Illegal PstI site found at 2124
Illegal PstI site found at 2285 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 760
Illegal XbaI site found at 2441
Illegal PstI site found at 191
Illegal PstI site found at 1129
Illegal PstI site found at 2124
Illegal PstI site found at 2285 - 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 success of the designed library consisting GEMS receptors containing PR3 as affinity domain (BBa_K4160009, BBa_K4160010 & BBa_K4160011) of theTU-Eindhoven team 2022 is dependent on the correct folding of the truncated PR3. Incorrect folding of PR3 prevents the anti-PR3 antibody from binding to the affinity domain and hence prevents activation of the GEMS receptor. Therefore, a library of GEMS receptor constructs containing a HA-tag at the C-terminus of PR3 was designed. With these receptor constructs, it could not only be investigated whether the GEMS receptor could be activated by antibodies, in general, using anti-HA but also by disease-related antibodies using anti-PR3.
This part is a member of a library that was created. Additional parts of this library are the GEMS receptor constructs containing the PR3 fused to HA-tag as affinity domain fused to EpoR with no (BBa_K4160012) and a 8 amino acid (BBa_K4160013) 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 fused to HA-tag 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
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