Part:BBa_K4768008
Split T7 RNA polymerase (Nterm) conjugated to Pertuzumab with a transmembrane linker
Part for Expression of the T7 RNA polymerase (Nterm) conjugated to Pertuzumab with a transmembrane linker ZipA
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 987
Illegal XbaI site found at 40
Illegal SpeI site found at 1147 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 987
Illegal SpeI site found at 1147 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 987
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 987
Illegal XbaI site found at 40
Illegal SpeI site found at 1147 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 987
Illegal XbaI site found at 40
Illegal SpeI site found at 1147
Illegal AgeI site found at 1200 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 21
Illegal SapI.rc site found at 1836
Introduction
The CALIPSO part BBa_K4768008 is composed of the N-terminal subunit of the T7 RNA polymerase (residues 1 to 180) fused to the anti-HER2 antibody Pertuzumab through an optimized linker sequence containing a transmembrane domain. This gene is under transcriptional control of an SP6 promoter and T7 terminator.
This part, coupled to the part BBa_K4768007 containing the C-terminal subunit of the T7 RNA polymerase, has been designed to develop a split T7 RNAP-based biosensor capable of recognizing HER-2, an epidermal growth factor that is overexpressed in cancer cells. This biosensor will be incorporated into the liposome membrane to detect cancer cells via HER2-targeting antibodies and activate the expression of a gene of interest inside the liposome.
The HER2-induced T7 RNAP complex was designed from two existing constructs: a split T7 RNAP-based biosensor for the detection of rapamycin and a split luciferase conjugated with antibodies capable of recognizing HER2. We decided to merge the relevant functionalities of these two constructs and created a new biosensor that transduces HER2 binding to gene expression activation.
The part BBa_K4768008 was only studied in silico by molecular modeling to optimize the transmembrane linker for adequate flexibility.
Molecular Modeling
As the biosensor encompasses both extracellular and intracellular domains, the challenge was to find a transmembrane linker that is flexible enough to enable complementation of the split T7 RNAP and simultaneous recognition of HER2.
We first studied the extracellular structure of our biosensor involving the simultaneous interaction of the two antibodies with the soluble extracellular domain of HER2. We retrieved the known structure from the Protein Data Bank (1S78 and 1N8Z) to verify that there were no steric hindrances when both antibodies were bound to HER2. Figure 2 shows the simultaneous binding of the antibodies to distinct epitopes of HER2. In addition, the two anchoring points of the linker sequences are located on the opposite side of the interface with HER2, and are thus accessible.
Characterisation
TXXXXXX.
Conclusion and Perspectives
TXXXXXX.
References
- article 1 xxxxxxxx
- article 2 xxxxxxx
| None |