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 [1] and 1N8Z [2]) 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.
As the structural conformation of the split T7 RNA polymerase is not known, we reconstructed the natural T7 RNAP structure from the two fragments of our split T7: N-term part from residue 1 to 180, and C-term part from residue 181 to 704. This was achieved using AlphaFold2 with MMseqs2, a relaxation step, and the structure of the full-length T7 RNAP from PDB (1CEZ) as a template, as shown in Figure 3 A and B.
Once these structures were validated, we focused on the optimization of the transmembrane (TM) linker to allow adequate flexibility without being too long. The chosen TM part was ZipA (membranome ID: ZIPA_ECOLI [3]), a cell division protein localized in the inner membrane of Gram negative bacteria through a single TM segment. We selected this TM domain because it is from an E. coli protein from which PURE system derives, it consists of a single helix, and its length is compatible with the lipid composition of our liposomes. We included additional residues between the transmembrane segment and the adjacent structural domains to promote flexibility.
The disordered amino acid sequence we used as a basis was: GAAASEGGGSGGPGSGGEGSAGGGSAGGGS
We simulated different potential conformations of our biosensor (part BBa_K4768008 coupled to part BBa_K4768007) using a statistical algorithm called MoMA-FReSa [4] that was developed in the Laboratoire d’Analyse et d’Architecture des systèmes (LAAS, Toulouse). It samples protein conformations, including disordered regions, and gives a set of conformations that are statistically the most probable. We tested different lengths for the linker regions flanking the TM segment and chose the optimal one of residues by comparing it to a standard disordered linker as a reference : 52 amino acids between the antibodies and TM, and 32 amino acids between TM and the T7 RNAP . The results of the simulation comforted us in the disordered characteristic of the chosen sequence.
Sequence of the transmembrane linker for NT7-ZipA-Pertuzumab:
NT7-SGGGASGGGASGEGGSGPGGSGGGESAAAGSGRLILIIVGAIAIIALLVHGFGASGEGGSGPGGSGGGESAAAGSGGGASGGGASGEGGSGPGGSGGGESAAAG-Pertuzumab
Sequence of the transmembrane linker for Trastuzumab-ZipA-CT7:
Trastuzumab-GAAASEGGGSGGPGSGGEGSAGGGSAGGGSGAFGHVLLAIIAIAGVIILILRGSGAAASEGGGSGGPGSGGEGSAGGGSAGGGS-CT7
Figure 4 shows different conformations adopted by the chosen sequence.
Conclusion and Perspectives
These results show that the part BBa_K4768008 coupled to the part BBa_K4768007 has the ability to create an anti-HER2 biosensor that promotes gene expression in the presence of the cancer biomarker HER2. In silico optimization of the linker sequences led to improved protein variants with greater flexibility on each side of the TM domain. We did not have time to study the engineered proteins experimentally. We encourage future iGEM teams to clone the selected HER2-induced RNAP variants and perform experimental assays of the biosensors, and to contact us for further details.
References
- article 1 xxxxxxxx
- article 2 xxxxxxx
| None |