Team:TU-Eindhoven/Scaffold Design Tetramer
The second scaffold protein we designed is a tetrameric version of T14-3-3. A tetramer is a protein formed from four monomers, which means that the tetrameric T14-3-3 can enable the assembly of four CT52 coupled proteins instead of just two. The tetrameric T14-3-3 is split into two different variants; The homotetramer (four equal monomers) and the heterotetramer (four non-equal monomers).
The homotetrameric T14-3-3 was designed by linking two wildtype T14-3-3 dimers to each other with a flexible GGS10-linker forming a tetramer. The GGS10-linker is a ten times repeating sequence of Glycine-Glycine-Serine amino acids.
The heterotetrameric T14-3-3 was designed in a similar way as the homotetrameric variant, the only difference is that it now consists of four different monomers with four different binding pockets. One monomer is maintained as a wildtype. In one monomer the E19R mutation is introduced, since the functioning of this mutation has already been confirmed. In the other two monomers mutations were introduced that we have found through computational modelling. Only one of the S71L-I72V and S71L mutations is chosen to prevent decreased orthogonality because of expected similarity between those mutations. The mutation introduced in the last monomer is the W237R mutation. Composition of the heterotetramer: T14-3-3-S71L-I72V - T14-3-3-W237R - T14-3-3-E19R - T14-3-3(wildtype).
The ability of the homotetramer to assemble four equal proteins can be used to create a kill switch. The caspase-9 protein is part of the caspase family; Proteases that have been associated with apoptosis. Activated caspase-9 cuts downstream caspases initiating a caspase cascade.1 Caspase-9 is essential for the induction of apoptosis, utilizing this feature we created a kill switch by linking caspase-9 to CT52. It is not quite understood how caspase-9 is activated, but there is evidence that procaspases activate each other.2 By letting four CT52-caspase-9 proteins assemble on the scaffold under influence of fusicoccin instead of two, the local concentration of caspase-9 will be higher, resulting in the activation of caspase-9.
The heterotetrameric variant could be used to assemble four non-equal proteins, therefore the BRET system was designed. The BRET system consists of three parts: mNeonGreen, SmallBiT and LargeBiT. mNeonGreen is linked to CT52-I947F and to CT52-S953K, SmallBiT is linked to CT52(wildtype) and LargeBiT is linked to CT52-K943D.
The two CT52 split NanoLuc fragments will bind next to each other on the tetrameric scaffold and lead to a functional NanoLuc system, emitting light at a wavelength of 460 nm. On the other free binding sites CT52 with linked mNeonGreen will bind. In the BRET system, NanoLuc will be the donor and mNeonGreen will be the acceptor that changes the wavelength of the light emitted by NanoLuc to a wavelength of 517 nm.
The pET28a vector has a single cloning site, so the pET28a vector can be used for the expression of one gene and thus the production of one type of protein. The pET28a vector is a plasmid that is often used, it contains Kanamycin resistance and can provide the target protein with a N- and C-terminal His-tag. The pET expression system is one of the most used vector systems for cloning and expression of proteins in E. coli. This is due to the high selectivity and activity of the T7 RNA polymerase, which is utilized in the pET system. The pET vectors contain a T7 promoter and a T7 termination sequence. To start expression of the target protein, a source of T7 RNA polymerase has to be added to the host. This gives the pET system another benefit, namely the ability to maintain target genes transcriptionally silent by inducing pET vectors in hosts, who contain a T7 RNA polymerase gene that requires the precence of a small molucule for their transcription. To provide the host with the T7 RNA polymerase, IPTG is used, IPTG is a molecular mimic of the allolactose molecule. The E.coli strain that is used, contains a lac promoter that initiates the transcription of the T7 RNA polymerase gene when activated. IPTG activates this promoter resulting in the production of T7 RNA polymerase, which leads to expression of the target genes in the pET vector.
- [1] Kuida, K. (2000). Caspase-9.The international journal of biochemistry & cell biology, 32(2), 121-124
- [2] Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., & Alnemri, E. S. (1998). Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Molecular cell, 1(7), 949-957.
