Part:BBa_K3428004
COP1(WD40)-VP16 fusion protein
COP1(WD40)-L-VP16 is a fusion protein which consists of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) binding domain fused to a VP16 activation domain via a 3x Gly-4-Ser3 linker. E3-ubiquitin ligase COP1 (from Arabidopsis thaliana) is the binding partner for the photoreceptor UV-B resistance locus 8 (UVR8) (BBa_K3428000). The ubiquitin ligase activity was removed from COP1 so that only the WD40 domain remains. Upon stimulation of UV-B light (~300 nm), UVR8 undergoes a conformational change allowing it to exit from its homodimer state. The monomeric UVR8 can then interact with COP1 and the complex accumulates in the nucleus, where it can regulate the expression of a particular gene. A flexible (Gly4Ser)3 peptide linker was used to connect these two proteins (BBa_K416001). Viral protein 16 (VP16) from Herpes simplex virus-1 (HSV-1) is a transcriptional activation domain (BBa_K1150001). Stony Brook 2020 took advantage of the UVR8_COP1 system’s ability to activate gene expression to induce transcription of a siRNA molecule.
To further mitigate the possibility of crosstalk between the COP1 fusion protein, a number of mutants were also produced, but are not listed to prevent redundancies. These mutants are as follows: COP1 - W467F, COP1 - R465E. Note that these mutations are labelled for their position in the full length COP1 protein. Within the coding sequence of these fusion proteins, the mutation W467F is equivalent to W133F, and the mutation R465E is equivalent to R131E. Respectively, these mutations are hypothesized to weaken interactions between COP1 and it’s binding partners and to abolish or weaken interactions between COP1 and STO, STH and CRY1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 435
Illegal SapI.rc site found at 376
| None |