Part:BBa_K5427002:Design
KerDZ
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
To optimize the expression of KerDZ in E. coli, we applied several strategies. Codon optimization was performed to enhance translational efficiency in the host. Repetitive sequences were manually removed to prevent issues with genetic instability. We ensured that the GC content was within the optimal range for E. coli expression (37%), and illegal restriction sites were removed to comply with Biobrick standards. Alongside this KerDZ has two domains: a pro-protein and the proteolytic domain. We considered keeping the pro-protein within our clones as it is involved in the in vivo folding of the proteolytic domain and is naturally cleaved following secretion. If we only cloned the proteolytic domain this had the potential of affecting the in vivo folding of the enzyme, resulting in a loss of function.
Source
Elhoul, M. B., Jaouadi, N. Z., Rekik, H., Benmrad, M. O., Mechri, S., Moujehed, E., ... & Jaouadi, B. (2016). Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50. International Journal of Biological Macromolecules, 92, 299-315. https://doi.org/10.1016/j.ijbiomac.2016.07.009