Part:BBa_K3581007
Hagfish intermediate filaments gamma subunit with BBa_K880005 part with HlyA signal peptide
This composite part includes composite part K880005, the gamma subunit BBa_K3581001, His tag BBa_K3581002, HlyA signal peptide BBa_K3581003 and terminator BBa_B0015. The aim of this part is to maximize protein expression of the gamma subunit of hagfish intermediate filaments through the use of strong promoters and RBS optimal for protein secretion. The His tag will be used for purifying the protein and TEV protease cut site can cleave out the tag once the protein is purified. The signal peptide is flanked by HindIII cut sites which will allow us to secrete the protein extracellularly, when we can also cut out the signal peptide using HindIII to secrete them as inclusion bodies to determine the best method with highest yield. The terminator is a double terminator which is commonly used for termination.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References:
1. Jing, F., A. Guerette, P., Pavesi, A., Horbelt, N., Chwee Teck, L., J. Harrington, M., & Miserez, A. (2017). Artificial hagfish protein fibers with ultra-high and tunable stiffness, 9(35), 12908–12915. The Royal Society of Chemistry.
2. England, N. (n.d.). TEV Protease | NEB. Retrieved October 13, 2020, from https://international.neb.com/products/p8112-tev-protease#Product%20Information
3. Wikipedia. (2020, October 14). TEV Protease. Wikipedia. Retrieved October 13, 2020, from https://en.wikipedia.org/wiki/TEV_protease
4. Palmer, I., & Wingfield, P. T. (2004). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Current protocols in protein science, Chapter 6, Unit–6.3. https://doi.org/10.1002/0471140864.ps0603s38
| None |