CFTR_pGHE FLAG Tag

CFTR_pGHE Plasmid Map

This is the CFTR_pGHE Plasmid we have in the lab. An epitope (FLAG) will be added to this plasmid in order to determine the receptor density with fluorescence microscopy.

CFTR_pGHE +FLAG Tag Plasmid Map

This is the CFTR_pGHE Plasmid map with the epitope FLAG added. FLAG was added into the CFTR protein coding sequence after N901 and before S902. This was between base pairs 2869 and 2870 in the CFTR_pGHE Plasmid. The insertion was between base pairs 2703 and 2704 in the CFTR protein coding sequence.

FLAG Tag

This is the FLAG Tag inserted into the CFTR_pGHE plasmid. The FLAG Tag is an 8 amino acid, 24bp epitope. The sequence is DYKDDDDK. FLAG is a common and well characterized hydrophilic tag. The TAG is between N901 and S902. It is in CFTR's 4th extracellular loop.

References

CFTR Domain Map:

Includes every amino acid and its position within the membrane, intracellular space and extracellular space. The 4th extra cellular loop is on the middle right portion of the image with 2 pitch forks (glycosylations).

Hildebrandt, E., Ding, H., Mulky, A., Dai, Q., Aleksandrov, A. A., Bajrami, B., ... & Kappes, J. C. (2015). A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface. Molecular biotechnology, 57(5), 391-405.

This article placed a FLAG epitope after N901 in CFTR and reported no problems with location. The scientists based their model on the next article.

Howard, M., DuVall, M. D., Devor, D. C., Dong, J. Y., Henze, K., & Frizzell, R. A. (1995). Epitope tagging permits cell surface detection of functional CFTR. American Journal of Physiology-Cell Physiology, 269(6), C1565-C1576.
- The addition of the FLAG epitope at position 901 does not compromise the function of CFTR. This was tested by analyzing the chlorine ion conductance response to cAMP.