Part:BBa_K1659002:Design
Artilysin Art-175 fused at N-terminal with DsbA signal peptide
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 417
Illegal AgeI site found at 616 - 1000COMPATIBLE WITH RFC[1000]
Design Notes and Sources
We fused the 2-19 peptide sequence of DsbA to the N-terminus of Art-175 (BBa_K1659000) sequence without any spacer/linker peptide in between, in view of how SMAP-29 was fused to endolysin KZ144 in the same fashion. A Hisx6 tag is added at the C-terminus for ease of protein purification using metal-affinity chromatography.
Briers et al created Art-175 by fusing the sheep myeloid protein SMAP-29, which in itself is a potent broad-spectrum antimicrobial albeit with significant cytotoxicity, to the N-terminus of Pseudonomas aeruginosa bacteriophage phiKZ endolysin KZ144 to first create artilysin Art-085. Art-085 formed oligomers due to intermolecular disulfide bridges and as such three cysteine residues (Cys14, Cys23, and Cys50) were mutated into serine residues to make Art-175 [1].
The original literature describing SMAP-29 and KZ144 can be found below at [2] and [3] respectively.
The gene sequence for DsbA 2-19 was obtained from BBa_K729004, a part comprising DsbA 2-19 fused to the N-terminal of micrococcal nuclease made by [http://2012.igem.org/Team:University_College_London Team UCL 2012]. The part sequence was not annotated, and as such we ran a [http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome Protein BLAST search] using the part's translated sequence to identify the bases that coded for our desired peptide. The sequence for DsbA 2-19 was adopted as-is, without further codon optimization.
References
[1] Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., … Lavigne, R. (2014). Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774–3784. http://doi.org/10.1128/AAC.02668-14
[2] Skerlavaj B, Benincasa M, Risso A, Zanetti M, Gennaro R. (1999). SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Lett. 46:58–62. http://dx.doi.org/10.1016/S0014-5793(99)01600-2
[3] Briers Y, Volckaert G, Cornelissen A, Lagaert S, Michiels CW, Hertveldt K, Lavigne R. (2007). Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL. Mol. Microbiol. 65:1334–1344. http://dx.doi.org/10.1111/j.1365-2958.2007.05870.x