Part:BBa_K1659003:Design
Artilysin Art-175 fused at N-terminal with porin-dependent YebF secretion signal protein
- 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 714
Illegal AgeI site found at 913 - 1000COMPATIBLE WITH RFC[1000]
Design Notes and Sources
We fused the protein YebF 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 YebF was obtained from the codon optimization (IDT Codon Optimization Tool) of its protein sequence, found at [4].
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.
[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.
[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.
[4] Zhang, G., Brokx, S. & Weiner, J.H., 2006. Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature biotechnology, 24(1), pp.100–104.