Part:BBa_K1659001:Design
Artilysin Art-175 fused at N-terminal with flagellin 26-47 peptide segment
- 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 525
Illegal AgeI site found at 724 - 1000COMPATIBLE WITH RFC[1000]
Design Notes and Sources
We fused the 26-47 peptide sequence of flagellin 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. More details about the use of flagellin 26-47 as a secretion signal can be found in the protocol book 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. 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
[4] Vondervizst, F., Sajó, R., Dobó, J., & Závodszky, P. (2012). The Use of a Flagellar Export Signal for the Secretion of Recombinant Proteins in Salmonella. In: Recombinant Gene Expression - Reviews and Protocols, Methods in Molecular Biology, 824, 131-143. http://doi.org/10.1007/978-1-61779-433-9_6