Coding
Art175

Part:BBa_K1659000:Design

Designed by: Wei Chung Kong   Group: iGEM15_Oxford   (2015-08-28)

Artilysin Art-175, an antibacterial fusion protein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 366
    Illegal AgeI site found at 565
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes and Sources

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]. We obtained the peptide sequence from their original publication and converted it into a nucleotide sequence using IDT's Codon Optimizer.

The original literature describing SMAP-29 and KZ144 can be found below at [2] and [3] respectively.

  • Note: The "GGC" codon which follows the first "ATG" was introduced by mistake due to a wrongly-designed primer. In the actual Art-175 the start codon is followed immediately by "CGC" which in this part is the nucleotides 7-9.

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