Difference between revisions of "Part:BBa K1659001:Design"

(Created page with "__NOTOC__ <partinfo>BBa_K1659001 short</partinfo> <partinfo>BBa_K1659001 SequenceAndFeatures</partinfo> ===Design Notes=== ===Sources=== The artilysin Art-175 is a fus...")
 
Line 7: Line 7:
  
  
−
===Design Notes===
+
===Design Notes and Sources===
  
 +
We fused the 26-47 peptide sequence of flagellin to the N-terminus of Art-175 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.
  
−
 
−
===Sources===
 
−
 
−
The artilysin Art-175 is a fusion protein (SMAP-29 fused to the N-terminus of endolysin KZ144) created by Briers et al as laid out in their publication in 2014 [1]. Briefly, endolysin KZ144 originates from Pseudomonas aeruginosa bacteriophage varphiKZ. SMAP-29 originates from sheep genomic DNA.
 
  
  
Line 21: Line 20:
  
 
[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
 
[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

Revision as of 18:28, 30 August 2015

Artilysin Art-175 fused at N-terminal with flagellin 26-47 peptide segment



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 525
    Illegal AgeI site found at 724
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes and Sources

We fused the 26-47 peptide sequence of flagellin to the N-terminus of Art-175 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.


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