Part:BBa_K4607007

Expression cassette for LysK-ABD-SH3B30 protein

Figure 1. LysK-ABD-SH3B30 protein diagram.

This part contains the linear sequence of the LysK-ABD-SH3B30 nucleotide optimized for E. coli. It incorporates some of the most efficient biobricks as described below: the T7 promoter with LacO regulations BBa_J435350, the medium strength RBS BBa_Z0262, the triple terminator BBa_J435371, and the high copy pUC ori /Kan R backbone BBa_J435330. It also contains the BBa_K4607001 that codifies for the fused endolysin from the CHAPk K bacteriophage, with a very efficient catalytic activity, the albumin binding domain that increases its lifetime, the SH3 B30 bacteriophage domain, and the x6 HisTag for their posterior purification.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 938
  Illegal XbaI site found at 96
  Illegal SpeI site found at 127
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 938
  Illegal NheI site found at 552
  Illegal SpeI site found at 127
  Illegal NotI site found at 1080
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 938
  Illegal BglII site found at 30
  Illegal BamHI site found at 465
  Illegal BamHI site found at 932
  Illegal XhoI site found at 1089
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 938
  Illegal XbaI site found at 96
  Illegal SpeI site found at 127
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 938
  Illegal XbaI site found at 96
  Illegal SpeI site found at 127
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

The endolysin Lys from the K bacteriophage, is composed of three domains. For the design of a novel antimicrobial enzyme, the CHAPk domain from the K bacteriophage was selected for their ability to cleave between the D-alanine and the first glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the peptidoglycan, with a high efficiency [1]. To increase the sensitivity of the enzyme for pathogenic bacteria, specifically Streptococcus uberis, Staphylococcus aureus, and Streptococcus agalactiae, the SH3 domain from the B30 bacteriophage was selected, which makes it completely safe for the host [2].

The use of enzybiotics represents an alternative to the misuse of antibiotics without loss of efficiency; it is a novel and environmentally friendly process. It supplies antibacterial protection to pathogenic bacteria but shows no toxic effects on mammalian cells. Our protein has an extra region, the albumin binding domain, that causes an important increase in the life-time of the fusion protein [3].

Table 1. LysK-ABD-SH3B30 protein parameters.

Results

Figure 2. 3D structure of the LysK-ABD-SH3B30 protein, obtained with AlphaFold2.

References

[1] Sanz-Gaitero, M., Keary, R., Garcia-Doval, C., Coffey, A., & van Raaij, M. J. (2013). Crystallization of the CHAP domain of the endolysin from Staphylococcus aureus bacteriophage K. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 69(12), 1393–1396. https://doi.org/10.1107/s1744309113030133

[2] Jarábková, V., Tišáková, L., Benešík, M., & Godány, A. (2020). SH3 BINDING DOMAINS FROM PHAGE ENDOLYSINS: HOW TO USE THEM FOR DETECTION OF GRAMPOSITIVE PATHOGENS. Www.muni.cz, 9(6). https://www.muni.cz/vyzkum/publikace/1674660

[3] Seijsing, J., Sobieraj, A. M., Keller, N., Shen, Y., Zinkernagel, A. S., Loessner, M. J., & Schmelcher, M. (2018). Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.029