Part:BBa_K4607001
LysK-ABD-SH3B30
Description
The biobrick consists of a fusion of the CHAP domain from the Lys of the bacteriophage K, with the ability to degrade the cell wall of antibiotic-resistant strains of Staphylococcus aureus [1] [2]; the albumin binding domain (ABD) from streptococcal protein G that is capable of increasing antibody, protein, and enzyme lifetimes; and the SH3 domain from the bacteriophage B30, which binds to the cell-wall of Streptococcus agalactiae, Streptococcus uberis, and Staphylococcus aureus. The ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. The domain consists of an affinity-maturated variant of the streptococcal protein G, which has been used for LysK expression with results of up to 34 hours in increasing the lifetime of the protein in mice [3]. The principle of the mechanism of the SH3B30 domain is to recognize and bind to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the S. aureus, S. agalactiae and S. uberis peptidoglycan, activating the catalytic domain [4][5]. The enzyme has a length of 262 amino acids and a molecular weight of 28.437 kDa. The average endolysin lifetime is 20 minutes [3]. The part is adapted to the Golden Gate cloning method. This part also contains a x6 HisTag in the C-terminal site, to facilitate its purification process.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 415
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 328
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 [3].
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 contains an extra region, the albumin binding domain, that causes an important increase in the life-time of the fusion protein [2].
Results
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] 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
[3] 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
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