Part:BBa_K4607000
LysCSA13-ABD
Description
This biobrick consists of a fusion protein based on the endolysin (Lys) from Staphylococcus aureus virulent bacteriophage CSA13 (BBa_K4607004) which is composed of two domains: the CHAP domain, with excellent catalytic activity, up to 90%, degrading almost 15 strains of Staphylococcus, including methicillin-resistant strains (MRSA) [1], and the SH3 domain, which recognizes and binds to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the S. aureus peptidoglycan, activating the catalytic domain [2]; and the albumin binding domain (ABD) (BBa_K4607002) from streptococcal protein G. It is capable of increasing the antibody, protein, and enzyme lifetimes. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This section consists of an affinity-maturated variant of the streptococcal protein G which has been used to Lys K expression in mice, with results of up to 34 hours in increasing the lifetime of the protein [3]. The part is adapted to the Golden Gate cloning method. This part also contains a TEV cleavage site for the removal of the x6 HisTag after the protein purification process. The enzyme has a length of 316 amino acids and a molecular weight of 35.098 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9, and it has extracellular protein expression. The average endolysin lifetime is about 30 hours [3].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The endolysin Lys from the Staphylococcus aureus virulent bacteriophage CSA1, is composed of two domains. The bacteriophage CSA13 CHAP domain has excellent catalytic activity, up to 90%, degrading almost 15 strains of Staphylococcus including methicillin-resistant strains. As with many of the endolysins, it cleaves to the cell wall by disrupting the peptidoglycan that composes the bacterial cell; for this to be possible, the bacteriophage CSA13 SH3 domain recognizes and binds to the glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the peptidoglycan, which makes it completely safe for the host [1].
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 [2].
Results
References
[1] Cha, Y., Son, B., & Ryu, S. (2019). Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13. Food Microbiology, 84, 103245. https://doi.org/10.1016/j.fm.2019.103245
[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| None |