Part:BBa_K4607006
Expression cassette for LysCSA13-ABD protein
Expression cassette for LysCSA13-ABD protein: Fusion endolysin against S. aureus bacteria who causes bovine mastitis
This part contains the linear sequence of the biobrick BBa_K4607000 optimized for Escherichia 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_K4607000 that codifies for a fusion protein based on the endolysin Lys from Staphylococcus aureus virulent bacteriophage CSA13 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) from streptococcal protein G. The ABD 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 for the expression of LysK 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. Other characteristics of the endolysin are that it requires the presence of calcium and manganese to reach its maximum catalytic activity. The average endolysin lifetime is about 30 hours [3].
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1100
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1100
Illegal SpeI site found at 127
Illegal NotI site found at 1242 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1100
Illegal BglII site found at 30
Illegal BamHI site found at 1094
Illegal XhoI site found at 1251 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1100
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1100
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 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 (MRSA). 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
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