Difference between revisions of "Part:BBa K4607001"

Revision as of 22:24, 27 July 2023

LysK-ABD-SH3B30

Figure 1.LysK-ABD-SH3B30 protein diagram.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 415
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 328
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
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 [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].

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] 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

@@ Line 5: / Line 5: @@
 <center>https://static.igem.wiki/teams/4607/wiki/parts/lysk-cds.jpg</center>
 <center><b>Figure 1.</b>LysK-ABD-SH3B30 protein diagram.</center>
+===Description===
 <p align="justify">
-This biobrick is a fusion protein based on the endolysin (Lys) from <i>Staphylococcus aureus bacteriophage K</i> [1], the albumin binding domain (ABD) from <i>streptococcal protein G</i> [2], and the SH3 domain from the <i>Streptococcus agalactiae, Streptococcus uberis y S. aureus bacteriophage B30</i> [3]. Its purpose is to recognize the <i>S. aureus, Streptococcus. agalactiae, Streptococcus uberis</i> cell wall, and degrade it. The part is adapted to the Golden Gate cloning method.</p>
+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 <i>Staphylococcus aureus</i> [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 <i>Streptococcus agalactiae</i>, <i>Streptococcus uberis</i>, and <i>Staphylococcus aureus</i>. 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 <i>S. aureus</i>, <i>S. agalactiae</i> and <i>S. uberis </i>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.
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