Cloning, expression and structure determination of SH3b cell wall binding domain of bacteriophage 812 endolysin
| Autoři | |
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| Rok publikování | 2011 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | In this study we focused on endolysin of staphylococcal bacteriophage 812, a member of unclassified SPO1-like viruses from family Myoviridae. The endolysin of phage 812 includes three domains: two catalytic domains and the SH3b-domain that is probably a cell wall targeting domain, which could be responsible for binding of endolysin to peptidoglycan. The gene sequence for the C-terminal SH3b domain was cloned in pET28 vector and expressed as a soluble protein in E. coli BL21 (DE3) to determine the 3D structure of the protein. A native variant of the SH3b protein was purified without the his-tag. Subsequently, three variants of this protein have been prepared: (i) non-labeled, (ii) single-labeled (15N) and (iii) double-labeled (13C, 15N) domain. The protein was purified to homogeneity using ammonium sulphate precipitation, anion exchange chromatography, cation exchange chromatography, and gel filtration. Functionality of the cell wall binding of the purified protein has been verified by a co-sedimentation test in using S. aureus peptidoglycan. |
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