Part:BBa_K3378000
Chimeric lysin ClyR with activity against Streptococcus mutans.
ClyR is a chimeric lysin with extended streptococcal host range and has been demonstrated high lytic activity against Streptococcal mutans. Its composed of a catalytic domain from PlyC and a cell-wall binding domain from PlySs2.
Usage and Biology
This chimeric lysin can bind to the S. mutans by a cell-wall binding domain and then lysis its cell wall from outside by a catalytic domain. ClyR has no activity for Gram-negative bacteria and can be expressed in Escherichia coli. With the guidance of appropriate signal peptide, ClyR can be secreted to the outside of the cells, killing the S. mutans in the environment. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 757
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 121
Illegal AgeI site found at 205
Illegal AgeI site found at 520
Illegal AgeI site found at 703 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| protein | -NA- |
To obtain the ClyR protein, we transferred pET-28a(+)-ClyR to E. coli BL21(DE3). The E. coli strain was cultured to OD600~0.6, induced with 0.2 mM IPTG, and allowed to grow overnight at 16 ℃. The harvested bacteria were resuspended with a Binding buffer containing 10 mM imidazole, and then the cell was lysis by ultrasonication. Purification was performed following the instructions of Ni-NTA Sefinose (TM) Resin (Sangon Biotech, Shanghai, China). By SDS-PAGE analysis, we can demonstrate that ClyR can be expressed as a soluble protein in E. coli at the condition described above (Figure 1). Purified ClyR was desalted and concentrated by Amicon® Ultra-4 Centrifugal Filter Units (Millipore). After quantitation by Bradford assay, the ClyR solution was stored at -20 ℃ until use.
S. mutans UA159 was used as a test strain to verify the activity of purified ClyR protein. Overnight cultured S. mutans UA159 was resuspended in PBS and then treated with 120 μg/ml ClyR, another tube was added the same volume of PBS as control. After shaking 1 h at 37℃, a significant decrease of turbidity was observed in the ClyR treated group (Figure 2).
To determine the dose-dependent and time-dependent lytic activity of ClyR, PBS resuspended S. mutans UA159 was adjusted to an OD600 of ~0.8. In the 96-well plates, 20 μl ClyR solution at different concentrations and 140 μl cell suspension were added in each well. The Synergy H1 microplate reader was used to measure the drop of OD600 at 37 ℃ for 1 h. The results obtain from triple independent experiments are shown below (Figure 3).
Besides, we also detected the viable counts of ClyR treated S. mutans. PBS resuspended S. mutans UA159 was treated with ClyR solution in 2 ml EP tubes for 10 min. At the end of the reaction, serial dilutions of each tube were plated on BHI agar and incubated at 37 ℃ overnight. The results obtain from triple independent experiments are shown in Figure 4.
| protein |