Development of a Magnetic Bead-Based Method for Specific Detection of Using C-Terminal Domain of ECP3 Phage Endolysin.

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and an enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes were determined using the bacterial colony counting from the magnetic separations after the binding reaction between bacteria and CBD-eMB complexes. could bind to CBD-eMB complexes, but other bacteria (such as , , , , , and ) could not. cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable cells were recovered. The recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for was >17 CFU/ml. We developed a simple method for the specific detection of using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for among other bacterial species.