Peptidoglycan Hydrolytic Activity of Bacteriophage Lytic Proteins in Zymogram Analysis

Zymogram or zymography is an electrophoretic technique based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which enables visualization of enzymatically active protein species separated by molecular mass. The strategy is to perform SDS-PAGE on the proteins in question while including an opaque substrate of the enzyme embedded within the polyacrylamide gel. Here, we describe a zymogram protocol for phage lytic proteins (peptidoglycan hydrolases) using peptidoglycan (or whole cells) from a susceptible gram-positive bacterial species as substrate. Proteins are prepared and analyzed simultaneously on two separate gels: First, standard denaturing SDS-PAGE followed by conventional protein staining (e.g., Coomassie) is run to identify the migration pattern of the protein species in the sample; second, the zymogram gel in which either cells or peptidoglycan from a susceptible bacterium have embedded in the SDS gel matrix is performed. After electrophoresis, the SDS is removed from the zymogram gel, allowing the proteins (now separated by molecular mass) to assume an active conformation and ultimately digest the opaque substrate (yielding a nonopaque product). This results in a cleared spot in an otherwise opaque gel which corresponds to the location of an enzymatically active protein species. This assay can be used to qualitatively assay the enzymatic activity of endolysins from cell extracts, or to identify virion-associated peptidoglycan hydrolases in phage particles.