Endolysin Expression, Purification and Activity Determination by Zymography

[Abstract] Endolysins are peptidoglycan-degrading (muralytic) enzymes produced by many bacteriophages for cell lysis of the host bacterium. The enzymatic activity of muralytic enzymes can be assayed qualitatively using a zymogram containing incorporated peptidoglycan. This protocol describes the expression of a recombinant 6x His-tagged endolysin using an Escherichia coli (E. coli) expression system and native affinity purification of the protein using Ni-NTA agarose. For the zymogram, the protocol details isolation of crude peptidoglycan from the Gram-negative bacterium Rhodobacter capsulatus and the zymography of purified protein and crude cell lysate. Construction of an E. coli BL21 (DE3) pET28-a(+)-derived endolysin-expression system is briefly described. The protocol described here was developed and optimized for the endolysin 555 utilized by the Rhodobacter capsulatus bacteriophage-like gene transfer agent (RcGTA) (Westbye et al., 2013) and to study the muralytic activities of protein P14 of RcGTA (Fogg et al., 2012), but should be transferrable as a general protocol to express and study a variety of endolysins.