Peptidoglycan isolation and binding studies with LysM-type pattern recognition receptors

Abstract

In the last decade, more and more plant receptors for complex carbohydrate structures have been described. However, studies on receptor binding to glycan ligands are often hampered due to the technical challenge to obtain pure preparations of homogeneous carbohydrate ligands such as bacterial peptidoglycan (PGN) in amounts suitable for studying protein–glycan interactions. Also, most approaches rely on the availability of defined soluble ligands, which in the case of glycans can rarely be synthesized but have to be purified from the respective microorganism. In this chapter, we describe the purification of complex PGN from sources such as gram-positive bacteria, from which PGN isolation is facilitated due to its larger content in their cell wall. Insoluble PGN can subsequently be used in simple carbohydrate pull-down assays to test for interaction with plant proteins. In this respect, lysin motif (LysM)-domain containing proteins are of particular interest. All plant receptors described to date to be involved in the perception of N-Acetylglucosamine-containing ligands (such as PGN or chitin) have been shown to belong to this protein class. Thus, this chapter will also include the production of recombinant LysM proteins to analyze their PGN interaction.