13 Identification, Characterization, and Phenotypic Analysis of Covalently Linked Cell Wall Proteins

Abstract

Publisher Summary This chapter discusses the identification, characterization, and phenotypic analysis of covalently linked cell wall proteins (CWPs). Proteins that are destined for the cell wall and become covalently attached to the cell wall polysaccharide network first have to traverse the secretory pathway, where they may undergo various posttranslational modifications. Generally, isolated cell walls are used as starting material for the release and isolation of covalently linked CWPs. To avoid heavy contamination with cytosolic proteins, cell wall preparations should be completely free from unbroken cells. The use of a FastPrep instrument is recommended for full cell breakage. To prevent contamination by ionically bound cytosolic proteins and entrapped membrane proteins, isolated walls are washed with concentrated salt solutions and extracted with hot detergent containing a reducing agent. The procedure for cell breakage and the isolation of cell walls is given in the chapter. Purified cell walls usually have a protein content of about 3%. Isolated walls are treated with hot alkali and the extract is used for assaying the protein content.