| The yeast cell wall acts as a scaffold to support and display GPI-anchored cell wall proteins (GPI-CWPs) at the cell surface. These proteins are required for normal cell growth and perform a variety of functions, interacting with the environment, in structural roles and as enzymes. In Part I of this dissertation we demonstrate a role for DCW1 in the processing of GPI-CWPs. Temperature sensitive dcw1 mutants show growth arrest, loss of viability, a significant reduction in crosslinking of a model GPI-CWP into the cell wall and persistence of this GPI-protein at the membrane. These phenotypes are seen both with and without osmotic stabilization, and are not simply a result of activation of the cell wall integrity pathway.;In Part II we discuss the construction of a 6-Histidine epitope tagged β-1,3-endoglucanase from the soil bacterium, Cellulosimicrobium cellulans. When expressed in BL21 cells under the control of an inducible promoter, the construct allows purification by immobilized metal affinity chromatography of a protease-free glucanase for digesting yeast cell wall glucan with subsequent analysis of the protein content.;Part III serves to discuss early work to manipulate GPI-CWP expression for use in immunological studies. We show efficient surface expression of GPI-anchored peptides from chick ovalbumin, an exogenous protein, in Saccharomyces cerevisiae. These peptides were affinity purified from the cell wall and shown to elicit an in vitro immune reaction divergent from larger cell wall fragments. |
