Expression of heterologous proteins at the S. cerevisiae cell surface using Pir4p as a cell wall anchor

In the past several years much efforts have been devoted to the study of expression systems for the display of heterologous proteins at the surface of microorganisms, opening new perspectives in biotechnology. Recently a number of surface – engineered yeasts, displaying different heterologous proteins interesting for biotechnological or medical applications, have been constructed. Yeast cell surface systems have the advantages of simplicity of genetic manipulation and ability for proper post-translational modifications and folding of mammalian proteins. Yeast whole-cell biocatalysts displaying enzymes on their cell surface can be produced at a low cost and show a high enzymatic activity without permeabilization treatment. S. cerevisiae cell wall proteins that are covalently bound to the carbohydrate components of the wall can be divided in two main groups. Majority of proteins of this class are bound at their C-termini through a remnant of the GPI-anchor. A smaller group of proteins are directly covalently bound at their N-termini to β -1, 3-glucan by the alkali labile ester linkage between the glutamic acid γ -carboxyl group and hydroxyl groups of glucoses (Pir – proteins). Almost all heterologous proteins constructed for yeast surface display are GPI-anchored to the cell wall. Most frequently used GPI-anchored yeast cell wall protein for this purpose is  -agglutinin. Some enzymes, whose active sites are located near their C-termini are not suitable for display through GPI anchor that must be fused at their C-terminal region. Possible approach for such enzymes is to use Pir – proteins as a cell wall anchor. In this work Pir4p was used as anchor for N-terminal immobilization of β -galactosidase and yeast lipase Tgl3p to the yeast cell surface.