COMPARISON OF CELL-WALL PROTEINS OF SACCHAROMYCES-CEREVISIAE AS ANCHORS FOR CELL-SURFACE EXPRESSION OF HETEROLOGOUS PROTEINS

The carboxyl terminal regions of five cell wall proteins (Cwp1p, Cwp2p , Ag alpha 1p, Tip1p, and Flolp) and three potential cell wall protein s (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha- galactosidase in the cell wall of Saccharomyces cerevisiae, The fracti on of the total amount of fusion protein that was localized to the cel l wall varied depending on the anchor domain used, The highest proport ion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, o r Sed1p as an anchor, Although 80% of these fusion proteins were incor porated in the cell wall, the total production of alpha-galactosidase- Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p a nd eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectab le. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production, alpha-Galactosidase immobilized by the carboxy l-terminal 67 amino acids of Cwp2p was most effective in the hydrolysi s of the high-molecular-weight substrate guar gum from Cyamopsis tetra gonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.