Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins

Abstract

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Agα1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing α-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Agα1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of α-galactosidase-Agα1p was sixfold lower than that of α-galactosidase- Cwp2p and eightfold lower than that of α-galactosidase-Sed1p. Differences in mRNA levels were nut responsible for this discrepancy, nor was an intracellular accumulation of α-galactosidase-Agα1p detectable. A lower translation efficiency of the α-galactosidase-AGα1 fusion construct is must likely to be responsible for the low level of protein production. α- Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the rise of a large anchoring domain lines not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.