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 Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-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, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and 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 detectable. 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 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 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.
Van der Vaart
J M
JM
Department of Molecular Cell Biology, University of Utrecht, The Netherlands. marcelvan-de.vaart@unilever.com
te Biesebeke
R
R
Chapman
J W
JW
Toschka
H Y
HY
Klis
F M
FM
Verrips
C T
CT
D002451:Cell Compartmentation; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D005685:Galactans / Q000378:metabolism; D006868:Hydrolysis; D008351:Mannans / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D053149:Plant Gums; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013499:Surface Properties; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D005685:Galactans; D008351:Mannans; D008565:Membrane Proteins; D053149:Plant Gums; D011993:Recombinant Fusion Proteins; C007894:guar gum; D000519:alpha-Galactosidase