Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae.
We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form.
Murai
T
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Ueda
M
M
Atomi
H
H
Shibasaki
Y
Y
Kamasawa
N
N
Osumi
M
M
Kawaguchi
T
T
Arai
M
M
Tanaka
A
A
D001230:Aspergillus / Q000201:enzymology*; D002473:Cell Wall / Q000201:enzymology*; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D000072235:Mating Factor; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D010446:Peptide Fragments / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D010446:Peptide Fragments; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C068142:glyceraldehyde 3-phosphate dehydrogenase (304-313); D000072235:Mating Factor; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002480:Cellulase