Secretion and glycosylation of Clostridium thermocellum endoglucanase A encoded by the celA gene in Saccharomyces cerevisiae.
The Clostridium thermocellum celA gene encoding endoglucanase A is expressed in Saccharomyces cerevisiae but the enzyme produced from the native celA gene is not secreted. After removal of the bacterial signal peptide-coding sequence, the gene was fused to the promoter and prepro segment of the S. cerevisiae MF alpha 1 gene. This construction directs secretion of active endoglucanase A into the culture medium when introduced in yeast on either replicating or integrating vectors. Secretion of endoglucanase A required growth of transformants on rich medium. The secreted enzyme is a 97,000 Da glycoprotein containing about half of its molecular weight as carbohydrate. This new gene fusion could facilitate further research on protein secretion in yeast by using a cellulase as a marker enzyme.
Benitez
J
J
Departamento de Genética, Centro Nacional de Investigaciones Cientificas, La Habana, Cuba.
Silva
A
A
Vazquez
R
R
Noa
M D
MD
Hollenberg
C P
CP
D015139:Blotting, Southern; D015153:Blotting, Western; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D006023:Glycoproteins; D002480:Cellulase