Endoglucanase A gene fusion vectors for monitoring protein secretion and glycosylation in yeast.
We have constructed a set of replicating and integrating vectors that allow expression and secretion of Clostridium thermocellum endoglucanase A in Saccharomyces cerevisiae using the alpha-factor or the invertase promoters and secretion signals. The enzyme expressed in yeast was enzymatically active regardless of its degree of glycosylation and was released into the culture medium. One advantage of using this experimental system is that secretion of the reporter enzyme can be detected in individual colonies facilitating the isolation of mutants. A second advantage is that transit through the secretory pathway, as judged from the extent of glycosylation, can be easily monitored by staining for enzyme activity in agar replicas of polyacrylamide gels.
Silva
A
A
Facultad de Biología de la Universidad de La Habana, Cuba.
Benitez
J
J
Hollenberg
C P
CP
D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics; D006031:Glycosylation; D000072235:Mating Factor; D010455:Peptides; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D043324:beta-Fructofuranosidase
D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D002480:Cellulase