Construction and Determination of an Episomal Secreting Expression Vector:pYES2/CT/?-factor for Saccharomyces cerevisiae
- VernacularTitle:一种酿酒酵母附加型分泌表达载体pYES2/CT/?-factor的构建及鉴定
- Author:
Yadong HU
;
Dongsheng YAO
- Publication Type:Journal Article
- Keywords:
Saccharomyces cerevisiae Signal peptide Secreting expression
- From:
China Biotechnology
2006;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
pPIC9 was used as a template and ?-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/?-factor(pYC?)was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man(pKLman) was cut by restriction enzymes and linked with pYC?.This recombinant vector pYC?-man was used to determine the secretory ability and stability of pYC?.The excellent secretory ability of pYC? was proved by two experiments.One showed that INVSc1/pYC?-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYC? clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYC? were not detected,while INVSc1/pYC?-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYC? was also very good proved by continuous cultivation for about 150h.
