title: High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast.
abstract: Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress) into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Hemant Kumar
authors.initials: HK
authors.lastname: Verma
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Poonam
authors.initials: P
authors.lastname: Shukla
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Md
authors.initials: M
authors.lastname: Alfatah
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Asheesh Kumar
authors.initials: AK
authors.lastname: Khare
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Udita
authors.initials: U
authors.lastname: Upadhyay
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Kaliannan
authors.initials: K
authors.lastname: Ganesan
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Jagmohan
authors.initials: J
authors.lastname: Singh
mesh_terms: D003001:Cloning, Molecular; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008156:Luciferases / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*
chemical_list: D029702:Schizosaccharomyces pombe Proteins; D008156:Luciferases