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.
Highlights
IntroductionThe methylotrophic yeast Pichia pastoris has emerged as a useful expression host for commercial scale production of therapeutic proteins
Among various yeast species, the methylotrophic yeast Pichia pastoris has emerged as a useful expression host for commercial scale production of therapeutic proteins
Subjecting the cells to oxidative stress showed no effect. The discrepancy of these results from the published microarray data is surprising. This may be due to lack of some further upstream regulatory sequences or presence of negative regulatory sequences inhibiting the induction of the existing promoter elements in the putative promoter region in response to oxidative stress
Summary
The methylotrophic yeast Pichia pastoris has emerged as a useful expression host for commercial scale production of therapeutic proteins. Reasons behind popularity of the Pichia system are availability of AOX1 promoter, a strong methanol-inducible promoter of alcohol oxidase I and ease of growth to very high cell density in an inexpensive, non-complex and chemically defined medium [1,2,3,4]. Despite these advantages, there are serious issues regarding safety aspects of Pichia expression system, which uses inflammable methanol for induction; methanol is added after every 24 hrs of growth phase and maximum expression is achieved only after 4–6 days of induction. Nmt promoter yielded only moderate level of expression of a therapeutic protein, indicating inadequacy of nmt for commercial scale expression of proteins [10]
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