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.

Figure 1. Schematic diagram of the Fluc-expression vectors used in this study.

Restriction map of Fluc-expression vectors; (A) pJH6c-Fluc, (B) pART1-Fluc, (C) pREP3X-Fluc and (D) pPIC3.5-Fluc. Approximately 1.0 kb upstream region of gene SPAC1F8.02C/lsd90 of S. pombe was PCR amplified and inserted into the plasmid pJH5 at SphI/NdeI sites as promoter Plsd90 respectively. The resulting vectors were designated as pJH6c (A). The strategy of Fluc cloning is described in the methods section.

Figure 2. Constitutive expression of the Firefly luciferase (Fluc) under lsd90 promoter.

Cultures of strain SPJ25 harboring the vector constructs pJH6c-Fluc and pJH6c were grown in selective media (PMA ura⁻) at 30°C and 200 rpm. Aliquots were taken at the indicated time points and subjected to protein extraction. (A) Luciferase activity was determined using the Luciferase Assay System (Promega, USA). Assays were done in triplicate and the average luminescence values were plotted against the indicated time points. (B) Graph showing growth kinetics of cultures up to early stationary phase. (C) The histogram shows the cell density in Cells/ml at the indicated time points of culture.

Figure 3. Time course of Fluc-expression under control of the adh1 promoter of S. pombe.

Cultures of strain SPJ25 harboring the vector constructs pART1-Fluc and pART1 were grown in selective media (PMA leu⁻) at 30°C and 200 rpm. (A) Luciferase activity measured in RLU and (B) growth kinetics of cultures.

Figure 4. Time course of Fluc-expression under control of the nmt1 promoter of S. pombe.

Cultures of strain SPJ25 harboring the vector constructs pREP3X-Fluc and pREP3X were grown in selective media (PMA leu⁻) at 30°C and 200 rpm. Initially the cultures were grown in medium containing thiamine and then sub-cultured in medium lacking thiamine for the indicated time points. (A) Luciferase activity measured in RLU and (B) growth kinetics of cultures.

Figure 5. Time course of luciferase expression under control of the AOX1 promoter of P. pastoris.

Cultures of recombinant strain GS115 harboring the Fluc-containing expression vector pPIC3.5-Fluc and the control vector pPIC3.5 were grown in suitable medium up to six days. Luciferase assay was performed in triplicate and activity was plotted against indicated time points.

Figure 6. Relative-qPCR analysis of Luciferase mRNA.

RNA isolated form cells harvested at time points showing maximum level of Fluc RNA under the control of different vectors was subjected to real time RTPCR analysis. Samples were analyzed in triplicate and displayed as histogram. The relative Fluc/act1 RNA levels expressed by different vectors are displayed after normalization with respect to the vector pJH6c-Fluc. The time points were: pJH6c-Fluc: 32 hrs; pART1-Fluc: 48 hrs; pREP3X-Fluc: 16 hrs, pPIC3.5-Fluc: 96 hrs.