Construction of an expression vector for the fission yeast Schizosaccharomyces pombe

摘要

We have isolated and characterized a S. pombe promoter using a functional heterologoues gene product assay. Random S. pombe genomic fragments were cloned upstream from the promoterless lacZ gene and tested in vivo for their efficiency to promote expression of the beta-galactosidase protein in the fission yeast. An efficient S. pombe promoter called 54/1 was isolated and shown to drive up to 5% of total protein synthesis as beta-galactosidase The structure and nucleotide sequence of this promoter were determined, precise localization of its mRNA transcriptional start points established. Translational fusion of the Pseudomonas putida XylE gene with the 54/1 gene was shown to allow expression of catechol oxydaee activity in S. pombe. An expression vector suitable for transcriptional fusions was then constructed from engineered 54/1 promoter sequences and used to drive expression of the E. coli Tn5 ble gene, thus confering resistance to the fission yeast against bleomycin and phleomycin antibiotics.