A copy-number-controlled expression vector for the fission yeast Schizosaccharomyces pombe.
A novel expression vector for the fission yeast Schizosaccharomyces pombe carries the neomycin-resistance-encoding gene regulated by the SV40 early promoter, and its copy number is controlled by the level of Geneticin (G418). Foreign gene expression is driven by the human cytomegalovirus (hCMV) promoter which is transcriptionally active in S. pombe. Moreover, the vector expresses foreign genes at high levels, due to the 5'-untranslated region (5'-UTR) containing an A + T-rich sequence of about 50 nucleotides located between the TATA box of the hCMV promoter and the start codon. Recombinant human lipocortin I was produced at levels of up to 50% of the total soluble protein in the presence of 100-200 micrograms/ml of G418 in the media. Southern and Northern blotting showed that this high level of expression was due to an increase in copy number induced by G418, the high transcriptional activity of the hCMV promoter and the high translational efficiency of the 5'-UTR. We modified the vector into an 'ATG vector', named pTL2M, that maintains the 5'-UTR optimized for gene expression and into which any foreign gene, whose exact sequence is known, can be easily inserted.
Tohda
H
H
Research Center, Asahi Glass Co., Ltd., Kanagawa, Japan.
Okada
H
H
Giga-Hama
Y
Y
Okayama
H
H
Kumagai
H
H
D017305:Annexin A1 / Q000096:biosynthesis*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003587:Cytomegalovirus / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009355:Neomycin; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012568:Schizosaccharomyces / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic
D017305:Annexin A1; D005839:Gentamicins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C010680:antibiotic G 418; D015500:Chloramphenicol O-Acetyltransferase; D009355:Neomycin