Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1999-06-30
2000-10-10
Mckelvey, Terry
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352542, 4353201, 536 231, 536 2374, 536 241, C12P 2106, C07H 2100, C12N 119, C12N 1552, C12N 1563
Patent
active
061300705
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to an inducible promoter gene and secretion signal gene for use in the fission yeast Schizosaccharomyces pombe (hereinafter referred to as S. pombe), an expression vector containing them and a process for producing a protein using them. In particular, it relates to a process for producing a desired protein wherein the S. pombe invertase promoter is used to make it possible to control the timing of the protein production by the presence or absence of a specific nutrient through regulated gene expression, and a process for secretory production of a desired protein by using the secretion signal gene for the S. pombe invertase precursor.
BACKGROUND ART
S. pombe, despite being a eukaryote, has been studied extensively for its high versatility in genetics, molecular biology and cellular biology as a unicellular organism (Nasim A. et al. eds., Molecular biology of the fission yeast, Academic Press, 1989). In its cultures, monosaccharides such as glucose and fructose are used as the main carbon sources. It is known that in a culture medium lacking these monosaccharides, expression of invertase, the enzyme that degrades sucrose into glucose and fructose, is induced to secure the carbon source necessary for its growth (Moreno S. et al., Arch Microbial. 142, 370, 1985).
S. pombe invertase is and is a high-molecular weight glycoprotein located on the cell surface with a molecular weight of about 205000, 67% of which is attributed to sugar chains composed of equimolar amounts of mannose and galactose residues. Molecular weight and amino acid studies of the protein moiety of the pure enzyme and experiments using antibodies have shown high similarlity between S. pombe invertase and the invertase from the budding yeast Saccharomyces cerevisiae from the viewpoint of protein chemistry (Moreno S. et al., Biochem. J. 267, 697, 1990). It is also known that a drop in glucose concentration de-represses synthesis of invertase (Mitchinson J. et al., Cell Sci 5, 373, 1969).
Induced invertase synthesis (de-repression) is also observed in Saccharomyces cerevisiae. Previous detailed studies on genetic regulation of invertase expression, the biosynthetic pathway and the structure of the sugar chain moiety have shown that Saccharomyces cerevisiae invertase is encoded by six overlapping genes, SUC1 to SUC5 and SUC7, on one chromosome and that activation of at least one of these SUC genes leads to utilization of sucrose and raffinose (Hohmann S. et al., Curr Genet 11, 217, 1986).
In contrast, with respect to S. pombe, although purification of the invertase protein has been reported (Moreno S. et al., 1985), no invertase genes had been identified until the present inventors and coworkers recently reported two overlapping invertase genes inv0.sup.+ and inv1.sup.+ in S. pombe. Because inv0.sup.+ is likely a pseudogene having an incomplete open reading frame, inv1.sup.+ is the only one gene encoding S. pombe invertase, which is supposed to confer the ability to grow on sucrose even in the absence of other carbon sources ("Kobogaku" edited by Yositaka Hashitani, Iwanami Shoten, 1967).
Analysis of the promoter region of the isolated gene suggested that a specific sequence between the 1st and 62nd base pairs is involved catabolite repression.
In Saccharomyces cerevisiae, the SUC2 gene is transcribed into two messenger RNAs (mRNAs) from different transcription initiation sites. The shorter one is a constitutive mRNA encoding the intracellular invertase, while the longer one is a mRNA encoding the catabolite-repressible secretory invertase with a de-repression ratio of not less than 200 (Carlson M. et al., Mol. Cell. Biol. 3, 439, 1983). Analysis of the promoter region for the longer mRNA suggested that the transcription initiation factor binds to a specific repeated sequence between positions -650 and -418 (Salokin L et al., Mol. Cell. Biol. 6, 2314, 1986). The region between positions -418 and -140 has been shown to be necessary for glucose repression.
These regions in the SUC2 gene showed no sig
REFERENCES:
patent: 5817478 (1998-10-01), Tohda et al.
patent: 5919654 (1999-07-01), Hama et al.
N. Tanaka et al., "Isolation and Characterization of an Invertase and its Repressor Genes from Schizosaccharomyces Pombe", Biochem. Biophys. Res. Commun. vol. 245, pp. 246-253, Apr. 1998.
S. Moreno et al., "Purification and Characterization of the Invertase from Schizosaccharomyces", Biochem. J., vol. 267, pp. 697-702, 1990.
S. Yoshioka et al., "Identification of Open Reading Frames in Schizosaccharomyces Pombe cDNAS", DNA Res., vol. 4, pp. 363-369, Dec. 1997.
J.A. Perez et al., "Cloning and Sequence Analysis of the Invertase Gene INV1 from the Yeast Pichia Anomala", Curr. Genet., vol. 29, pp. 234-240, 1996.
L. Salokin et al., "Short Repeated Elements in the Upstream Regulatory Region of the SUC2 Gene of Saccharomyces Cerevisiae", Mol. Cell. Biol., vol. 6, pp. 2324-2333, 1986.
Hama Yuko
Kumagai Hiromichi
Tohda Hideki
Asahi Glass Company Ltd.
McKelvey Terry
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