Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-10-07
1998-11-10
Degen, Nancy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 43525411, 43525421, 536 231, 536 241, C12P 2106
Patent
active
058342370
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the biotechnology field, in particular to an improvement made to the production of a heterologous protein of commercial or therapeutic importance in a eukaryotic cell, and especially a yeast of the genus Schizosaccharomyces. It relates in the first place to the use of a gene coding for a product capable of activating a thiamine-regulable promoter region, thiamine governing the expression of the protein of interest, and in the second place to an expression cassette in which the DNA fragment coding for the protein of interest is placed under the control of a promoter region isolated or derived from the Schizosaccharomyces pombe pho4 gene.
For some years now, numerous expression cassettes for the production of proteins of interest in eukaryotic cells have been described in the literature. These cassettes comprise, in particular, a promoter region which is functional in the cell in question. Generally speaking, a promoter region is located in the 5' flanking region of the genes, and comprises the set of elements permitting the transcription of a DNA fragment placed under their control. A promoter region may also consist of the assembly of elements of various origins which are functional in the host cell, such as, in particular: startsite. While it appears to be necessary for a correct initiation of transcription, it cannot on its own provide for an effective transcription; and for an effective level of transcription, either constitutively (transcription level constant throughout the cell cycle, irrespective of the culture conditions), or regulably (so-called regulatory regions permitting an activation of transcription in the presence of an activator and/or repression of transcription in the presence of a repressor), according to the gene from which they originate.
When it is desired to obtain a large amount of a protein of interest, a strong and constitutive promoter region is generally used to control the expression of the DNA fragment coding for the protein of interest, for example the 5' flanking regions of the Saccharomyces cerevisiae PGK (3-phosphoglycerate kinase) gene (Hitzeman et al., 1983, Science, 219, 620-625) and the Schizosaccharomyces pombe and (alcohol dehydrogenase) gene (Russel and Hall, 1983, J. Biol. Chem., 258, 143-149).
However, the use of a strong and constitutive promoter region is not suited to the production of proteins of interest displaying some degree of toxicity with respect to the host cell. In effect, the production of toxic proteins affects cell growth and risks bringing about the selection of spontaneous mutations leading to a loss or a significant reduction in the level of production. In extreme cases, cell viability may be affected.
For these reasons, it can be advantageous to have at one's disposal regulable promoter regions enabling the production of the protein of interest to be varied in accordance with the culture conditions or the cellular growth phase. Generally speaking, such promoter regions are isolated or are derived from regulable genes.
Over the last few years, a number of regulable genes have been demonstrated in numerous eukaryotes, and in particular in Schizosaccharomyces pombe. A wide variety of mechanisms govern the regulation of these genes. As examples of regulable Schizosaccharomyces pombe genes, there may be mentioned the heat shock genes whose expression increases with temperature (Gallo et al., 1991, Mol. Cell. Biol., 11, 281-288), and the gene coding is repressed in the presence of glucose and induced under conditions of glucose deficiency (Hoffman and Winston, 1989, Gene, 84, 473-479). Other members of this category include the thiamine-regulable genes, namely the pho4 (Yang and Schweingruber, 1990, Curr. Genet., 18, 269-272), nmt1 (Maundrell, 1990, J. Biol. Chem., 265, 10857-10864) and thi2 (Zurlinden and Schweingruber, 1992, Gene, 117, 141-143) genes whose expression is regulated at transcriptional level by thiamine, more precisely repressed in the presence of thiamine and induced or derepressed in its absence.
The
REFERENCES:
DATABASE EMBL, X77512, 7 Feb. 1994, H. Frankhauser et al, Thiamine repressible genes regulatory protein THI1 (Transcription factor NTF1), & Journal of Biological Chemistry, vol. 269, 1994 MD US, pp. 11921-11926.
J. BIOL. CHEM. (1994), 269(16), 11921-6, CODEN; JBCHA3;ISSN: 0021-9258, Apr. 1994, Tang, Carol S.L. et al, "ntfl+ encodes a 6-cysteine zinc finger-containing transcription factor that regulates the nmtl promoter in fission yeast".
DATABASE WPI, Section CH, Week 9340, Derwent Publications Ltd., London, GB; Class B04, AN 93-314182 & IT-B-1 236 844, 22 Apr. 1993.
FEBS Lett. (1992), 305(3), 244-8 CODEN: FEBLAL;ISSN: 0014-5793, K. Nosaka et al, "Upstream activation element of ther PH03 gene encoding thiamin -- repressible acid phosphatase in Saccharomyces cerevisiae".
GENETICS (1992), 130(3), 445-9 CODEN: GENTAE;ISSN: 0016-6731, Anne Marie Scheingruber et al, "Isolation and characterization of regulatory mutants from Schizosaccharomyces pombe involved in thiamine-regulated gene expression".
GENE (1993), 123(1), 127-30 CODEN: GENED6;ISSN: 0378-1119. Kinsey Maundrell, "Thiamin-repressible expression vectors pREP and pRIP for fission yeast".
FEBS Letters, vol. 297, No. 1,2, Feb. 1992 AMSTERDAM NL, pp. 155-158, Hiroshi Nishimura et al, "Cloning and characteristics of a positive regulatory gene, THI2(PH06), of thiamin biosynthesis in Saccaromyces cerevisiae".
J. BACTERIOL. (1994), 176(21), 6631-5 CODEN: JOBAAY;ISSN: 0021-9193, Andreas Zurlinden et al, "Cloning, nucleotide sequence, and regulation of Schizosaccharomyces pombe thi4, a thiamine biosynthetic gene".
GENE (1994), 147(1), 141-4 CODEN: GENED6;ISSN: 0378-1119, Hans Fankhauser et al, "Thiamine -repressible genes in Schizosaccharomyces pombe are regulated by a Cys6 zinc-finger motif-containing protein".
Basi, G., et al., 1993, Gene, vol. 123, pp. 131-136, 1993.
Giniger, E., et al., 1985, Cell, vol. 40, pp. 767-774, 1985.
Yang, J., et al., 1990, Current Genetics, vol. 18, pp. 269-272, 1990.
Jacobs Eric
Schweinbryber Ernst
Silvestre Nathalie
Degen Nancy
Transgene S.A.
Yucel Irem
LandOfFree
Combined use of two expression cassettes for the production of a does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Combined use of two expression cassettes for the production of a, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Combined use of two expression cassettes for the production of a will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1515471