Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-02-01
1997-04-01
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 701, 536 231, 536 241, 935 22, 935 23, C12P 2100, C07H 2104, C12N 1563, C12N 1567
Patent
active
056164742
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the field of molecular biology. More particularly, it relates to a new DNA sequence having a transcriptional promoter activity, expression vectors containing this sequence, and its use for the production of recombinant proteins, for example heterologous proteins. The invention also relates to recombinant cells containing this DNA sequence.
The progress achieved in the field of molecular biology has enabled microorganisms to be modified to cause them to produce heterologous proteins. In particular, several genetic studies have been carried out on the E. coli bacterium. However, industrial use of these new production methods is still limited, in particular by problems of the effectiveness of gene expression in these recombinant microorganisms. In addition, in order to increase the performance of these production systems, investigations have been carried out to isolate strong promoters to enable high levels of expression of heterologous proteins to be obtained. With respect to E. coli, tryptophan and lactose operon promoters may be mentioned in particular.
More recently, studies on the yeast S. cerevisiae have related to promoters derived from genes involved in the glycolysis. The works on the promoter of the 3-phosphoglycerate kinase gene PGK (Dobson et al., Nucleic Acid Res. 10, 1982, 2625; Hitzeman et al., Nucleic Acid Research 1982, 7791), on that of the glyceraldehyde 3-phosphate dehydrogenase gene GAPDH (Holland et al., J. Biol. Chem. 254, 1979, 9839; Musti et al., Gene 25, 1983, 133), on that of the alcohol dehydrogenase 1 gene ADH1 (Bennentzen et al., J. Biol. Chem. 257, 1982, 3018; Denis et al., J. Biol. Chem. 25, 1983, 1165), on that of the enolase 1 gene ENO1 (Uemura et al., Gene 45, 1986, 65), on that of the GAL1/GAL10 gene (Johnston and Davis, Mol. Cell. Biol. 4, 1984, 1440) or on that of the CYC1 gene (Guarente and Ptashne, PNAS 78 (1981) 2199) may be mentioned in particular.
Genetic tools have recently been developed in order to make use of the yeast Kluyveromyces as the host cell for production of recombinant proteins. The discovery of a plasmid of the 2-micron type originating from K. drosophilarum (plasmid pKD1- EP 241 435) has enabled a very efficient host/vector system to be established for production of recombinant proteins (EP 361 991). However, the promoters used in this system have not been optimized to date. In particular, they are essentially heterologous promoters, that is to say originating from other microorganisms, such as, in particular, S. cerevisiae. This situation may result in various disadvantages, and in particular limit the activity of the promoter due to the absence of certain elements of the transcriptional machinery (for example trans-activators), cause a certain toxicity to the host cell because of an absence of regulation, or affect the stability of the vector.
Under these conditions, the lack of strong homologous promoters in the case of Kluyveromyces constitutes a limiting factor in the industrial use of this expression system.
The Applicant has now identified, cloned and sequenced a genome region of Kluyveromyces lactis having a transcriptional promoter activity (SEQ ID No. 1). More precisely, this region corresponds to a gene promoter coding for a transaldolase of K. lactis (called KlTAL1 gene). This region or derivatives or fragments thereof can be used in a very effective manner for production of recombinant proteins in yeasts of the genus Kluyveromyces. It is understood that this sequence may also be used in other host organisms.
Furthermore, one advantage of the promoter region obtained rests in the absence of repression by glucose, enabling use in the conventional and industrial culture media.
The present invention thus relates to a DNA sequence comprising all or part of the sequence SEQ ID No. 1 (nucleotide sequence of the fragment of 1.3 kb corresponding to the promoter KlTAL1 of K. lactis) or of its complementary strand, or of a derivative thereof, and having a transcriptional promoter activity.
In the context of the prese
REFERENCES:
patent: 5223408 (1993-06-01), Goeddel et al.
Schaff et al. "Molecular Analysis of the Structural Gene for Yeast Transaldolase" Eur. J. Biochem 188(3): 597-604 1990.
Jacoby et al. "Transaldolase Mutants In The Yeast Kluyveromyces . . . " Mol. Microbiol. 10(4) 867-876 1993.
Chen et al. "A Gene Cloning System for Kluyveromyces lactis . . . " J Basic Microbiol. 28(4) 211-220 1988.
Sun et al. "Purification & Crystallization of Transaldolase Isozyme I . . . "Arch. Biochem Biophys 178: 69-78 1977.
Bolotin Monique
Menart Sandrine
Degen Nancy J.
Fleisher Mindy
Rhone-Poulenc Rorer S.A.
Savitzky Martin F.
Smith Julie K.
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