Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-02-22
1997-05-06
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...
536 231, 536 241, C12N 1511, C12P 2102
Patent
active
056270461
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the field of molecular biology. More especially, it relates to a novel DNA sequence possessing transcription promoter activity, to expression vectors containing this sequence and to its use for the production of recombinant proteins, e.g. heterologous proteins. The invention also relates to the recombinant cells containing this DNA sequence.
The progress made in the field of molecular biology has enabled microorganisms to be modified in order to make them produce heterologous proteins. In particular, a large number of genetic studies have focused on the E. coli bacterium. However, the industrial application of these novel production methods is still limited, especially by problems of efficacy of expression of the genes in these recombinant microorganisms. Thus, with the aim of increasing the performance of these production systems, research has been performed in order to isolate strong promoters, enabling high levels of expression of heterologous proteins to be obtained. In E. coli, the promoters of the tryptophan and lactose operons may be mentioned in particular.
More recently, in S. cerevisiae yeast, studies have focused on promoters derived from genes involved in glycolysis. Examples include promoters from the 3-phosphoglycerate kinase PGK gene (Dobson et al., Nucleic Acid Res. 10, 1982, 2625: Hitzeman et al., Nucleic Acid Research 1982, 7791), the glyceraldehyde-3-phosphate dehydrogenase GAPDH gene (Holland et al., J. Biol. Chem. 254, 1979, 9839; Musti et al., Gene 25, 1983, 133), the alcohol dehydrogenase 1 ADH1 gene (Bennentzen et al., J. Biol. Chem. 257, 1982, 3018; Denis et al., J. Biol. Chem. 25, 1983, 1165), and the enolase 1 ENO1 gene (Uemura et al., Gene 45, 1986, 65).
Recently, genetic tools have been developed in order to make use of Kluvveromyces yeast as a host cell for the production of recombinant proteins. The demonstration of a 2-micron type plasmid originating from K. drosoohilarum (plasmid pKD1-EP 241,435) has enabled a very effective host/vector system for the production of recombinant proteins to be established (EP 361,991). However, the promoters used in this system have never been optimised. In particular, the promoters in question are essentially heterologous ones, i.e. originating from other microorganisms such as, notably, S. cerevisiae. This situation may lead to various disadvantages. The activity of the promoter may be limited because of the absence of certain elements of the transcription machinery (e.g. transactivators). A certain toxicity to the host cell may occur due to an absence of regulation, or the stability of the vector may be affected.
The lack of strong homologous promoters in Kluyveromyces constitutes a limiting factor in the industrial exploitation of this expression system.
The Applicant has now identified, cloned and sequenced a region of the Kluvveromyces lactis genome possessing transcription promoter activity (see FIG. 1). More specifically, this region corresponds to the promoter of the K. lactis ADH4 gene (K1ADH4) coding for alcohol dehydrogenase IV. This region, or derivatives or fragments thereof, may be used very efficaceously for the production of recombinant proteins in yeasts of the genus Kluyveromvces. This sequence may also be used in other host organisms.
Moreover, analysis of the region of the Kluvveromvces genome obtained has enabled a bidirectional promoter activity to be demonstrated. The complementary strand of the region presented in FIG. 1 also possesses promoter activity acting in the other direction.
Furthermore, another advantage of the promoter activity obtained lies in its regulable character. Depending on the conditions of use (medium, strain), it is possible to control the activity of the promoter and hence to trigger or repress the expression of a recombinant gene.
Another advantage of the promoter region obtained lies in the absence of repression by glucose. This result is surprising, since it represents the first example of a promoter derived from an ADH gene induced by ethanol and not represse
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Falcone Claudio
Fleer Reinhard
Saliola Michele
Fleisher Mindy
Goodman Rosanne
Ketter James
Rhone-Poulenc Rorer S.A.
Savitzky Martin F.
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