K. lactis RP28 ribosomal protein gene promoter and use thereof

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...

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435243, 4352542, 4353201, 435325, 435419, 536 231, 536 232, 536 234, 536 235, 536 2352, 536 2372, 536 241, C12P 2106, C07H 2104, C12N 100, C12N 1563

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056270496

DESCRIPTION:

BRIEF SUMMARY
CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a 371 of PCT/FR93/00695, filed Jul. 6, 1993.


BACKGROUND OF THE INVENTION

The present invention relates to the field of molecular biology. More especially, it relates to a novel DNA sequence possessing transcriptional promoter activity, to expression vectors containing this sequence and to its use for the production of recombinant proteins, and for example 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 carried out 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. There may be mentioned, in particular, the work on the 3-phosphoglycerate kinase PGK gene promoter [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 GAPDH gene [Holland et al., J.Biol.Chem. 254, 1979, 9839; Musti et al., Gene 25, 1983, 133], on that of the alcohol dehydrogenase 1 ADH1 gene [Bennentzen et al., J.Biol.Chem. 257, 1982, 3018; Denis et al., J.Biol.Chem. 25, 1983, 1165], on that of the enolase 1 ENO1 gene [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].
Recently, genetic tools have been developed in order to make use of Kluyveromyces yeast as a host cell for the production of recombinant proteins. The discovery of a 2-micron type plasmid originating from K.drosophilarum [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 not been optimized up until now. In particular, the promoters in question are essentially heterologous ones, i.e. originating from other microorganisms such as, notably, S.cerevisiae. This situation can give rise to various drawbacks, and can, in particular, limit the activity of the promoter on account of the absence of certain elements of the transcriptional machinery (e.g. trans-activators), exhibit some degree of toxicity for the host cell due to an absence of regulation or affect the stability of the vector.
Under these conditions, the lack of strong homologous promoters in Kluyveromyces constitutes a limiting factor in the industrial exploitation of this expression system.


SUMMARY OF THE INVENTION

The Applicant has now identified, cloned and sequenced a region of the Kluyveromyces lactis genome possessing transcriptional promoter activity (SEQ ID NO. 1). More specifically, this region corrresponds to the promoter of the gene for the K.lactis ribosomal protein rp28 (KIrp28). This region, or derivatives or fragments thereof, may be used very efficiently for the production of recombinant proteins in yeasts of the genus Kluyveromyces. It is understood that this sequence may also be used in other host organisms.
Moreover, one advantage of the promoter region obtained lies in the absence of repression by glucose, which permits its use in conventional and industrial culture media.
One subject of the present invention therefore lies in a DNA sequence comprising all or part of the sequence SEQ ID. NO. 1 or of its complementary strand,

REFERENCES:
Heusterspreute et al, DNA, 1984, vol. 3(3): pp. 259-268.

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