Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1988-02-29
1990-02-06
Teskin, Robin
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 691, 435320, 435255, 435256, 536 27, 935 28, 935 37, 935 69, C12N 1500, C12N 120, C12N 500
Patent
active
048988235
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a DNA sequence. In particular it relates to an upstream activator sequence of the yeast phosphoglycerate kinase (PGK) gene, a vector containing the upstream activator sequence and a method for increasing the expression level from a yeast vector using the upstream activator sequence.
The recent advances in recombinaot DNA technology have been such that there are now many examples of the expression of heterologous gene products by host organisms. The commercial viability of such products is limited by the level of gene expression and there has been much research effort directed towards increasing expression levels.
In our copending European patent application EP-A2-0073635 we describe a number of plasmid vectors suitable for the expression of genetic material in yeasts. A particular vector described uses the promoter of the yeast PGK gene and is useful for the synthesis of commercially important polypeptides in yeast. The yeast PGK gene is itself expressed efficiently by a yeast cell and indeed the PGK promoter directs efficiently the expression of heterologous genetic material.
The analysis of the 5' region of other yeast genes e.g. CYC1 and HIS3 has lead to the discovery of sequences of DNA capable of potentiating the efficiency of promoter sequences of yeast genes. These sequences are known as upstream activator sequences (Faye et al. 1981 PNAS 78 2258; Lowry et al. 1983 PNAS 80, 151; Struhl 1982 PNAS 79, 7385). However all the genes analysed to date are expressed at low levels in yeast and consequently their promoter regions are of limited use for the construction of yeast vectors that direct the high level production of heterologous gene products.
Prior to the present invention it was not known whether efficiently expressed genes such as PGK contained upstream activator sequences and indeed, if such sequences did exist, whether they are primarily responsible for the efficiency of the expression of genes encoding abundant products. Furthermore if an upstream activator sequence from an efficiently expressed gene could be isolated it was not known whether it would function in a similar way to the `Potentiator` sequences of higher organisms which are capable of activiating heterologous promoters (Wasylyk et al. 1983 Cell 32, 503).
According to the present invention we provide an upstream activator sequence derived from the yeast PGK gene. The upstream activator sequence is contained in the 5' region of the yeast PGK gene between nucleotides -324 and -456. The upstream activator sequence may be used in a DNA "cassette" to activate any yeast promoter. In such an application the upstream activator sequence may have synthetic linker sequences attached at either end to facilitate attachment to a vector.
Further according to the present invention we provide a yeast expression vector including the upstream activator sequence of the yeast PGK gene. Preferably the yeast expression vector contains a promoter sequence heterologous to the PGK gene.
Further according to the present invention we provide a method for increasing the expression level of a yeast expression vector comprising inserting the upstream activator sequence of the yeast PGK gene into the vector, upstream of a heterologous promoter sequence in the vector.
The heterologous promoter sequence may be any promoter sequence known to the art other than the PGK promoter sequence. For example, the promoter may be derived from the TRP1, ADH1, URA3, HIS3, or CYC1 gene sequences.
The invention is now illustrated by the following Example, with reference to the accompanying drawings in which:
FIG. 1 is a schematic diagram of plasmid pMA213,
FIG. 2 is a schematic diagram of the construction of plasmids pMA318-325,
FIG. 3 shows the nucleotide sequence of a portion of the 5' region of the yeast PGK gene from necleotide -820 to nucleotide -220 inclusive,
FIG. 4 shows the nucleotide sequence and amino acid sequence about the pM22a deletion end point,
FIG. 5 shows a subjective estimate of the blue colour caused by .beta.-galactosidase act
REFERENCES:
Efficient Expression of the Saccharomyces Cerevisiae PGK Gene Depends on an Upstream Activation Sequence but Does Not Require TATA Sequences, Ogden et al., Molecular and Cellular Biology, Dec. 1986, pp. 4335-4343.
Nucleic Acids Research, vol. 15, No. 17, 1987, Stanway et al., pp. 6855-6873.
Kingsman Alan J.
Kingsman Susan M.
Celltech Limited
Teskin Robin
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