Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-10-19
2001-07-24
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S440000, C435S483000, C435S254110, C435S254200, C435S320100, C536S024100
Reexamination Certificate
active
06265185
ABSTRACT:
FIELD OF INVENTION
Background of the Invention
The advent of recombinant DNA techniques has made it possible to select single protein components with interesting properties and produce them on a large scale. This represents an improvement over the previously employed production process using micro-organisms isolated from nature and producing a mixture of proteins which would either be used as such or separated after the production step. However, the conventional cloning techniques have the drawback that each protein component has to be purified and characterised by its (partial) amino acid sequence before it is possible to prepare synthetic oligonucleotide probes for hybridisation experiments. Since this is a rather time-consuming process, the cloning of novel proteins might be considerably expedited by using a screening method involving selecting clones expressing a desired protein activity, i.e. expression cloning.
Recently, a novel method for cloning of fungal enzyme genes by expression cloning in yeast was developed by Dalbøge and Heldt-Hansen (A novel method for efficient expression cloning of fungal enzyme genes. Mol. Gen. Genet. 243: 253-260. (1994), WO 93/11249).
This expression cloning technique combines the ability of a yeast strain (e.g.
Saccharomyces cerevisiae
) to express heterologous genes with the utilisation of sensitive enzyme plate assays. The principle in expression cloning is outlined in FIG.
1
.
This method makes it possible to clone enzyme genes independently of knowledge of the amino acid sequence and has proven successful in cloning a number of new enzymes.
Even though the above described method already have proven successful, there is still room for improvement.
Improvement of the expression cloning technique can be done by identifying new improved promoters, e.g. to increase expression of naturally low expressed enzymes and thereby facilitating the subsequent screening.
EP 220864 describes a
Yarrowia lipolytica
yeast promoter XPR2. The XPR2 yeast promoter is only active at pH above 6.0 on media lacking preferred carbon and nitrogen sources and full induction requires high levels of peptone in the culture medium (Ogrydziak, D. M., Demain, A. L., and Tannenbaum, S. R. (1977) Biochim. Biophys. Acta. 497: 525-538.; Ogrydziak, D. M. and Scharf, S. J. (1982). Gen. Microbiol. 128: 1225-1234.).
The demand for pH above 6.0 in the medium makes it difficult to screen directly for secreted enzymes that are active only in an acidic environment.
Therefore, an object of the present invention, is to provide new improved yeast promoters, especially for use in expression cloning in yeast, but also for heterologous expression of a desired polypeptide in an expression system of choice.
SUMMARY OF THE INVENTION
The present invention is based on the cloning and characterisation of two DNA sequences shown in SEQ ID NO 1 and 2, respectively, which both:
1) have yeast promoter activity, and
2) have improved properties for expression cloning in yeast.
Further deletion studies on both yeast promoter sequences have identified the most important regions for each yeast promoter.
For the yeast promoter shown in SEQ ID NO 1, the most important region is from position −241 to −41 and for the yeast promoter shown in SEQ ID NO 2, it is from position −163 to −3. For further details see example 8.
Accordingly, in a first aspect the invention relates to a cloned yeast promoter DNA sequence, which comprises
a) the DNA sequence from position −241 to −41 shown in SEQ ID NO 1, or
b) an analogue of the DNA sequence defined in a) which
i) is at least 90% homologous with said DNA sequence, or
ii) hybridises with the same nucleotide probe as the DNA sequence defined in a).
In a second aspect the invention relates to a cloned yeast promoter DNA sequence, which comprises
a) the DNA sequence from −163 to −3 shown SEQ ID NO 2 , or
b) an analogue of the DNA sequence defined in a) which
i) is at least 90% homologous with said DNA sequence, or
ii) hybridises with the same nucleotide probe as DNA sequence defined in a).
In a further aspect the invention relates to an expression vector comprising a cloned yeast promoter according to the invention.
In a further aspect the invention relates to the use of said expression vector for expression cloning in yeast.
Further the invention relates to a process for producing a polypeptide of interest in a yeast host cell, the process comprising transforming a suitable yeast host cell with a recombinant expression vector comprising i) a yeast promotor of the invention and ii) a DNA sequence coding for a polypeptide of interest, culturing the transformed cells under suitable conditions to express the polypeptide, and recovering the expressed polypeptide from the culture.
Finally the invention relates to the use of a polypeptide produced as described above for various industrial applications.
REFERENCES:
patent: 0 220 864 A1 (1987-05-01), None
patent: 0 220 864 B1 (1987-05-01), None
patent: 0 402 226 (1990-12-01), None
patent: WO 93/11249 (1993-06-01), None
patent: WO 95/06739 (1995-03-01), None
Dalboge et al., Mol Gen Genet, vol. 243, pp. 253-260 (1994).
Dalbøge Henrik
Muller Sven
Garbell Esq. Jason I.
Lambiris Esq. Elias
Novozymes A/S Patents
Yucel Remy
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