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
1999-12-03
2003-02-25
McKelvey, Terry (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...
C435S254100, C435S410000, C536S024100
Reexamination Certificate
active
06524816
ABSTRACT:
The present invention relates to an expression element suitable for increasing the levels of expression of polypeptides in cells or organisms. In particular, the invention relates to an expression element having the sequence (SEQ ID NO: 7) CGGCAGGGTCTC.
Xylan, a heterogeneous polysaccharide commonly found in plant cell walls, is one of the most common polysaccharides in nature. Xylanase is one of the major enzymes involved in the breakdown of xylan, catalysing the digestion of xylan into oligoxylose subunits.
Xylanase enzymes and corresponding genes have been isolated from a very large number of different organisms. Examples include xylanases A and B from Penicillium, xylanase A from
Thermotoga maritima
and
Bacillus subtilis,
xylanases B and D from
Ruminococcus flavefaciens,
and many others.
It is generally established that it is desirable to direct expression of a heterologous nucleotide sequence in an organism, such as a filamentous fungus (e.g.
Aspergillus niger
), or yeast. The resultant protein or enzyme may then be used in industry. Alternatively, the resultant protein or enzyme may be useful for the organism itself. For example, it may be desirable to produce fungal protein products with an optimised amino acid composition and so increase the nutritive value thereof. For example, the fungus may be made more useful as a feed. In the alternative, it may be desirable to isolate the resultant protein or enzyme and then use the protein or enzyme to prepare, for example, food compositions. In this regard, the resultant protein or enzyme can be a component of the food composition or it can be used to prepare food compositions, including altering the characteristics or appearance of food compositions. It may even be desirable to use the organism, such as a filamentous fungus or a yeast, to express heterologous genes, such as for the same purposes.
Filamentous fungi in particular are attractive hosts for the large-scale production of proteins in industry. They have the capacity to secrete a large amount of heterologous and/or homologous protein into their growth medium, they have been extensively studied and are well known, and moreover they are considered safe for use in the preparation of products useful in the food, feed and beverage industries. They are known for their ability to secrete several hydrolytic enzymes, amongst which number the xylanases. A number of different xylanases are known and have been characterised and/or cloned (for example, see Ito, et al., (1992) Biosci. Biotech. Biochem. 56:906-912), including xylanases from
A. awamori, A. kawachii
and xylanases A, B and C from
Aspergillus tubingensis.
Of the latter, xylanase B is known to be more weakly expressed (see WO 94/14965). In general, xylanases belong to one of two families of glycosyl hydrolases, family H and family F. There is little homology between the two families, but within each family there is some sequence identity. For example, xylanases A and B of
A. tubingensis
belong to family H and share approximately 45% sequence identity at the amino acid level.
For heterologous expression in filamentous fungi, as well as the vast majority of other organisms, it is desirable to use a strong promoter to direct expression of the gene in question. Usually it is assumed that highly expressed genes contain a strong promoter and consequently promoters derived from highly expressed genes are frequently used for this purpose. Examples of expression systems for use in filamentous fungi include systems employing the glucoamylase promoter (U.S. Pat. No. 5,198,345) and the
A. awamori
xylanase A (xlnA) promoter (Gouka, et al., (1996) Appl. Microbiol. Biotechnol. 46, 28-35).
De Graaff et al., in xylans and xylanases, J. Visser et al. (eds.), 1992:235-246, Elsevier, and De Graaff et al., (1994) Molecular Microbiology 12:479-490, have identified a 158 bp upstream regulatory region of the
A. tubingensis
xlnA gene which is responsible for activation of transcription from the xlnA promoter. This region contains inter alia a sequence which comprises three repeats of the element (SEQ ID NO: 23) GTCCATTTAGCCA. De Graaff et al. showed that the entire 158 bp region was capable of activating an
A. niger
glucose oxidase gene (goxC) core promoter. It was not, however, determined whether the element (SEQ ID NO: 23) GTCCATTTAGCCA was itself responsible for the activity of the upstream region or the activating effect.
According to a first aspect of the present invention there is provided the use of a nucleic acid element having the sequence (SEQ ID NO: 7) CGGCAGGGTCTC to modulate transcription of a nucleotide sequence from a promoter.
Preferably, the promoter is a core promoter.
The present invention is concerned with the use of the sequence (SEQ ID NO: 7) CGGCAGGGTCTC as a control element for modulating transcription of a nucleotide sequence or nucleotide sequences from a promoter. The invention accordingly provides nucleic acid constructs in which at least one heterologous copy of the element is operatively linked to a promoter which is itself operatively linked to a nucleotide sequence, vectors containing such nucleic acid constructs and host cells transformed with such vectors andlor expressing DNA constructs according to the invention. Moreover, the invention concerns the use of multiple copies of the activating element operatively linked to the promoter, in order to provide further activation of transcription. In a further aspect, the invention concerns a sequence variant of the
A. tubingensis
xlnB gene which possesses enhanced expression characteristics as a result of the presence of three, rather than two, copies of the element of the invention upstream of the TATA box, as well as the use of this variant in nucleic acid constructs as above.
The element according to the invention is preferably placed upstream of a promoter which is operatively linked to a nucleotide sequence. The nucleotide sequence is preferably a heterologous nucleotide sequence.
Where a promoter already contains, in its upstream sequences, one or two copies of the regulatory element of the invention, an exogenous copy or copies is added in accordance with the invention in order to further activate transcription from this promoter.
The optimum number of elements is three. The presence of more than three elements is detrimental to transcriptional activation. The use of four or more elements is applicable where a lower, but still significant, level of transcription is desired. Thus, the invention may be applied to the modulation (downregulation as well as the upregulation) of transcription from a promoter. Preferably, the element of the invention is used to upregulate transcription from the promoter, such that the rate of transcription is increased.
Where more than one copy of the element of the invention is present, the elements may overlap. Thus, for example, the initial nucleotide of one element may also be the terminal nucleotide of another element. Preferably, the elements overlap by 1, 2 or 3 nucleotides. Preferably, the sequence of an overlapping element is (SEQ ID NO: 16) GGCAGGGTCTCGGCAGGGTCTC.
Preferably, the activated promoter linked to the nucleotide sequence according to the invention is incorporated into a nucleic acid construct, which may be a plasmid vector or the like. Advantageously, a vector according to the invention is an expression vector.
As stated above, three copies of the element provide the strongest transcriptional activation, being highly preferred to two elements. The addition of further elements, above three, leads to a progressive reduction in the activation level, as described in further detail in the accompanying examples.
Expression vectors according to the invention are useful for transforming cells. The invention accordingly provides cells transformed with vectors according to the invention, for the expression of polypeptides encoded by the heterologous nucleotide sequence.
The transformed cells may be cultured cells, for example in tissue culture or organ culture, but also cells which form all or part of
Danisco A/S
Foley & Lardner
McKelvey Terry
Sandals William
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