Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1994-10-20
1996-09-24
Elliott, George C.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4353201, 536 241, C12N 510, C12N 1511, C07H 2104
Patent
active
055590276
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to the generation of improved inducible mammalian expression systems.
BACKGROUND TO THE INVENTION
Mammalian expression systems are being widely used in the production, by recombinant techniques, of proteins that are extensively modified after translation. These systems can be either constitutive or inducible. It is advisable to use inducible systems for the expression of potentially cytotoxic proteins.
A key element in determining whether an expression system is constitutive or inducible is the promoter. Several mammalian promoters that can be induced in experimental systems have been characterized and promoters present in the metallothionein (MT) genes and in the mouse mammary tumour virus/long terminal repeat (MMTV-LTR) have been used extensively.
The best inducers for the MT promoter are heavy metal ions, such as cadmium (Cd) and zinc (Zn). The induction of the promoter is mediated by transcription factors which, after activation by metals, bind to the inducible metal responsive elements (MREs) that are present in the MT promoter. This promoter also contains several constitutive (non-inducible) elements that bind transcription factors which do not need to be activated and that are responsible for a basal level of gene expression. As a result of the presence of these constitutive elements, the non-induced level of expression of the MT promoter is significant and the induction ratio (the ratio between the inducible expression and the basal level of expression) is usually no greater than 5- to 10-fold. Attempts have been made to reduce the basal level of expression by removing some of the constitutive elements of the MT promoter. The removal of these elements, however, also reduces the inducible level of expression.
The native human MT-IIA promoter, besides having the MREs and the constitutive elements, contains a single inducible glucocorticoid responsive element (GRE) and glucocorticoids, such as dexamethasone (dex), induce low levels of expression from the MT-IIA promoter in its native context.
The native MMTV-LTR promoter contains four inducible GREs and can be strongly induced by glucocorticoids. The basal level of expression is lower than that obtained with the human MT-IIA promoter but the absolute level of inducible expression is not as high.
Nucleic acid sequences, such as inducible elements, involved in the regulation of gene expression, may be located 5' to, 3' to, or within the regulated gene.
SUMMARY OF INVENTION
In accordance with the present invention, there is provided a synthetic inducible eukaryotic promoter for the regulation of transcription of a gene, comprising at least two different classes of inducible elements. Classes of inducible elements with which the invention is concerned include hormone-responsive elements (including GREs), metal-responsive elements (MREs), heat shock-responsive elements, interferon-responsive elements and cytokine responsive elements.
In one embodiment, the synthetic promoter provided herein is derived from a native promoter and one of the different classes of inducible elements is a native inducible element while another of the different classes of inducible elements is provided, such as by insertion into the native promoter or by activation of a normally-inactive element in the native promoter. While, in general two different classes of inducible elements are present in the novel synthetic promoter of the invention, combinations of three or more may be present, if desired.
The utilization of different classes of inducible elements in the synthetic promoters enables synergistic induction of a expression of a gene product in a eukaryotic expression system, particularly a mammalian expression system. That is, the level of gene expression obtained by induction of multiple classes of inducible element is greater than the sum of the individual gene expressions achieved by separate induction of the individual classes of inducible elements. In addition, overall levels of gene expression may be enhanced.
The
REFERENCES:
Hu et al; Molecular & Cellular Biology, vol. 10, No. 12; pp. 6141-6151; Dec. 1990.
Wright et al.; Proceedings of The National Academy of Sciences USA, vol. 88, pp. 8283-8287; Oct. 1991.
Allan et al.; The Journal of Biological Chemistry, vol. 266, No. 9, pp. 5905-5910; Mar. 25, 1991.
Filmus Jorge
Klein Michel
Connaught Laboratories Limited
Elliott George C.
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