Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1990-06-04
1993-01-26
Weimar, Elizabeth C.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 697, 435192, 4353201, 935 10, 935 14, 935 34, 935 47, C07H 1512, C12P 2106, C12N 908, C12N 1500
Patent
active
051823764
DESCRIPTION:
BRIEF SUMMARY
This invention relates to synthetic genes coding for horseradish peroxidase.
Horseradish peroxidase C (E.C.1.11.1.7) (HRP) is the major peroxidase isozyme isolated from the horseradish (Armoracia rusticana). It is a monomeric glycoprotein of 308 amino acids the polypeptide chain having a MW of 33,980 D. There are three neutral carbohydrate side chains and 4 disulphide bridges. The amino acid sequence of the mature protein has been determined. The presence of a pyrrolidonecarboxylyl amino terminus indicates that the protein is probably produced as a precursor form that is processed on secretion. The active form of the enzyme contains a hemin prosthetic group.
The enzyme is particularly stable and is amenable to crosslinking and derivitisation without excessive loss of activity. This together with its wide range of chromogenic substrates, some of which give rise to insoluble, chemiluminescent or flourescent products, and the low background activities observed in most applications, have made horseradish peroxidase an invaluable tool for diagnostic and research applications in the fields of immunology, histochemistry, cytology and molecular biology. A further advantage it presents over other enzymatic markers is that some some substrates for the enzyme give rise to electron dense products that allow correlation of peroxidase location with cellular ultrastructure using electron microscopy. In addition, horseradish peroxidase is electron dense itself by virtue of the Fe it contains and as a result can act as an E.M. marker in its own right. Particular applications have been found in immunochemistry, where peroxidase cross linked to immunoglobulin is widely used in both ELISA based assay systems and immunocytochemistry. Methods have been described that use either direct crosslinking of peroxidase to the immunoglobulin or indirect crosslinking of biotin labelled immunoglobulin to a streptavidin/horseradish peroxidase complex. Such streptavidin complexes have also found widespread application in nucleic acid hybridisation methods where biotinylated probe sequences can be localised by sequential incubation with the streptavidin/peroxidase complex and a suitable chromogenic peroxidase substrate.
The amino acid sequence of horseradish peroxidase is taught by Welinder, K. G. (Eur. J. Biochem. 96, 483-502 (1979)). The cloning of the cDNA or natural gene for horseradish peroxidase has not been described.
In order to facilitate the dissection of the structure/function relationships of HRP, its incorporation into expression vectors and the production of novel chimeric proteins containing HRP functionality an improved novel synthetic gene for the peroxidase C produced by Armoracia rusticana is sought.
It is by no means easy to predict the design of an improved HRP gene, since the factors that determine the expressibility of a given DNA sequence are still poorly understood. Furthermore, the utility of the gene in various applications will be influenced by such considerations as codon usage and restriction sites. The present invention relates to a synthetic HRP gene which has advantages in the ease with which it can be modified due to the presence of useful restriction sites.
When synthesising and assembling genes, problems have been encountered when there are inverted or direct repeats greater than eight bases long in the genetic sequence. In addition, areas of unbalanced base composition such as G/C or A/T rich regions or polypurine/polypyrimidine tracts have been found to lead to inefficient expression. :The present invention seeks to overcome or at least alleviate these difficulties.
According to a first aspect of the invention, there is provided DNA coding for HRP and having restriction sites for the following enzymes: ##STR2## The DNA may also contain a 5' HindIII site and/or a 5' NdeI site and/or a 3' BamHI site and/or a 3' EcoRI site.
According to a second aspect of the invention, there is provided DNA including the following sequence: ##STR3## The above sequence may be immediately preceeded by an initiation codon (AT
REFERENCES:
Welinder (1976) Covalent Structure of the . . . Horseradish Peroxidase, FEBS Lett. 72, 19-23.
Suggs et al. (1981) Proced. Natl. Acd. Sci. 78, 6613-6617.
Maniatis (1983) Molecular Cloning, 412-413.
Kunkel (1985) Proced. Nat'l Acad. Sci. 82, 488.
Burke Julian F.
Edwards Richard M.
British Bio-technology Limited
Crouch Deborah
Weimar Elizabeth C.
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