Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1992-07-28
1995-01-17
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435 691, C12N 900, C12P 2100
Patent
active
053825187
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a novel protein possessing urate oxidase activity; the invention also concerns the drugs containing this protein as well as the genetic engineering tools for producing that protein and notably the recombinant gene coding for that protein, the expression vector carrying that gene and the eukaryotic cells or the prokaryotic microorganisms transformed by this expression vector.
Urate oxidase (EC 1.7.3.3.), which is also called uricase, is an enzyme of the purine degradation pathway. This enzyme does not exist in primates (such as man), birds, a few reptiles or most insects. It is also non-existent in some dogs (such as the dalmatian).
In man, the purine bases--adenine and guanine are--converted to xanthine. The xanthine is oxidized by xanthine oxidase to form uric acid according to the following reaction:
The O.sub.2 - radical, which is the substrate for superoxide dismutase, is converted by the latter to hydrogen peroxide.
Uric acid, a metabolite present in blood, is normally found essentially in the form of the soluble monosodium salt. However, in certain people, it may happen that the uric acid precipitates and forms calculi. Hyperuricemia, which is an increase in the amount of uric acid circulating in the blood, causes uric acid to deposit in the cartilaginous tissues, leading to gout. Hyperuricemia can also have consequences on the kidneys: an excess of uric acid in the urine and in the kidneys can result in uric acid nephrolithiasis, i.e. the accumulation of renal calculi, which are very painful and can damage the kidney. These calculi are composed of uric acid possibly associated with phosphate and oxalate salts. Overproduction of uric acid can have a variety of origins: congenital metabolic defects, Lesch-Nyhan syndrome, excess ingestion of purine or proteins, treatments with uricosuric drugs, treatments of the hemopathies, particularly the cancerous hemopathies by cytolytic agents (chemotherapy) or by radiotherapy. (Gutman, A. B. and YU, T. F. (1968) Am. J. Med. 45-756-779).
Urate oxidase, the enzyme which catalyzes the degradation of uric acid to allantoin (a compound which is much more soluble than uric acid and does not crystallize at the concentrations reached in biological fluids), therefore has therapeutic value. Used in injections, it has a large number of advantages in the treatment of hyperuricemia and nephrolithiasis: speed of the hypouricemic effect (reduction of hyperuricemia of the order of 50% in less than 24 h), better protection of the kidney against lithiasis compared with other drugs such as allopurinol (a xanthine oxidase inhibitor), etc. At the present time, this enzyme is mainly used as adjuvant for the cytolytic agents in chemotherapy.
The urate oxidase currently used as a drug is obtained by a method comprising the culture of a mycelium of Aspergillus flavus and isolation of the urate oxidase from the culture medium by extraction, together with several steps for purifying this protein. This method, which makes it possible to obtain urate oxidase of high purity, nevertheless has disadvantages. In fact, the physiology and especially the genetics of A. flavus are not easy to work with (WOLOSHUK et al. (1989) Applied environ. microbiol., vol. 55, p. 86-90). It is therefore impossible to obtain strains which produce this enzyme in substantial amounts. Furthermore, A. flavus is liable to produce aflatoxins, which are sometimes difficult to separate off. The purified product should consequently be checked to ensure that it is free from these toxins.
There is therefore a need for a purer urate oxidase of A. flavus as well as for genetic engineering tools and techniques whereby these disadvantages can be overcome.
The Applicant purified the urate oxidase extracted from A. flavus, named thereafter the urate oxidase extract, up to a purity degree higher than that already known for this protein; the Applicant also determined the partial sequence of that protein and built two pools of labelled probes able to hybridize with the nucleotides coding for two portions of
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Caput Daniel
Ferrara Pascual
Guillemot Jean-Claude
Kaghad Mourad
Larbre Elisabeth
Sanofi
Schmickel David
Wax Robert A.
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