Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-09-09
2001-11-13
Slobodyansky, Elizabeth (Department: 1657)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S252300, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06316238
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the production of a human ALT (alanine aminotransferase), and particularly, an active human ALT maintaining a sufficient enzyme activity and having a similar property to that of the native enzyme.
More precisely, the present invention relates to a novel gene encoding an amino acid sequence of a human ALT, a novel plasmid having an altered-type human ALT gene having restriction sites added at the upstream and downstream of said gene, and
Escherichia coli
transformed with said plasmid, as well as a process for production in which the human ALT is expressed as an active enzyme using said
Escherichia coli.
DESCRIPTION ON THE RELATED ART
Human ALT is an enzyme that is leaked into serum in liver diseases such as viral hepatitis, hepatic cirrhosis and the like, and is important in clinical chemistry. In the serum diagnosis, standardization such as minimization of the difference between laboratories on measured values of ALT activity and the like is one of important problems. While a crude product originated from porcine heart is presently used as the standard for such purpose, this product is different from human enzyme in enzymological properties such as substrate specificity, Km values and the like, and therefore, there has been a demand for commercialization of an active enzyme originated from human.
On the other hand, the production of a protein derived from a heterologous organism in a microorganism became possible and it has been put to the practical application, thanks to the recent genetic engineering technology. For example, the production of an animal protein in
Escherichia coli
into which a plasmid formed by ligating a lactose promoter to plasmid pBR 322 is introduced is described in Science, 198, 1056, 1978. While, in the case of human ALT, the gene is cloned and its expression in
Escherichia coli
is attempted, there has not been an article reporting the expression of the ALT protein maintaining a sufficient activity.
The present inventors have also attempted previously the expression of a recombinant human ALT as an active holoenzyme in
Escherichia coli
, but fails to successfully obtain a high expression of the desired recombinant enzyme (Japanese Patent Publication (A) Hei 8-103278).
In the field of clinical laboratory test, standardization such as minimization of the difference between laboratories on measured values of ALT activity and the like is one of important problems. While a crude product originated from porcine heart is presently used as the standard for such purpose, this product is different from the human enzyme in enzymological properties such as substrate specificity, Km values and the like. Therefore, there has been a demand for an active enzyme originated from human, but a large supply of the enzyme from human tissues has been difficult.
REFERENCES:
patent: 5952211 (1999-11-01), Nakamura et al.
patent: 5068548 (1993-03-01), None
patent: 08103278 (1996-04-01), None
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Eto Akira
Funatsu Masahiko
Matsuo Yushi
Nakamura Atsuo
Tanaka Toshio
Browdy and Neimark
Oriental Yeast Co. Ltd.
Slobodyansky Elizabeth
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