High expression system of proteins

Details High expression system of proteins High expression system of proteins

2000-03-02

2003-05-06

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S254300, C435S205000, C435S201000, C536S023200, C536S024100

Reexamination Certificate

active

06558920

ABSTRACT:

DETAILED DESCRIPTION OF THE INVENTION
1. Field of the Invention
The present invention relates to a homologous or heterologous gene expression system in which at least a part of a promoter region of a tyrosinase-encoding gene (melO) of
Aspergillus oryzae
is used as a promoter. More specifically, in the present invention, an expression mechanism of a tyrosinase-encoding gene of
Aspergillus oryzae
has been investigated, and it has been consequently found that this gene is expressed in large quantities in liquid medium for a long period of time. It relates to a construction of a system in which homologous or heterologous genes are expressed in large quantities with
Aspergillus oryzae
using this property, making possible the high production of useful proteins.
2. Description of the Related Art
The development of the genetic recombination technology in recent years has enabled useful human proteins to be produced with
Escherichia coli
or yeast. However, when genes derived from eucaryotes such as humans are expressed using
Escherichia coli
as a host, such problems have been pointed out that normal processing is not conducted and a sugar chain is not adhered. Further, when secretory production of heterologous proteins is conducted with yeast, sugar chain linkage is conducted, but there is a defect that the secretion amount thereof is very small. Accordingly, fungi having a high protein secretion ability have attracted considerable attention as a host of eucaryotic protein expression. Of these,
Aspergillus oryzae
has been long used in the brewing industry of sake and miso, and it has been therefore positively applied to the heterologous gene expression. An acid protease of
Mucor miehei
has been already industrially produced using
A. oryzae
as a host.
Since
Aspergillus oryzae
provides large amounts of amylase proteins such as &agr;-amylase and glucoamylase, the promoters of these amylase genes are used for the production of heterologous proteins. The use of a gene promoter such as a protease or a 3-phosphoglycerate kinase has been studied. However, the expression amount thereof is far smaller than the expression amount of the promoters of amylase genes, especially the &agr;-amylase gene. Accordingly, in order to highly express heterologous protein genes with
Aspergillus oryzae
, there is actually no way but to use the promoters of amylase genes. Problems that the Inveniton is to Solve.
The promoters of amylase genes, especially the promoter of &agr;-amylase gene exhibits quite high expression even in submerged culture, and its usefulness in the heterologous protein production has been admitted (Japanese Patent Laid-Open No. 51067/1995), but there is a need to add an inducer such as starch or oligosaccharides to a medium. Further, when proteins producing glucose, such as glucoamylase and glycosidase, are expressed as heterologous proteins, glucose is produced in a large amount by the proteins expressed, so that a phenomenon of decreasing the production amount through glucose repression is observed. Accordingly, in order to widen a possibility of producing recombinant proteins with
Aspergillus oryzae
, it is required to study a promoter having a high expression ability in a control system different from the amylase system.
A glucoamylase that
Aspergillus oryzae
produces in solid-state culture is an important enzyme in the enzyme industry using a high sugar content. However, since glaB gene (Japanese Patent Laid-Open No. 84968/1998) which is its gene is little expressed in submerged culture, it cannot be mass-produced in submerged culture. Further, when recombinant proteins are produced using a high expression promoter, a large amount of glucose is formed by the recombinant proteins, so that the promoter of amylase genes does not allow the high production. The discovery of a promoter which shows a gene control system having a high expression ability, other than the amylase-type gene control system enables the production of glaB-type glucoamylase which was difficult so far in submerged culture.
In view of these circumstances, upon studying genes that can be highly expressed in submerged culture with
Aspergillus oryzae
, the present invention aims at high production of useful proteins such as glucoamylase using the promoter of high expression gene.


REFERENCES:
patent: 7-51067 (1995-02-01), None
patent: 10-84968 (1998-04-01), None
Davies et al. (1994) Prog Ind Microbiol 29:527-560.*
Tsuchiya et al. (1994) Biosci Biotechnol Biochem 58(5):895-899.*
Jefferson et al., “&bgr;-Glucuronidase fromEscherichia colias a gene-fusion maker”,Proc. Natl. Acad. Sci, vol. 83, pp. 8447-8451, (1986).
Unkles et al., “The development of a homologous transformation system forAspergillus oryzae, based on the nitrate assimilation pathway: A convenient and general selection system for filamentos fungal transformation”,MGG, vol. 218, pp. 99-104, (1989).
Fujita et al., “Molecular cloning and nucleotide sequence of the protyrosinase gene melO, fromAspergillus oryzaeand expression of gene in yeast cells”,BBA, vol. 1261, pp. 151-154, (1995).

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