Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Isomerase
Reexamination Certificate
1999-08-05
2002-05-14
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Isomerase
C435S069100, C435S252300, C435S320100, C435S942000, C536S063000, C530S350000
Reexamination Certificate
active
06387683
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a recombinant protein of a yeast-derived enzyme, protein disulfide isomerase (hereinafter referred to as “PDI”), and a process for production thereof.
According to the present invention, a region within yeast PDI gene encoding the endoplasmic reticulum localization signal at the C-terminal is modified, the modified gene is incorporated into an expression vector, and is expressed. The recombinant yeast PDI then can be secreted outside cells, with its enzymatic activity being fully retained, and thus can be produced in a large amount. Further, the process for production according to the invention provide a yeast PDI recombinant protein under culture conditions of pH close to neutrality, by employing host cells which can be cultured at nearly neutral pH.
PDI is an enzyme (EC 5.3.4.1.), which catalyzes disulfide exchange in a protein. This enzyme promotes exchange of disulfide bonds in a protein in the presence of an appropriate oxidizing or reducing agent. The PDI protein is localized in the lumen of an endoplasmic reticulum in an eukaryote, and has the activity of catalyzing the binding of disulfide bonds of a secretory protein, which has been translated by a membrane-attached ribosome and transported to the endoplasmic reticulum. It is expected that PDI can be used to act on various proteins produced by gene recombination, and to make these proteins take stereostructures with which they can show activity. Commercially, this enzyme can be applied, for example, to improving the quality of protein-containing foods, such as dough, ham, sausages, fish paste products, and soybean curd. As noted from these facts, PDI is a useful enzyme.
Genetic engineering techniques in recent years have resulted in the disclosure of genes encoding PDI. Japanese Patent Public Disclosure No. 197176/92 describes the physicochemical properties of PDI derived from the yeast Saccharomyces cerevisiae, and a gene sequence encoding the protein. The Saccharomyces cerevisiae-derived PDI protein has a molecular weight of about 70,000 and an isoelectric point of about 4.0 to 4.1. Its optimal pH is about 8.5 to 8.75 (J. Biochem., 108, 846 (1990), Japanese Patent Public Disclosure No. 197176/92).
Usually, yeast PDI is present in cells. In order to obtain this enzyme, it was necessary to perform a complicated process comprising culturing the cells, grinding the cells, extracting and purifying cell extract and obtaining a final product. This complicated extraction process simply led to a build up of cost. Furthermore, the yeast PDI is an enzyme having the unstable property of being easily deactivated by heat or heavy metal ions. Because of this unstable property, in addition to said complexity of the extraction step, a careful, tiresome manipulation is required for maintaining the activity during the entire process.
It is one of the important challenges to produce the PDI protein, which is a useful substance, stably and in a large amount with its enzyme activity being fully retained, and supply it to the market in a fully active state. To attain this purpose, the extraction step needs to be simplified. Means for allowing the enzyme, an intracellular substance, to be secreted and produced extracellularly is effective for its simplification. Namely, if the enzyme can be continuously produced in a culture medium outside cells, which have once been cultured, the frequency of culturing the cells will be decreased, and the cell grinding step will become unnecessary. Purification from the culture medium with few impurities could simplify the purification step as well, reduce influence on the activity, and result in efficient mass production.
Yeast PDI, at the stage of a precursor, retains an extracellular secretion signal, and is produced in an endoplasmic reticulum, which is the starting point of a secretion pathway. However, even if expression is attempted so that yeast PDI will be secreted outside the cell, the wild type is not secreted extracellularly (M. Lamantia et al., Cell, 74, 899, 1993). It is speculated that this is because an endoplasmic reticulum localization signal at the C-terminal (the amino acid sequence HDEL in the case of Saccharomyces cerevisiae, for example) is retained. The number of PDI genes per cell of yeast is only one, and the amount of PDI produced per cell is minuscule. Even if the PDI is secreted in a culture medium, it cannot be obtained in sufficient concentration.
Further, the yeast PDI activity is unstable in an acidic region, while the optimum pH for ordinary yeast is in a weakly acidic region. In this weak acidity (pH 6.0 or lower), PDI is deactivated. At pH in a region in which PDI is not deactivated, yeast cells, usually, cannot remain active. In addition, culture medium components ordinary used for culture of yeast may contain substances which decrease or destroy PDI activity. The use of a culture medium containing such components would deactivate PDI produced extracellularly. For these various reasons, a method for mass production of yeast PDI in an active state had not been developed before the present invention.
SUMMARY OF THE INVENTION
The present invention provides a process suitable for mass production of recombinant yeast protein disulfide isomerase which is biologically active. Specifically, the process for production of the present invention is characterized by producing the yeast PDI protein by a genetic engineering technology using a yeast PDI gene, and causing its extracellular secretion.
The invention also provides recombinant yeast protein disulfide isomerase produced by the process for production of the present invention.
REFERENCES:
patent: 197176 (1992-07-01), None
patent: 30967 (1993-02-01), None
patent: 289267 (1995-11-01), None
Denecke et al., Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope, The EMBO Journal, vol. 11, No. 6, pp. 2345-2355, 1992.
J. Biochem., 108, p. 846-851 (1990).
Cell 74, 899, 1993, M. Lamantia et al.
J. Biochem., 110, p. 306-313 (1991).
Agric. Biol. Chem. 54(4), p. 1043-1044, 1990.
Ishii Nobuyoshi
Matuo Yushi
Suzuki Yasuo
Tanaka Hideo
Uchida Kohji
Oriental Yeast Co. Ltd.
Saidha Tekchand
LandOfFree
Recombinant yeast PDI and process for production thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant yeast PDI and process for production thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant yeast PDI and process for production thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2875943