Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-02-24
2002-05-14
McGarry, Sean (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023600
Reexamination Certificate
active
06388068
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for producing a foreign polypeptide of interest by secreting the foreign polypeptide into a plant cell or plant body using a plant genetic recombination technique, a promoter for use in the method, and the like.
DESCRIPTION OF RELATED ART
Recombinant DNA techniques have enabled, through the use of appropriate transformants, the production of a foreign peptide by an organism that cannot produce the peptide naturally. Production systems using plants as hosts are also included in such techniques. Up to now, however, the use of plants as hosts for the production of foreign polypeptides have few practical applications.
For the production of a foreign polypeptide in a plant by a genetic engineering technique, the following method is known: a chimeric gene, in which the structural gene for the foreign polypeptide that is to be expressed in the plant is ligated at a site downstream of a promoter, is introduced in a plant cell to give a transformant plant cell; and the transformant plant cell is then regenerated by a conventional plant cell culture technique to generate a transgenic plant body capable of expressing the foreign polypeptide. A typical example of the promoter used in such a method is cauliflower mosaic virus 35S promoter (hereinafter, simply referred to as “35S promoter”). 35S promoter is known to direct the expression of a polypeptide of interest in a plant cell in a tissue-non-specific manner and, therefore, has been widely used as a promoter applicable to relatively general purposes particularly for laboratorial studies. However, in the tissue-non-specific expression, the expressed polypeptide is required to be isolated and purified from all kinds of tissues of the plant, which makes it difficult to produce the polypeptide at low cost, with good efficiency and in a large quantity. That is, in the case of production of a foreign polypeptide in various organs or tissues of a plant (such as fruit, tuber (root), leaf, or stem), polypeptides other than the polypeptide of interest are likely to contaminate as impurities in a large quantity. Therefore, such a production method involves higher cost for purification of the polypeptide compared to the conventional processes employing microbial fermentation.
Higher plants have adapted to their environments by developing into multi-cellular organisms and differentiating their tissues and even organs through a long process of evolution. The cells constituting the tissues and organs are individually surrounded by a cell wall, and the spaces between their cell walls are filled with an intercellular fluid (i.e., an apoplastic fluid). In particular, transport of water or other nutrient ingredients to various tissues becomes more important as plant bodies grow larger, and an intercellular fluid plays an important role in this transport. A vascular bundle found in a higher plant, particularly a pteridophyte, gymnosperm or angiosperm, which is composed of xylem (vessel and tracheids) and phloem (sieve tubes), is a representative organ having this transporting function. Xylem sap, which is one of the vascular bundle saps, contains water and inorganic nutrient ingredients absorbed from the soil, as well as other various substances synthesized in the root (e.g., plant hormones and carbohydrates). For example, it has been reported that when xylem sap from squash is subjected to SDS-polyacrylamide gel electrophoresis, several polypeptide bands are observed, demonstrating the presence of several kinds of polypeptides in the xylem sap in the relatively pure forms (S. Satoh et al., Plant Cell Physiol., 33, 841, 1992). If a foreign polypeptide can be secreted into a plant intercellular fluid in a tissue-specific manner, then a more economical processs for production of a foreign peptide compared to conventional tank fermentation methods with microbial or animal cultured cells will be realized. A recently developed related method is a method for the expression of a protein of interest in vascular bundles of the root using a promoter capable of directing the expression of the protein in a root-specific manner (Japanese Patent Application Laid-open No. 10-52273).
SUMMARY OF THE INVENTION
Under these situations, the present invention is directed to the production of a foreign polypeptide with high efficiency by secreting it into a plant intercellular fluid. That is, the object of the present invention is to provide a method for producing a foreign polypeptide in a plant without the need of a complicated purification, by utilizing the tissue-specific biofunction of the plant; and to provide a promoter, a signal peptide and the like for use in the method.
The present inventors have made extensive and intensive studies for overcoming the aforementioned problems. As a result, the inventors have found a method for producing a foreign polypeptide in a plant by utilizing the function to secret the polypeptide specifically into xylem sap (which is one of the plant intercellular fluids). The inventors have also found, as the intercellular fluid secretion function elements, a promoter and a signal sequence for the secretion of the polypeptide into xylem sap. Based on these findings, the inventors have succeeded in the establishment of a method for producing a foreign polypeptide in a plant intercellular fluid with high efficiency by constructing the xylem sap secretion promoter gene, a gene encoding a foreign polypeptide of interest and the plant secretion signal coding region, which led to the accomplishment of the invention.
According to the present invention, there is provided a method for producing a foreign polypeptide of interest in a plant, comprising secreting the foreign polypeptide into an intercellular fluid of the plant and then recovering the foreign polypeptide therefrom.
In this method, the intercellular fluid may be a vascular bundle sap. In this case, the vascular bundle sap may be a xylem sap.
In the method, the plant may be a plant cell or plant body. In this case, the plant cell or plant body may be of a monocotyledonous or dicotyledonous plant, or may be of a plant selected from the group consisting of poaceous, leguminous, solanaceous, brassicaceous, cucurbitaceous, umbelliferous and asteraceous plants.
In the method, the foreign polypeptide of interest may be human serum albumin.
The present invention also provides a promoter DNA capable of directing the expression of both a signal peptide for the secretion of a foreign polypeptide of interest into an intercellular fluid of a plant and the foreign polypeptide.
The promoter DNA may be:
(a) a promoter DNA comprising the nucleotide sequence depicted in SEQ ID NO: 1; or
(b) a promoter DNA having addition, deletion or substitution of one or a plurality of nucleotides in the nucleotide sequence depicted in SEQ ID NO: 1 and capable of directing the expression of a foreign polypeptide that is secreted into an intercellular fluid of a plant.
The promoter DNA may be:
(a) a promoter DNA comprising the nucleotide sequence depicted in SEQ ID NO: 2; or
(b) a promoter DNA having addition, deletion or substitution of one or a plurality of nucleotides in the nucleotide sequence depicted in SEQ ID NO: 2 and capable of directing the expression of a foreign polypeptide that is secreted into an intercellular fluid of a plant.
The present invention also provides a recombinant DNA comprising the above-described promoter DNA and a structural gene encoding the foreign polypeptide of interest.
The present invention also provides a recombinant DNA comprising the above-described promoter DNA, a structural gene encoding the foreign polypeptide of interest and a gene encoding a signal peptide.
The present invention also provides a signal peptide gene encoding:
(a) a peptide comprising the amino acid sequence depicted in SEQ ID NO: 3; or
(b) a peptide having addition, deletion or substitution of one or a plurality of amino acid residues in the amino acid sequence depicted in SEQ ID NO: 3 and having a signal peptide activity.
The present invention also pr
Masuda Susumu
Satoh Shinobu
Foley & Lardner
McGarry Sean
Noda Institute for Scientific Research
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