Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-05-04
2001-04-10
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S294000, C800S287000, C435S069800, C435S204000, C435S431000, C435S469000, C435S430100, C435S429000
Reexamination Certificate
active
06215051
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for producing a gene product, in particular to a method for the mass production of a desired gene product by expressing a gene encoding said gene product in plant host cells, whereby said desired gene product can be recovered from the culture medium of said plant host cells.
BACKGROUND OF THE INVENTION
The plant cell culture expression system has several advantages over the bacterial, yeast or Baculovirus expression systems. Bacteria do not, and yeasts only limitedly, carry out post-translational modifications of the expressed proteins. Plant cells are eukaryotic and able to perform sophisticated protein modifications which are often necessary for the proper function of proteins.
Although Baculovirus is a potent transformation vehicle for higher eukaryotes and generally performs satisfactory modifications of proteins, the cost for culturing baculovirus is much higher than that for plant cells. In addition, the host cells are eventually lysed by Baculovirus and thousands of host proteins along with the expressed transformation protein are mixed and released into the culture medium, which makes purification of the expressed transformation protein difficult.
The culture medium for plant cells contains mainly salts and vitamins and therefore, it costs much less than that used to culture insect cell lines which are used for the Baculovirus transfection. Moreover, the culture medium for plant cells will not need a supply of serum, whereas almost all animal cell cultures cannot survive without serum. In addition, since plant cells are eukaryotes, the expressed proteins therein will be appropriately post-translationally modified so as to render said proteins capable of functioning and being secreted out of the plant cells. Although no one has yet made a deeper understanding of the mechanism of protein secretion in plant cells, the common belief at present is that it could be similar to the secretory mechanism in animals.
Plant cell cultures are a potential commercial source of medicines, dyes, enzymes, flavoring agents and aromatic oils. Plant cell culture production of such compounds are sought when (1) they are produced by the plant in small quantities or in fleeting or unharvestable developmental stages of the plant's life cycle; (2) when they are produced by plants which are not amenable to agriculture or are native to vanishing or inaccessible environments; and (3) when the compounds cannot be satisfactorily synthesized in vitro or by other biosynthesis systems.
Attempts to produce products by plant cell culture, however, are often commercially unsuccessful due to such factors as insufficient production and secretion of the desired product, poor cell growth, and difficulties in maintaining the appropriate cell type in culture.
The callus alpha-amylase (&agr;-amylase) expression system has features which make it of potential use to plant cell fermentation technology, namely its high level of expression, sustained expression, expression irrespective of either the tissue of origin of the cell culture or tissue formation in the cell culture, and its product secretion. Although rice callus itself may not be an ideal source of commercial &agr;-amylase, the gene regulatory regions responsible for the high expression could be used, with the aid of recombinant DNA technology and plant transformation, to achieve high expression of other valuable proteins (Carl R. Simmons, et al (1991), Biotechnology and Bioengineering, 38: 545-551).
Starch includes straight-chain starch and branched starch, two types of polysacchardies, and is the basic stored nutrient component in cereal grains (T. Akazawa et al (1985), Ann. Rev. Plant Physiol., 36: 441-472). During the initial germinating period of cereal seeds, the aleurone layer cells will synthesize &agr;-amylase. Alpha-amylase, &agr;-glucosidase and enzymes restricting dextrinase are secreted into the endosperm and together hydrolyze starch to form glucose and maltose, so as to provide the nutrients needed for the growth of the germ (J. C. Rogers and C. Milliman, J. Biol. Chem., 259 (19): 12234-12240, 1984; Rogers, J. C., J. Biol. Chem., 260: 3731-3738, 1985). Other enzymes contributing to starch hydrolysis include &bgr;-amylase which can hydrolyze starch to form maltose and a small amount of glucose. In a dry seed, &bgr;-amylase normally exists in an inactive form in the endosperm due to protein disulfide bonding. When the seed germinates, the aleurone layer cells will be subjected to the induction by gibberellic acid (GA
3
) to produce protease, which can destroy the disulfide bond and release the active form of &bgr;-amylase. The above four enzymes take part in the hydrolysis of starch during the germination of seeds. However, &agr;-amylase is the most active and holds the most important role (Akazawa, T., et al (1985), Ann. Rev. Plant Physiol., 36: 441-472).
It is known that GA
3
exerts a direct influence over the expression of &agr;-amylase (Chandler, P. M., et al (1984), Mol. Biol., 3: 401-418). When rice seeds are treated with GA
3
, the new synthesis of &agr;-amylase mRNA by the aleurone layer cells increases to 50 to 100-fold of the control value (no GA
3
) (O'Neill, S. D., et al (1990), Mol. Gene. Genet., 221: 235-244). In reality, the regulation of &agr;-amylase gene expression by GA
3
has provided a very ideal model for studying the mechanism of hormonal regulation of gene expression in plants (Ho, T. H. D., et al (1987), “Regulation of gene expression in barley aleurone layers,” In:
Molecular Biology of Plant Growth Control,
pp.35-49. St. Louis, Mo.: Alan R. Liss, Inc.).
Hitherto, &agr;-amylase genes from rice, barley and wheat have been cloned and subjected to further study and analysis. The results show that these cereal-type &agr;-amylase isozymes or isoforms are all manufactured by several types of &agr;-amylase genes (Baulcombe, D. C., et al (1987) Mol. Gen. Genet., 209: 33-40); Huang, N., et al (1990a), Plant Mol. Mo. Biol., 14: 655-668; Knox, C. A. P., et al (1987) Plant Mol. Biol., 9: 3-17).
The &agr;-amylase secreted from the aleurone layer cells during the germinating period of the seed of barley and wheat comprises typo classes, the high isoelectric point and low isoelectric point. In barley, there are around 7 &agr;-amylase genes which belong to the high isoelectric point and 3-4 genes which belong to the low isoelectric point (B. Khursheed and J. C. Rogers, J. Biol. Chem., 263: 18593-18960, 1988).
Currently, 7 &agr;-amylase cDNA and 9 &agr;-amylase genomic DNA groups of barley have been cloned (Chandler, P. M., et al (1984), Plant. Mol. Biol., 3: 401-418; J. Deikman and R. L. Jones, Plant Physiol., 78: 192-198, 1985; Khrusheed & Rogers (1988), supra; Knox, C. A. P., et al (1987), supra). The &agr;-amylase genes of wheat are grouped into &agr;-Amy1, &agr;-Amy2 and &agr;-Amy3. Alpha-Amy1 has a high isoelectric point while &agr;-Amy2 has a low isoelectric point, and each has more than 10 genes which are expressed in germinating seeds. Alpha-amylase &agr;-Amy3 includes 3-4 genes which are expressed in immature seeds (Baulcombe et al (1987), supra). With regard to the study of rice &agr;-amylase genes, the &agr;-amylase genes thereof have not been classified into the high isoelectric point group and the low isoelectric point group as was done in the study of barleys and wheats. In reality, MacGregor, A. W., et al (Cereal Chem., 65: 326, 1988) applied the analytical method of isoelectric point electrophoresis and found that rice 2-amylase isomers had a pI value of less than 5.5.
Therefore, it is possible that rice does not have any isoform of high isoelectric point. Huang, N., et al (Nucl. Acids. Res., 18: 7007-7014, 1990b) grouped the 10 rice &agr;-amylase genes into 5 groups by cross hybridization experiment and confirmed their distribution in 5 chromosomes (Ranjhan et al, the original manuscript is still under preparation). O'Neill et al (Mol. Gene. Genet. 221: 235-244, 1990) made the first more detailed study of the cDNA pOS103 and pOS137 of rice &ag
Chan Ming-Tsair
Liu Li-Fei
Yu Su-May
Fox David T.
Marshall O'Toole Gerstein Murray & Borun
National Science Council of R.O.C.
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