Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
2000-03-13
2003-04-08
Bui, Phuong T. (Department: 1638)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C435S410000, C435S320100, C435S252300, C436S024000, C436S023000, C800S278000, C800S295000
Reexamination Certificate
active
06544789
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to materials and methods for the controlled expression of polynucleotides in plant cells.
BACKGROUND OF THE INVENTION
Biological methods for the production of economically valuable compositions of matter in the form of polypeptides have shown promise as alternatives to traditional chemical syntheses. Although several biological systems have been successfully explored as potential sources of polypeptides, including proteins, each system has been found to have its limitations.
The simplest and most thoroughly investigated biological methods for chemical production are microbiological systems. Primitive prokaryotic cells have been amenable to investigation and have been found to produce a variety of small organic and inorganic compounds, as well as a variety of complex biomolecules, such as homologous and heterologous polypeptides and proteins. The most extensively characterized prokaryote,
Escherichia coli
, synthesizes complex biomolecules using a relatively straightforward process of gene expression requiring minimal expression control elements and an uninterrupted coding region. Further, genetic elements encoding heterologous polypeptides can be introduced and expressed in
E. coli
without much difficulty. With these advantages, a wide variety of polypeptides have been expressed in a controlled manner in this organism. However,
E. coli
cultures do require the costly inputs of energy and nutrients. The organism also does not readily secrete the produced polypeptides, adding to the time and expense required to isolate the desired compound. Although other microbes, e.g., species of Bacillus, do secrete polypeptides into growth media, cultures of these organisms also require costly inputs of energy and nutrients. Moreover, all of these primitive prokaryotic systems exhibit additional shortcomings such as the expensive effort to avoid culture contamination and the inability of the microbes to properly process or derivatize many expressed polypeptides to fully biologically active forms.
Yeast and fungi are fairly primitive eukaryotic cells that have also been used to produce polypeptides, including heterologous polypeptides. Although these cells may do a better job of reproducing the natural derivatization of most commercially desirable (i.e., eukaryotic) polypeptides, the reproduction is imperfect. Additionally, cultures of yeast or fungal cells are susceptible to contamination and the cells themselves require valuable resources in the forms of energy and nutrients, Efforts to obtain the desired chemicals, such as heterologous polypeptides, are also burdened by the frequent need to extract the chemical from the cell and purify that compound from the chemically complex contents of the yeast cell released during extraction.
Animal cells, e.g. mammalian cells, although expected to closely approximate the native derivation of many important polypeptides (e.g., human polypeptides), are very costly to culture, due to their sensitivity to contaminants, their requirements for energy, gases, and nutrients, and their limited lifespans. Isolation of the produced chemicals also would be it relatively expensive in view of the typical inability of mammalian cells to secrete products and the relative chemical complexity of the intracellular environment of these cells.
Plants, as photoautotrophic organisms, provide an alternative to heterotrophic animals as life forms for the production of chemicals. Transgenic plants have been generated, albeit typically to improve the characteristics of the plants themselves (e.g., to confer resistance to disease, to improve the yield of edible foodstuffs). Nevertheless, some transgenic plants have been used to produce chemicals such as heterologous polypeptides. Some expression control sequences (e.g., regulatory elements, signal peptide sequences) have been found to function in plant cells, or to preferentially function in the cells of particular plant tissues and organs. For example, Sijmons et al. (U.S. Pat. No. 5,650,307) expressed Human Serum Albumin (HSA) by fusing the HSA coding region to the leader sequence from Alfalfa Mosaic Virus. This fused coding region was placed under the control of the Cauliflower Mosaic Virus 35S promoter and the Nopaline Synthase terminator. The HSA was expressed in transgenic potato plants and transgenic tobacco cells. Sijmons et al. further disclosed the secretion of HSA by potato plant cells and recovery of the heterologous HSA from the intercellular space of those plants. Of course, this recovery method involved the destruction of the potato plants.
U.S. Pat. No. 5,580,768 also discloses the production and secretion of heterologous protein by a plant. In particular, the '768 Patent discloses a transgenic rubber tree, with the expressed transgene protein being collected from wounds as a part of the latex. This system is highly specialized for use with Hevaea species (perennial tree species with slow growth), and the tree must be damaged by wounding to recover the heterologous polypeptide in the form of a latex mixture.
Transformed plant material has also been used to express heterologous polypeptides. Wongsamuth et al., Biotech. and Bioengineer. 54:401-415 (1997), report the use of hairy root cultures to express murine IgG
1
monoclonal antibody. Further, some antibody activity was found in the medium of the hairy root cultures maintained under axenic conditions as heterotrophic biomasses requiring costly energy and nutrient inputs.
In plant expression systems, as in other biological expression systems, maximal utility is realized by an expression system that is controllable. Control of the timing and extent of polypeptide expression reduces the costs involved in maintaining the typically transformed host cells because recovery can be initiated at times that are suitable for the polypeptide being expressed. For example, recovery can be coincident with the period during which expression is elevated when attempting to produce and purify a labile polypeptide. To maximize the yield of stable polypeptides required in quantity, the recovery period may lag the expression period. In still other cases, the production of toxic polypeptides is delayed until optimal numbers of producing cells are present, with little, if any, lag in the recovery period.
A variety of controllable expression systems have been identified in animal, bacterial, yeast, or fungal cells, and some of these systems are also found in plant cells. However, the majority of these systems suffer from disadvantages in terms of the simple, versatile and economic production and recovery of polypeptides from plant cells. Frequently, the small molecule effector responsible for controlling expression is difficult to make or costly to obtain and, for those effectors that are available, problems associated with toxicity are frequently encountered. These toxicity considerations include the toxic potential of the effector on the host cell, as well as the deleterious presence of the effector in the isolated polypeptide preparation.
Phosphorus is a nutrient that plays a central role in energy metabolism and, in the form of phosphate, is found in the nucleic acids of all living organisms. Much of the phosphorus available in the environment is not in a bioavailable form such as orthophosphate, however. Consequently, diverse organisms have developed capacities for transforming environmental phosphorus into bioavailable forms that are assimilated. These capacities are evident in the number and diversity of genes that respond to phosphorus levels. One class of genes encodes phosphatase enzymes, which can generally be divided into alkaline and acid phosphatases. Within a given organism, there is variation in the number and characteristics of acid phosphatases that are expressed.
Acid phosphatase expression has been studied most extensively in lower organisms such as yeast, fungi and bacteria. Phongdara et al., Appl. Microbiol. Biotechnol. 50:77-84 (1998), characterized a yeast acid phosphatase sequence and reported that e
Haran Shoshan
Raskin Ilya
Board of Trustees, Rutgers The State University of New Jersey
Bui Phuong T.
Marshall Gerstein & Borun
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