Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-06-10
2001-12-18
Campbell, Bruce R. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S173300, C435S320100, C435S252300, C530S387300, C536S023400, C536S023100
Reexamination Certificate
active
06331416
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to a process of expressing and isolating recombinant proteins and recombinant protein products from plants, plant derived tissue or cultured plant cells, which process exploits (i) the high affinity between cellulose binding peptides and cellulose; (ii) the inherent abundance of cellulose in planta; and/or (iii) the simplicity associated with cellulose isolation from plants, plant derived tissue and cultured plant cells.
More particularly, the present invention relates to a process expressing and isolating recombinant proteins and recombinant protein products from plants, plant derived tissue or cultured plant cells, which process employs the expression of a fusion protein including a recombinant protein and a cellulose binding peptide fused thereto, plant homogenization, isolation of a fusion protein cellulosic matter complex and optional subsequent isolation of the fusion protein and/or the recombinant protein from the complex. The present invention further relates to nucleic acid molecules and to genetically modified or viral infected plants or plant cells which are useful while implementing the process, and further to a novel composition of matter which results from the process.
Citation or identification of any reference in this section or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
With the advent of recombinant technology, techniques for the genetic transformation of various host organisms, such as bacteria, yeasts, fungi, plants and animals, for the purposes of producing specific proteins through the expression of heterologous or foreign genes have been extensively developed.
Using these recombinant techniques and hosts, numerous commercially important recombinant proteins (examples of which are included hereinbelow) have been expressed and purified. Expression and isolation of a protein of interest on a commercial scale, neccesitate the selection of a suitable expression host. This selection largely depends on the economics of production and purification, as well as the ability of the host to accomplish the post-translational modifications needed for full biological activity of the recombinant protein.
Much of the early work in biotechnology was directed toward the expression of recombinant or “heterologous” proteins in prokaryotes like
Escherichia coli
and
Bacillus subtilis
because of the relative ease of genetic manipulation, growth in batch culture and large-scale fermentation of prokaryotes.
Although
E. coli
can in certain cases perform some post translational modifications and events, such as, protein folding and disulfide bond formation, it cannot secrete proteins extracellularly nor can it glycosylate, gamma carboxylate, beta hydroxylate, acetylate or process pre- and pro- peptides.
B. subtilis
suffers from the same limitations as
E. coli
except that it is capable of extracellular secretion.
Furthermore,
E. coli
and other bacteria are pathogens and therefore, depending on the application, contaminants such as pyrogens and endotoxins expressed along with the recombinant protein must be removed In addition, extensive post-purification chemical and enzymatic treatments (e.g., to refold the protein into an active form) are sometimes required in order to obtain a biologically active protein.
Because proteins are not secreted from prokaryotes like
E. coli,
bacterial cells must be disrupted for product recovery. The subsequent release of bacterial contaminants and other proteins make product purification more difficult and expensive. Because purification accounts for up to 90% of the total cost of producing recombinant proteins in bacteria, proteins like Insulin can cost several thousand dollars per gram when recombinantly produced in, and subsequently isolated from,
E. coli.
Because of the many limitations associated with prokaryotic hosts, the biotechnology industry has looked for eukaryotic host cultures such as, yeast, fungi, insect cells, and mammalian cell tissue culture, to properly and efficiently express recombinant proteins.
For most of the proteins requiring extensive post-translational modifications for therapeutic and/or functional activity, mammalian cell culture is the most common alternative to
E. coli.
Although mammalian cells are capable of correctly folding and glycosylating bioactive proteins, the quality and extent of glycosylation can vary with different culture conditions among the same host cells. Furthermore, mammalian culture has extremely high fermentation costs (60-80% of total production expense), requires expensive media, and poses safety concerns from potential contamination by viruses and other pathogens. Yields are generally low and in the range of 0.5-1.5% of cellular protein, or micrograms per liter (up to 300-400 milligrams per liter).
Yeast, other fungi, and insect cells are currently being used as alternatives to mammalian cell culture. Yeast, however, produces incorrectly glycosylated proteins that have excessive mannose residues and are generally limited in eukaryotic processing. Further, although the baculovirus insect cell system can produce high levels of glycosylated proteins, these are typically not secreted, making purification complex and expensive. Fungi represent the best current system for high-volume, low-cost production of recombinant proteins, but they are not capable of expressing many target proteins.
In addition, eukaryotic cultures, require the maintenance of suitable conditions for efficient commercially viable expression of proteins. As such, the ambient temperature, pH value and aeration level of such cultures need to be carefully controlled, while nutrients must be added to the culture medium in carefully regulated doses and waste products removed. In addition, rigorous aseptic practices must be observed in order to avoid contamination by extraneous microbes. Such cultures are thus normally grown in sophisticated fermentors or bioreactors which are housed in expensively maintained factories. Such overheads are reflected in the high price of the recombinant protein end-products.
To a lesser extent, animals have also been utilized for the production of recombinant proteins. Although large amounts of protein can be produced and relatively easily recovered from such animals (e.g., proteins specifically produced in mammary glands and secreted with the milk), production in such host is limited to the expression of proteins which do not interfere with the host physiology. In addition, transgenic animals are subject to lengthy lead times to develop herds with stable genetics, high operating costs, contamination by animal viruses and a relatively slow rate of biomass generation substantially prolonging the time period following which recovery of commercial amounts of the protein can be effected.
The biochemical, technical and economic limitations on existing prokaryotic and eukaryotic expression systems has created substantial interest in developing new expression systems for the production of recombinant proteins.
Plants represent the most likely alternative to existing expression systems. With the availability and on going development of plant transformation techniques, most commercially important plant species can now be genetically modified to express a variety of recombinant proteins.
Such transformation techniques include, for example, the Agrobacterium vector system, which involves infection of the plant tissue with a bacterium (Agrobacterium) into which the foreign gene has been inserted. A number of methods for transforming plant cells with Agrobacteriumn are well known (Klee et al., Annu. Rev. Plant Physiol. (1987) 38:467-486; Schell and Vasil Academic Publishers, San Diego, Calif. (1989) p. 2-25; and Gatenby (1989) in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. p. 93-112).
The biolistic or particle gun method, which permits genetic material to be delivered directly i
Shani Ziv
Shoseyov Oded
Campbell Bruce R.
CBD Technologies Ltd.
Woitach Joseph T.
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