Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-01-19
2002-11-05
Prouty, Rebecca E. (Department: 1633)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S004000, C435S007910, C435S183000, C435S023000
Reexamination Certificate
active
06475763
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to genetically modified cells overexpressing recombinant heparanase, to methods of overexpressing recombinant heparanase in cellular systems and to methods of purifying recombinant heparanase. The invention further relates to nucleic acid constructs for directing the expression of modified heparanase species to which a protease recognition and cleavage sequence has been introduced, to the modified heparanase species expressed therefrom and to their proteolytic products. The invention further relates to in vivo methods of inhibiting heparanase activity.
The extracellular matrix (ECM) acts both as a structural scaffold and as an informational medium. Its dynamic status is determined by cells that secrete both its constituent molecules and enzymes that catalyze the degradation of these molecules. A stasis between ECM degrading enzymes and their inhibitors maintains the integrity of the matrix. While controlled ECM remodeling is fundamental to normal processes, uncontrolled disruption underlies diverse pathological conditions.
Among the integral constituents of basement membrane and ECM are cell adhesion molecules such as laminin and fibronectin, structural components like collagen and ellastin, and proteoglycans including sydecan, serglican, proteoglycan I and II versican (1-2).
Brief Overview on Recombinant Gene Expression
For biochemical characterization of a protein and pharmaceutical applications, it is often necessary to overproduce and purify large quantities of the protein. A major consideration when setting up a production scheme for a recombinant protein is whether the product should be expressed intracellularly or if a secretion system can be used to direct the protein to the growth medium. The inherent properties of the protein and the intended applications dictate the expression system of choice. Another consideration when attempting the production of recombinant eukaryotic proteins are the folding and post translational modification processes associated with their natural expression.
Preferably, production is carried out in a cellular system that supports appropriate transcription, translation, and post-translation modification of the protein of interest. Thus, cultured mammalian cells are widely used in applied biotechnology as well as in different disciplines of basic sciences of cellular and molecular biology for producing recombinant proteins of mammalian origin.
One of the most widely used cells for recombinant protein expression, particularly for biotechnological applications, is the Chinese hamster ovary cell line (CHO). Alternatively, baby hamster kidney cells (BHK21), Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-Hep1 cells, MDCK
1
cells, MDBK
1
cells, Vero cells, Cos cells, CV-1 cells, NIH3T3 cells, L929 cells and BLG cells (mouse melanoma) have also been shown to consecutively express large quantities of recombinant proteins.
These cells are easily transfected with foreign DNA, that can integrate into the host genome to create stable cell lines, with new acquired characteristics (i.e. expression of recombinant proteins). These new cell lines originate from a single cell that has undergone foreign DNA incorporation and are therefore referred to as “cellular clones”.
Since integration of foreign DNA in host cell genome is relatively inefficient, the isolation of cellular clones requires a selection system that discriminates between the stably transformed and the primary cells.
Dihydrofolate reductase deficiency in CHO cells (CHO dhfr- cell line) offers a particularly convenient selection system for cellular clones. Transfection of the dhfr gene along with the gene of interest, results in the survival of clones in a growth medium containing methotrexate (MTX). The higher the number of foreign dhfr gene copies in the cellular clone, the higher the MTX concentration the cells can survive. It has been demonstrated that integration events of foreign DNA into host cell genome often maintain all the components of the transfected DNA. e.g., the selection marker as well as the gene of interest (67).
In contrast to mammalian expression systems, that inherently express limited quantities of recombinant proteins, other expression systems, such as bacteria, yeast, and virus infected insect cells are widely used.
Using such cellular gene expression systems, large amounts of either active or non-active protein can be obtained and used for biochemical analysis of protein properties, structure function relationship, kinetic studies, identification of, screening for, or production of specific inhibitors, production of poly- and monoclonal antibodies recognizing the protein, pharmaceutical applications and the like.
Bacteria are the most powerful tool for the production of recombinant proteins. A recombinant protein that is overproduced in a bacterial system might constitute up to 30% of the total protein content of the cells. The recombinant protein accumulates in inclusion bodies where it is relatively pure (comprises up to 50% of the protein content of the bodies) and protected from protease degradation.
Inclusion bodies enable the accumulation of up to 0.2 grams of protein per liter fermentation culture.
Using specific expression vectors, bacteria can also be directed to produce and secrete proteins into the periplasm and therefrom into the growth medium. Although the reported production quantities are not as high as in inclusion bodies, purification of the expressed protein may be simpler (68).
These advantages and the relative simple growth conditions required for bacteria to thrive, made bacteria a powerful and widely used cellular expression system for the production of recombinant proteins of interest (e.g., human &agr;-interferon, human &bgr;-interferon, GM-CSF, G-CSF, human LNF-&ggr;, IL-2, IL-3, IL-6, TNF, human insulin, human growth hormone, etc.).
Furthermore, non-active bacterialy produced recombinant proteins due to inappropriate folding and disulfide bonding may be reduced and/or denatured and thereafter deoxidized and/or refolded to acquire the catalytically active conformation.
However, when glycosylation of the protein is essential for its activity or uses, eukaryotic expression systems are required.
Yeasts are eukaryotic microorganisms which are widely used for commercial production of recombinant proteins. Examples include the production of insulin, human GM-CSF and hepatitis B antigens (for vaccination) by the yeast
Saccharomyces cerevisiae
. The relatively simple growth conditions and the fact that yeasts are eukaryotes make the yeast gene expression system highly suitable for the production of recombinant proteins, primarily those with pharmaceutical relevance.
In recent years methylotrophic yeasts (e.g.,
Pichia pastoris, Hansenula polymorpha
) became widely used, thus replacing in many cases the more traditionally used yeast
Saccharomyces cerevisiae.
Methylotrophic yeasts can grow to a high cellular density, and express and if appropriately, secrete, high levels of recombinant proteins. Quantities of the secreted, correctly-folded recombinant protein can accumulate up to several grams per liter culture. These advantages make
Pichia pastoris
suitable for an efficient production of recombinant proteins (69).
One aspect of the present invention thus concerns the expression of recombinant heparanase in cellular systems.
Heparan Sulfate Proteoglycans (HSPGs)
HSPGs are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (3-7). The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups (3-7). Studies on the involvement of ECM molecules in cell attachme
Ayal-Hershkovitz Maty
Ben-Artzi Hanna
Gilboa Ayelet
Mimon Madlene
Miron Daphna
G. E. Ehrlich Ltd.
Insight Strategy & Marketing Ltd.
Prouty Rebecca E.
Rao Manjunath N.
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