Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
1999-07-02
2003-06-03
Fredman, Jeffrey (Department: 1655)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C800S278000, C800S288000, C800S298000, C800S305000, C800S306000, C800S312000, C800S317200, C800S317000, C800S317300, C800S320000, C800S320100, C800S320200, C800S320300, C800S317400, C800S322000, C435S320100, C435S410000, C435S411000, C435S412000, C435S414000, C435S415000, C435S416000, C435S417000, C536S023100, C536S023500
Reexamination Certificate
active
06573431
ABSTRACT:
The present invention relates to the production, by plants, of recombinant preduodenal lipases, in particular recombinant gastric lipases, and to other polypeptide derivatives of these which have a lipase activity, and to their uses, in particular as functional foods or in pharmaceutical compositions or in enzymatic formulations for agro-alimentary or industrial applications.
Dog gastric lipase (DGL) is a glycoprotein of 379 amino acids (AA) having a molecular weight of about 50 kilodaltons (kDa), which is synthesized in the form of a precursor containing a signal peptide at the amino-terminal (NH
2
-terminal) end and is secreted by median cells of the mucosa of the fundus of the stomach of the dog (Carrière F. et al., 1991).
Human gastric lipase (HGL) is naturally synthesized in the form of a precursor and is described in the publication by Bodmer et al., 1987. The mature HGL protein is constituted by 379 amino acids. Its signal peptide (HGLSP) is composed of 19 amino acids.
These enzymes belong to a family of lipases called “preduodenal”, some members of which have already been purified and in some cases even cloned (Docherty A.J.P. et al., 1985; Bodmer M. W. et al., 1987; Moreau H. et al., 1988; European Patents no. 0 191 061 and no. 0 261 016).
For a long time it has been taken for granted that hydrolysis of food lipids took place in the small intestine by the action of enzymes produced by the pancreas (Bernard C., 1849).
However, findings have suggested that the hydrolysis of triglycerides could have taken place in the stomach by the indirect means of preduodenal enzymes (Volhard, F., 1901; Shonheyder, F., and Volquartz, K., 1945). These enzymes, and in particular dog gastric lipase, have enzymatic and physico-chemical properties which differentiate them from mammalian pancreatic lipases. These differences between gastric and pancreatic lipases essentially relate to the following points: molecular weight, amino acid composition, resistance to pepsin, substrate specificity, optimum pH of action and stability in an acid medium.
Moreover, in vitro, under certain conditions, it is possible to demonstrate a synergistic action between gastric and pancreatic lipases on the hydrolysis of long-chain triglycerides (Gargouri, Y. et al., 1989).
Several pathological situations (cystic fibrosis, exocrine pancreatic insufficiency) where patients are totally or partly lacking in exocrine pancreatic secretion and therefore in enzymes necessary for hydrolysis of foods (amylases, lipases, proteases) are known. Non-absorption of fats in the intestine, and in particular of long-chain triglycerides, manifests itself by a very significant increase in steatorrhoea in these patients and by a very considerable slowing down in weight increase in young patients. To correct this, porcine pancreatic extracts are administered to these subjects at mealtimes. The therapeutic efficacy of these a extracts could be distinctly improved by co-prescription of DGL due to the specificity of its action on long-chain triglycerides.
The article by Carrière et al. (1991) describes the purification and determination of the NH
2
-terminal sequence of DGL. A process for extraction of this enzyme from dog stomachs is also described in this publication. This process essentially comprises steeping the stomachs of dogs in an acid medium (pH 2.5) in the presence of water-soluble salts which promote the salting out of lipase in the said medium. The DGL can be purified to homogeneity by stages of filtration over a molecular sieve and ion exchange chromatography. The purified DGL obtained by these processes is a glycoprotein having a molecular weight of 49,000 daltons, 6,000 of which correspond to sugars and 43,000 to the protein part.
Obvious reasons of the difficulties of procurement of the stomachs of dogs prevent any development of this process both in the laboratory and industrially. This results in the need to discover a process which allows production of DGL in a large amount, dispensing with the use of the stomachs of dogs.
The nucleotide and peptide sequences of DGL were determined with the aim of industrial production of DGL by a process using genetic engineering. These works have been the subject of the international application no. WO 94/13816, filed on Dec. 16, 1993.
The process for the production of recombinant DGL described in this international application claims
Escherichia coli
(
E. coli
) as the transformed host cell which can produce DGL.
Some difficulties encountered during production of recombinant DGL by
E. coli,
in particular the need to culture large quantities of
E. coli
in a fermenter, with high costs, have led to inventors seeking other processes for the production of this DGL.
Mammalian cells are, a priori, more suitable for expression of mammalian genes. However, their use poses problems of maturation of proteins. The enzymatic equipment which realises post-translational maturation differs from one tissue, one organ or one species to another. For example, it has been reported that post-translational maturation of a plasma protein may be different if it is obtained from human blood or if it is produced by a recombinant cell, such as ovarian cells of the Chinese hamster or in the milk of a transgenic animal. Furthermore, the low expression levels obtained with mammalian cells involve cultures in vitro in very large volumes at high costs. The production of recombinant proteins in the milk of transgenic animals (mice, sheep and cows) allows production costs to be reduced and the problems of the level of expression to be overcome. However, ethical problems and problems of viral and subviral contamination (prions) remain.
For these reasons, transgenesis of mammalian genes into a plant cell could provide a route for production of new recombinant proteins in large quantities, at a reduced production cost and without risk of viral or subviral contamination.
In 1983, several laboratories discovered that it was possible to transfer a heterologous gene into the genome of a plant cell (Bevan et al., 1983; Herrera-Estrella et al., 1983 a and b) and to regenerate transgenic plants from these genetically modified cells. All the cells of the plant thus have the genetically modified characteristic, which is transmitted to the descendants by sexed fertilization.
As a result of these works, various teams concerned themselves with the production of mammalian recombinant proteins in plant cells or in transgenic plants (Barta et al., 1986; Marx, 1982). One of the first truly significant results is in this field was the production of antibodies in transgenic tobacco plants (Hiatt et al., 1989).
To express a heterologous protein in the seed, the protein storage site in plants, Vandekerckhove's team (1989) fused the sequence which codes for leu-enkephalin to the gene which codes for the 2S albumin of
Arabidopsis thaliana.
With this construction, transgenic rape plants which express the leu-enkephalin specifically in the seeds at expression levels of the order of 0.1% of the total proteins were produced. In 1990, Sijmons and colleagues transferred the gene of human serum albumin into cells of tobacco and potato. Whatever the origin of the signal peptides (human or plant), human serum albumin levels of the order of 0.02% of the total proteins were obtained in potato leaves, stems and tubers.
Other mammalian recombinant proteins have also been produced in plants: the surface antigen of hepatitis B (Mason et al., 1992), interferons (De Zoeten et al., 1989; Edelbaum et al., 1992; Truve et al., 1993); a murine anti-Streptococcus mutans antibody, the agent of dental caries (Hiatt and Ma, 1992; Ma et al., 1994), fragments of the scFV anti-cancer cell antibody (Russel D., 1994), an anti-herpes antibody (Russel D., 1994), hirudin (Moloney et al., 1994), the cholera toxin (Hein R., 1994) and human epidermal growth factor (EGF) (Higo et al., 1993).
All of these researches have demonstrated that the production of mammalian recombinant proteins in plant cells is possible and that the mechanisms of synthesis of proteins fro
Baudino Sylvie
Benicourt Claude
Cudrey Claire
Gruber Veronique
Lenee Philippe
Biochem S.A.
Einsmann Juliet C.
Fredman Jeffrey
Palmer & Dodge LLP
Williams Kathleen M.
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