Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the subject of the present invention is the production by plants of recombinant pancreatic lipases and colipases, in particular recombinant human pancreatic lipase (LPH) and / or human pancreatic colipase (COLPH), in a combined or separate manner, and other derivatives of the latter having a lipase activity or a lipase cofactor activity if appropriate, as well as their uses, in particular as functional foods, as pharmaceutical compositions, or in enzymatic formulations for agrifood or industrial applications.
• LPH human pancreatic lipase
• COLPH human pancreatic colipase
• Patent WO 93004216 which relates to a pancreatic mammalian lipase and its variants, is known in the prior art. The description is based on the cloning and sequencing of the pancreatic guinea-pig lipase and the mode of production described is expression in the fungus. filamentous Aspergillus oryzae. However, no indication is given in WO 9300426 to overcome the unforeseeable peculiarities and difficulties specific to transgenesis and to such production in plants, for example in tobacco, corn or rapeseed.
• pancreatic lipase and colipase allows the hydrolysis of fats in the duodenum.
• the natural substrate of pancreatic lipase consists of triglycerides with long chains dispersed in the solution of micellar bile salts.
• lipase is strongly inhibited by bile salts.
• this inhibition is overcome.
• the pancreatic lipase-pancreatic colipase complex is formed, catalyzed by long chain fatty acids that increase the binding by a factor of 100.
• the lipase: colipase stoichiometry is 1: 1, the concentration ratio lipase-colipase is variable depending on the species considered (Erlanson-
• LPH Human pancreatic lipase
• AA amino acid glycoprotein with an apparent molecular mass of approximately 50 kilodaltons (kDa) synthesized as a 465 AA precursor containing a signal peptide from 16 AA to 1 N-terminus
• lipases proteins or C
• the amino acid sequences have a homology percentage of at least 80%, especially from at least 80.6% to about 84.6 (Table 1).
• the nucleotide sequences have between them a percentage of homology of at least 79%, in particular from at least 79.3% to about 87% (Table 1).
• pancreatic lipases are as follows: they are activated by the lipid-water interfaces (interfacial activation), they are inhibited by bile salts but reactivated by colipase (activating protein); they do not significantly hydrolyze phospholipids
• Pancreatic lipase (triacylglycerol acylhydrolase, EC 3 1 1 3)) plays a key role in the absorption of edible fats by the hydrolysis of triglycerides to diglycerides, then monoglycerides and free fatty acids.
• pancreatic lipase The hydrolysis of triglycerides by pancreatic lipase is inhibited by physiological concentrations of bile acids This inhibition is overcome by the addition of colipase which binds to lipase and lipid micelles
• the three-dimensional structure of human pancreatic lipase has been determined by X-ray crystallography (Winkler et al 1990) It made it possible to identify the catalytic residue Ser 152 belonging to the Asp-His-Ser triad which is chemically analogous but structurally different from serine proteases. This catalytic site is covered by a surface loop, and therefore inaccessible to the solvent.
• pancreatic lipase consists of two domains, an N-terminal domain comprising residues 1 to 335, and a C-terminal domain
• the N-terminal domain contains the active site, a glycosylation site (Asn 166), a Ca 2 binding site + , and probably an intestinal pheparin binding site
• the C-terminal domain contains the colipase binding site
• Pancreatic colipase is secreted from the pancreas in the form of a precursor, procolipase (Borgstrom et al 1979) It facilitates the hydrolysis of interfacial lipids catalyzed by pancreatic lipase Pancreatic colipase, of apparent molecular mass of lOkDa, confers catalytic activity to pancreatic lipase under physiological conditions (high concentrations of bile salts) Procolipase
• pancreatic colipases have been isolated from mammals For example, pancreatic rabbit colipase has 82.7% AA sequence homology with human colipase Crystallographic study of the structure of colipase and its interaction with lipase pancreatic have been studied (Egloff et al, 1995): The surface of the colipase can be divided into a relatively hydrophilic part interacting with lipase and a more hydrophobic part at the ends of protein loops The interaction between colipase and the C- domain lipase terminal is stabilized by 8 hydrogen bonds and approximately
• transgenesis of mammalian genes in a plant cell could offer a production route in large quantities of new recombinant proteins, at a reduced production cost and without the risk of viral or subviral contamination.
• 1983 several laboratories discovered that it was possible to transfer a heterologous gene into the genome of a plant cell and to regenerate transgenic plants from these genetically modified cells. All the cells of the plant then have the genetically modified character which is transmitted to the offspring by sexual fertilization. Thanks to this work, various teams have taken an interest in the production of recombinant mammalian proteins in plant cells or in transgenic plants (Barta et a, 1986).
• One of the first truly significant results in this area was the production of antibodies in transgenic tobacco plants.
• hepatitis B surface antigen hepatitis B surface antigen
• interferons a mouse antibody to -Streptococcus mutons
• caries agent fragments of anti- cancer cells
• an anti-Herpes antibody an anti-Herpes antibody
• cholera toxin human epidermis growth factor (EGF).
• pancreatic lipase in particular recombinant LPH or proteins or polypeptides derived from the latter, having sufficient enzymatic activity for be developed in an industrial application.
• the aim of the present invention is to provide a new process for obtaining recombinant pancreatic lipases from mammals, and more particularly from LPH, by plants, or from proteins or polypeptides derived from the latter, having enzymatic activity, and more particularly lipase activity, such as said recombinant lipases, or their derived polypeptides can be used industrially.
• Another object of the present invention is to provide the tools for the implementation of such a method, in particular new recombinant nucleotide sequences, genetically transformed plant cells, plants or parts of plants (in particular leaves, stems, fruits , seeds or seeds, roots) genetically transformed, and fragments of these genetically transformed plants or parts of plants.
• the object of the invention is also to provide novel recombinant pancreatic lipases from mammals, in particular LPH, or any protein or polypeptide derived therefrom, enzymatically active and as obtained from plant or plant cells. , genetically transformed.
• the invention also aims to provide new enzymatic compositions which can be used in the context of the implementation of enzymatic reactions, in particular on an industrial scale.
• the invention also aims to provide new pharmaceutical compositions, in particular in the context of the treatment of pathologies associated with a deficit in lipase production in the body, such as cystic fibrosis as well as the treatment of pathologies associated with an eating disorder, such as obesity.
• Another object of the present invention is to provide new fuels, also known as biofuels, having the advantage of being less polluting than petroleum-based fuels, and of being of lower cost price.
• the subject of the present invention is the use of a recombinant nucleotide sequence containing, on the one hand, a cDNA coding for a pancreatic lipase of mammals or for a derived protein or polypeptide, and on the other hand, the elements allowing a plant cell to produce the pancreatic lipase, or the protein or the derived polypeptide, coded by said cDNA, in particular a promoter and a transcription terminator recognized by the transcriptional machinery of plant cells, for the transformation of plant cells with a view to obtaining, from these cells, or from plants obtained from the latter, a recombinant pancreatic lipase from mammals, or a derived protein or polypeptide.
• the subject of the present invention is the use of a recombinant nucleotide sequence containing, on the one hand, a cDNA coding for any pancreatic lipase from mammals or for a protein or a derived polypeptide, namely proteins or polypeptides whose nucleotide sequences coding for the latter have between them a percentage of homology of at least about 75%, in particular of at least about 77% to about 85%, and whose amino acid sequences have between them a percentage of homology of at least about 75%, in particular of at least about 80% to about 90%, and have a lipase activity of the pancreatic type, in particular a cDNA coding for any pancreatic mammalian lipase, or a cDNA coding for any protein or polypeptide derived from the aforementioned pancreatic lipases by addition and / or deletion and / or substitution of one (or more) amino acid (s), this protein or this polyp derived eptide having
• a more particular subject of the present invention is the use of a recombinant nucleotide sequence containing on the one hand, the cDNA coding for the LPH (Lowe ME et al 1989), or a nucleotide sequence derived from this cDNA, in particular by addition and / or deletion and / or substitution of one (or more) nucleotide (s), said derived sequence being capable of coding for a polypeptide whose amino acid sequence is identical to that of LPH, or for a polypeptide derived from LPH by addition and / or deletion and / or substitution of one (or more) amino acid (s), this derived polypeptide having a pancreatic type lipase activity, and on the other hand, the elements allowing a plant cell to produce the polypeptide encoded by said cDNA or by a derivative sequence mentioned above, in particular a promoter and a transcription terminator recognized by the machinery transcription of plant cells (and more particularly by the RNA polymerases of the latter), for the
• the invention also relates to a recombinant nucleotide sequence, characterized in that it contains on the one hand the coding sequence for a pancreatic lipase or a derived protein or polypeptide and on the other hand, the elements allowing a plant cell to produce a pancreatic pancreatic lipase or a derivative protein or polypeptide encoded by said sequence, in particular a promoter and a transcription terminator recognized by the transcriptional machinery of plant cells,
• a recombinant nucleotide sequence characterized in that it contains on the one hand the coding sequence for a colipase or a derived protein or polypeptide and on the other hand, the elements allowing a plant cell to produce a pancreatic colipase or a protein or derived polypeptide encoded by said sequence, in particular a promoter and a transcription terminator recognized by the transcription machinery of plant cells,
• a recombinant nucleotide sequence characterized in that it contains on the one hand the sequences coding for a pancreatic lipase and a colipase or the proteins or polypeptides derived and on the other hand, the elements allowing a plant cell to produce a pancreatic lipase and a colipase or the proteins or polypeptides derived encoded by said sequence, in particular a promoter and a transcription terminator recognized by the transcriptional machinery of plant cells.
• the invention also relates to any recombinant nucleotide sequence as described above, in particular that containing, as cDNA, that of the LPH or a nucleotide sequence derived therefrom as defined above.
• the recombinant nucleotide sequences according to the invention contain one (or more) sequence (s) coding for a peptide responsible for the addressing of the recombinant polypeptides of the invention (namely recombinant LPH or the above-mentioned derived polypeptides) in a determined compartment of the plant cell, in particular in the endoplasmic reticulum or in the vacuoles, or even outside the cell, in the pectocellulosic wall or in the extracellular space also called apoplasm.
• a peptide responsible for the addressing of the recombinant polypeptides of the invention namely recombinant LPH or the above-mentioned derived polypeptides
• transcription terminators which can be used for the transformation of plant cells in the context of the present invention, mention may be made of the polyA 35S terminator of the cauliflower mosaic (CaMV), or the polyA NOS terminator , which corresponds to the 3 ′ non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium lumefaciens nopaline strain.
• CaMV cauliflower mosaic
• NOS terminator which corresponds to the 3 ′ non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium lumefaciens nopaline strain.
• the invention relates to any recombinant nucleotide sequence as described above containing downstream of said cDNA or its derived sequence, the polyA 35S terminator of CaMV, or the polyA NOS terminator of Agrobacterium tiimefaciens.
• transcription promoters capable of being used for the transformation of plant cells in the context of the present invention, there may be mentioned:
• the chimeric PSP super-promoter (Ni M et al., 1995), consisting of the fusion of a triple repetition of a transcriptional activating element of the promoter of the octopine synthase gene from Agrobacterium tumefaciens, of a transcriptional activating element of the promoter of the mannopine synthase gene and of the mannopine synthase promoter of Agrobacterium tumefaciens,
• the subject of the invention is any recombinant nucleotide sequence as described above, containing upstream of said cDNA or its derived sequence, the constitutive double promoter 35S (Pd35S) of CaMV, or the promoter PCRU of the gene radish cruciferin, or the PGEA1 or PGEA6 promoter from Arabidop is thaliana, or the PSP super-promoter from Agrobacterium tumefaciens, or the PAR-IAR promoter from rice, the HMGW promoter from barley or the p ⁇ zein promoter from corn.
• sequences coding for an addressing peptide used in the context of the present invention can be of plant, human or animal origin.
• nucleotide sequence of 69 nucleotides (indicated in the examples which follow) coding for the prepeptide (signal peptide) of 23 amino acids of sporamine A in sweet potato, this signal peptide allowing the entry of the recombinant polypeptides of the invention in the secretion system of plant cells transformed according to the invention (namely mainly in the endoplasmic reticulum),
• nucleotide sequence of 42 nucleotides (indicated in the examples which follow) coding for the N-terminal propeptide for vacuolar addressing of 14 amino acids of sporamine A in sweet potato, allowing the accumulation of the recombinant polypeptides of the invention in the vacuoles of plant cells transformed according to the invention,
• the invention has for ob
• the subject of the invention is also
• the subject of the invention is also any recombinant nucleotide sequence as described above, containing a sequence
• the recombinant nucleotide sequences of the invention also contain a nucleotide sequence which can be used as a marker for said recombinant sequences, in particular for differentiating (and thus selecting) those of plant cells which are transformed by said recombinant sequences from those which are not. not.
• such a nucleotide sequence which can be used as a marker for said recombinant sequences is chosen from the antibiotic resistance genes, in particular the kanamycin resistance gene.
• the subject of the invention is also any vector, in particular plasmid, containing a recombinant nucleotide sequence according to the invention, inserted at a site which is not essential for its replication.
• the invention also relates to any cell host, in particular any bacterium such as Agrobacterium tumefaciens, transformed by a vector as defined above.
• the invention also relates to a process for obtaining recombinant pancreatic lipase, or a protein or a derived polypeptide, characterized in that it comprises:
• the subject of the present invention is any process for obtaining a recombinant human pancreatic lipase in the form of an active enzyme, and / or a (or more) polypeptide (s) derived therefrom , in particular by addition and / or deletion and / or substitution of one (or more) amino acid (s), this (or these) derived polypeptide (s) having lipase activity, characterized in that 'He understands:
• the transformation of plant cells can be carried out by transfer of the recombinant nucleotide sequence of the invention into the protoplasts, in particular after incubation of the latter in a polyethylene glycol (PEG) solution ) in the presence of divalent cations (Ca- + )
• PEG polyethylene glycol
• the transformation of plant cells can also be carried out by electroporation
• the transformation of plant cells can also be carried out by using a gene cannon allowing the projection, at very high speed, of metallic particles covered with recombinant nucleotide sequences according to the invention, thus delivering genes to the cell nucleus.
• Another method of transformation of plant cells is that of cytoplasmic or nuclear micro-injection
• plant cells are transformed by bringing the latter into contact with a cellular host transformed by a vector according to the invention, as described above, said host cellulan e being capable of infecting said plant cells by permitting integration into the genome of the latter, recombinant nucleotide sequences of the invention initially contained in the genome of the abovementioned vector
• the above-mentioned cell host used is Agrobacterium tumefaciens, in particular according to the methods described in the articles by Bevan,
• rapeseed tobacco, but, peas, tomato, carrot, wheat, barley, potato, soy, sunflower, lettuce, rice and alfalfa
• the plant cells transformed according to the invention are cultured in vitro, in particular in bioreactors, in a liquid medium, or in immobilized form, or even by cultivation of transformed roots / // viii o
• the culture media /// vili mentioned above are then recovered to extract, and if necessary purify, in particular by chromatography, the pancreatic lipase, in particular LPH, the ecombinant and / or the proteins or polypeptide (s ) derivative (s) defined above pi oduits by said transformed cells cultured /// vitro
• the ti ans oi mation of plant cells is followed by a step of obtaining transformed plants by culturing said transf cells formed in an appiated environment Pancre
• the transformed plants used for the recovery of pancreatic lipase, in particular of the recombinant LPH, and / or of the protein (s) or polypeptide (s) derived in the context of the aforementioned process are those of the TO generation, namely those obtained from culture of transformed cells of the invention on an appropriate medium, or advantageously those of the following generations (T1, T2, etc.) obtained, for example by self-fertilization of plants of the previous generation and in which the recombinant nucleotide sequences of the invention reproduce according to Mendel's laws.
• the invention relates more particularly to a process for obtaining, as described above, the recombinant LPH, characterized in that it comprises:
• the plant cells transformed in the methods described above are advantageously chosen from those of tobacco, rapeseed, but, peas, tomato, carrot, wheat, barley, potato, soy, sunflower, lettuce, rice and alfalfa.
• the invention relates more particularly to a process for obtaining recombinant LPH, characterized in that it comprises
• the invention has also poui ob
• the invention also relates to any transgenic cell as described above, containing one (or more) protein otein (s) or recombinant polypeptide (s) according to the invention, such as the LPH said plant cell still being designated plant cell with enzymatic activity, and more particularly with lipase activity as defined below
• the invention also relates to genetically transformed seeds containing one (or more) nucleotide sequence (s) recombinant (s) as (s) as dec ⁇ ⁇ te (s) above, according to the invention, integrated (s) stably in their genome
• the invention also relates to the transgenic seeds described above, and containing one (or more) protein (s) or recombinant polypeptide (s) according to the invention, such as LPH u-tombin me. said seeds also being designated seeds with enzymatic activity, and more particularly with activity as defined below
• the seeds transformed according to the invention are those harvested from plants genetically transformed according to the invention, these transformed plants being either those of the TO generation mentioned above and obtained by culturing cells transformed according to the invention, or those of the generations following (Tl, T2 etc) obtained for example by auto-fertilization or by crossing plants of previous generations (as indicated above)
• the invention also relates to plants and parts of plants (in particular explants, stems, leaves, seeds, acines, seeds, pollen, etc.) genetically transformed, characterized in that they (they) contain one (or more) overlapping nucleotide sequence (s) according to the invention, integrated so stable in their genome
• the invention also relates to the transgenic plants or parts of plants described above, containing one (or more) protein (s) or recombinant polypeptide (s) according to the invention, such as LPH, the said (e) s plants or parts of plants still being designated plants or parts of plants with enzymatic activity, and more particularly with lipase activity as defined below.
• the invention has more particularly for obi and, the aforementioned transformed plants, as obtained by culturing cells or seeds as described above, according to the invention
• the plants, or parts of plants, transferred according to the invention are advantageously chosen from rapeseed, tobacco, corn, peas, tomatoes, carrots, wheat, barley, apple, soia, sunflower, rice, lettuce, alfalfa and beetroot, or parts of these plants
• the present invention has poui ob
• the lipase (or lipolytic) activity of plants or parts of plants, and plant extracts with enzymatic activity of the invention can be measured in particular according to the method of Duan R et al 1991 using a short chain triglyceride (such as t ⁇ butynne) as a substitute or by Egloff's method
• the enzymatic activity is i provided in U units, a U unit corresponding to the quantity of enzyme required to release one ⁇ mole of free fatty acids per minute at 37 ° C. under optimal pH conditions
• the plant extracts with enzymatic activity of the invention are advantageously such that the percentage by weight of enzymatically active recombinant polypeptides, represents approximately 0.1% to 20%, in particular approximately 1% to approximately 15%, relative to the total weight of pi oteines pi esentes in these extracts
• the invention relates more particularly to the following plant extracts with enzymatic activity - extracts of leaves and / or of seeds and / or seeds of plants, as obtained by transformation of the cells of explants of these plants with the sequence Pd35S-PSLPH- LPH, or the sequence Pd35S-PS- LPH, or the sequence Pd35S-PPS-LPH, or the sequence Pd35S-PSLGL-LPH, according to one of the methods described above, and containing the recombinant LPH.
• the tobacco leaf extract as obtained by transformation of tobacco leaf explant cells with the sequence Pd35S-PS- LPH, or the sequence Pd35S-PPS- LPH, according to the method described above,
• the tobacco leaf extract as obtained by transformation of tobacco leaf explant cells with the sequence Pd35S-PSLGL-LPH or the sequence Pd35S-PSLPH-LPH, according to the method described above,
• rapeseed extract as obtained by transformation of explant cells from rapeseed leaves with the PCRU-PS-LPH sequence, or the PCRU-PPS-LPH sequence, or the PGEA I - PSLPH sequence - LPH, or the sequence PGEA6- PSLPH -LPH, according to the method described above,
• the subject of the present invention is a recombinant pancreatic lipase or derivative protein or polypeptide characterized in that it is obtained according to the method according to the invention.
• the present invention relates in particular to any enzymatically active recombinant pancreatic lipase, in particular LPH, or proteins or polypeptides derived therefrom, in particular by addition and / or deletion and / or substitution of one (or more) amino acid (s) , these derived polypeptides having lipase activity, as obtained in essentially pure form by implementing one of the methods described above of the invention, these methods comprising a step of purifying the recombinant polypeptides of the invention, in particular by chromatography carried out from the enzymatic extracts described above.
• pancreatic lipase or derived polypeptides exhibiting lipase activity, according to the invention, is understood to mean any recombinant polypeptide capable of exhibiting lipase activity of the pancreatic type, for example as measured according to the Duan method or the Egloff method.
• the invention relates more particularly to recombinant LPH as obtained by purification of the enzymatic extract of tobacco leaves or seeds, these leaves or seeds coming from transformed tobacco plants, themselves obtained from transformed tobacco cells with the sequence Pd35S-PSLGL-LPH or sequence Pd35S-PSLPH-LPH according to the method described above, said recombinant LPH exhibiting lipase activity as described above.
• the invention relates to antibodies directed against the recombinant polypeptides of the invention, and more particularly those directed against the recombinant LPH according to the invention.
• Such antibodies can be obtained by immunization of an animal with these polypeptides followed by the recovery of the antibodies formed. It goes without saying that this production is not limited to polyclonal antibodies.
• the invention also relates to the use of plants, parts of plants, plant cells, or seeds transformed according to the invention, for obtaining one (or more) proteins or recombinant polypeptide (s) according to the invention.
• invention such as recombinant LPH, or its derived polypeptides as defined above, in particular by implementing one of the above-mentioned methods of the invention, said recombinant polypeptides being in essentially pure form or contained in plant extracts with enzymatic activity as defined above.
• the invention relates in particular to:
• the invention relates more particularly to the use as food of plants, or parts of plants, in particular leaves, fruits, seeds with enzymatic activity according to the invention.
• the subject of the invention is more particularly functional food consisting of a plant with enzymatic activity as described above, or of parts of this plant, in particular of leaves or fruits or of seeds derived from the latter, and likely (s) to have an edible character in l 'Man or animal
• the invention also relates to any functional food containing one (or more) plant (s) with enzymatic activity as described above, and / or parts of this (these ) plant (s), in particular leaves and / or seeds and / or fruits of this (these) plant (s), and / or one (or more) plant extract (s)
• plants with enzymatic activity as described above, and / or one (or more) protein or recombinant polypeptide (s) of the invention, if necessary in combination with a ( or more) other edible compound (s).
• the subject of the invention is more particularly any food composition mentioned above containing recombinant LPH obtained according to the invention in essentially pure or partially pure form or in the form of plants and / or enzymatic extracts as described above possibly associated with one or more other enzymatic activities useful for digestion, preferably an enzymatic activity of the amylase, protease type (in particular trypsin and chymotrypsin and / or elastase)
• the plants or parts of plants, contained in the aforementioned food composition are in the form of ground materials
• the foods according to the invention also referred to as functional foods, or the food compositions according to the invention are more particularly intended to facilitate the absorption of animal or vegetable fats ingested by a healthy individual or suffering from one or more pathologies affecting or not The rate of production of gastric and / or pancreatic lipase
• the foods or food compositions of the invention are advantageously used as nutritional supplements
• the invention also relates to the use of plants, or parts of plants, especially leaves and / or fruits and / or seeds, or plant cells with enzymatic activity according to the invention, or plant extracts with enzymatic activity as defined above, or contain recombinant polypeptides according to invention, such as the recombinant LPH, or its derived polypeptides as defined above, for obtaining medicaments (or compositio pharmaceuticals) intended to facilitate the absorption of animal or plant gi aisses ingested by a healthy individual or suffering from one or more pathologies affecting or not affecting the rate of production
• compositions according to the invention are also more particularly intended for the treatment of pathologies linked to the insufficiency of lipases (in particular gastric and / or pancreatic episodes) in the organism, and more particularly of pathologies such as cystic fibrosis, exocrine pancreatic insufficiency, and P obesity
• the invention relates more particularly to any pharmaceutical composition, characterized in that it comprises plants, or parts of plants according to the invention, and / or plant extracts according to the invention, and / or proteins or of polypeptides or of association thereof according to the invention, where appropriate in combination with one or more pharmaceutically acceptable vehicles or excipients
• the invention relates more particularly to any pharmaceutical composition mentioned above containing recombinant LPH in essentially pure form or in the form of enzymatic extracts as described above.
• the subject of the invention is more particularly any pharmaceutical composition mentioned above containing recombinant LPH in essentially pure or partially pure form or in the form of enzymatic extracts as described above associated with one or more other enzymatic activities useful for digestion, preferably an enzymatic activity of the amylase, protease type (in particular trypsin and chymotrypsin and / or elastase).
• the pharmaceutical compositions according to the invention are preferably administrable by oral route, and are presented in particular in the form of capsules, tablets or powders to be diluted.
• the daily dosage in humans is advantageously from approximately 200 mg to approximately 1000 mg, preferably distributed at the time of the main meals, when said pharmaceutical compositions contain enzymatic extracts as described above, and from approximately 100 mg to about 500 mg, when said pharmaceutical compositions contain recombinant polypeptides according to the invention in essentially pure form.
• a subject of the invention is also the use of plants, or parts of plants, in particular leaves and / or fruits and / or seeds, or plant cells with enzymatic activity according to the invention, or plant extracts with activity enzymatic as defined above, or of recombinant polypeptides according to the invention, such as recombinant LPH, or its derived polypeptides as defined above, for the implementation of enzymatic reactions in the industrial, agro- food or agro-industrial, in particular in the fat industry, lipochemistry and the dairy industry.
• the invention relates to any process, in particular of enzymatic bioconversion, or of biocatalysis, by implementation of one or more enzymatic reactions in the industrial, agro-food or agro-industrial field, in particular in the industry of fatty substances, lipochemistry and the dairy industry, these enzymatic reactions being carried out by means of plants, or parts of plants, in particular leaves and / or fruits and / or seeds, or plant cells with enzymatic activity according to • l invention, or plant extracts with enzymatic activity as defined above, or of recombinant polypeptides according to the invention, such as recombinant LPH, or its derived polypeptides as defined above.
• a more particular subject of the invention is enzymatic preparations for industrial, food-processing or agro-industrial use, capable of being used in the context of the implementation of a process as described above, and comprising plants, or parts of plants according to the invention, and / or extracts of plants according to the invention, and / or proteins or polypeptides or association of these according to the invention if necessary in combination with one (or more) additives) or other enzyme (s) capable of being used in the context of the aforementioned industrial application .
• the invention relates more particularly to the use of plants, or parts of plants, in particular leaves and / or fruits and / or seeds, or plant cells with enzymatic activity according to the invention, for the implementation, with industrial scale, reactions of enzymatic bioconversions, or biocatalysts, such as hydrolyses, or enzymatic trans-esterifications.
• the plants with enzymatic activity, or parts of these plants, in particular the leaves and / or fruits and / or seeds, or of plant cells, according to the invention are used both as an enzymatic source and reaction substrate.
• the subject of the invention is also any biocatalysis process using plants, or parts of plants, in particular leaves and / or fruits and / or seeds, or plant cells with enzymatic activity according to the invention, and more particularly plants containing LPH, said plants, or parts of plants, being used both as an enzymatic source and reaction substrate.
• the invention relates more particularly to the use of plants with enzymatic activity, or of parts of these plants, according to the invention, for obtaining biofuels.
• the subject of the present invention is any process for obtaining biofuel by adding alcohol, in particular methanol or ethanol, to a ground material of all or part of plants transformed according to the invention, advantageously to a ground material rapeseed, sunflower or soybean seeds, transformed according to the invention, and recovery of biofuel, in particular by filtration.
• the invention also relates to the esters of vegetable fatty acids as obtained by implementing the aforementioned process, in particular the methyl ester of oleic acid.
• the subject of the invention is also any biofuel as obtained by implementing a method as described above, and more particularly any aforementioned biofuel comprising esters of vegetable fatty acids.
• the invention relates more particularly to any biofuel as obtained by implementing the aforementioned method from rapeseed, and comprising a methylester of oleic acid.
• the invention also relates to the use of the above-mentioned antibodies directed against the recombinant polypeptides of the invention, for the implementation of a method for detecting or assaying LPH in a biological sample likely to contain it
• the invention relates more particularly to the use of these antibodies for the implementation of a method of /// in vitro diagnosis of pathologies linked to overproduction, or conversely, to insufficiency, or absence of lipase production in the body
• This diagnostic method /// vnio carried out from a biological sample taken from a patient, includes a step of bringing this sample into contact with one or more anticoips according to the invention, followed by a step of detecting possible antibody-LPH complexes formed during the previous step.
• the invention also relates to a kit for carrying out a method of detection or diagnostic /// vitro mentioned above, comprising
• Antibodies as described above, advantageously labeled in a radioactive or enzymatic manner, as well as the active agents for the constitution of a medium suitable for carrying out the immunological leaction between these antibodies and LPH,
• the invention also relates to the association of the lipase activity of pancreatic type obtained according to the invention with an activity of colipase type and the use of the product of this association especially in the context of the treatment of pathologies associated with a deficit of lipid pioduction in the organism, such as cystic fibrosis or in the context of the treatment of food disorders, such as obesity
• the colipase type activity can come from plant extracts expressing a recombinant colipase (for example human colipase) or a derivative of this colipase, a derivative being any protein or polypeptide derived from the abovementioned colipase by addition and / or suppiession and / or substitution of one (or more) amino acid (s), this protein or this polypeptide derived having a colipase type activity More particularly, the colipase type activity can come from a colipase or from a derivative thereof purifies or partially purified from plants or plant extracts expressing a recombinant colipase (for example human pancreatic colipase) or a nve of this colipase
• the activity of colipase type for example human colipase can therefore be produced from a transgenic plant according to the methods described above for the production of pancreatic lipase Colipase can in particular be co-expressed in has the same transgenic plant as pancre
• the invention therefore also relates, if appropriate analogously to what is described for the pancreatic lipase activity according to the invention, the use of a recombinant nucleotide sequence containing, on the one hand, a cDNA coding for a colipase mammals or for a derived protein or polypeptide, and on the other hand, the elements allowing a plant cell to produce the colipase, or the derived protein or polypeptide, encoded by said cDNA, in particular a promoter and a transcription terminator recognized by the transcriptional machinery of plant cells, for the transformation of plant cells with a view to obtaining, from these cells, or from plants obtained from the latter, a recombinant colipase from mammals, or from a derived protein or polypeptide, the use of the sequences for the co-transformation of plant cells with a view to obtaining, from these cells, or from plants o obtained from the latter, from a recombinant mammalian pancreatic lipase and co
• the recombinant nucleotide sequence characterized in that it contains on the one hand the coding sequence for a colipase or a derived protein or polypeptide and on the other hand, the elements allowing a plant cell to produce a pancreatic colipase or a protein or derived polypeptide encoded by said sequence, in particular a promoter and a transcription terminator recognized by the transcription machinery of plant cells,
• the recombinant nucleotide sequence characterized in that it contains on the one hand the sequences coding for a pancreatic lipase and a colipase or the proteins or polypeptides derived therefrom and on the other hand, the elements allowing a plant cell to produce a lipase pancreatic and a colipase or the proteins or polypeptides derived encoded by said sequence, in particular a promoter and a transcription terminator recognized by the transcriptional machinery of plant cells.
• a vector in particular a plasmid, containing a nucleotide sequence as defined above, inserted at a site which is not essential for its replication.
• a cell host in particular any bacterium such as Agrobacterium tumefaciens, transformed by a vector as defined above, a process for obtaining recombinant colipase, or a protein or a derived polypeptide, characterized in that it understands
• a process for the co-production of recombinant pancreatic lipase and colipase, or of derived protein or polypeptides characterized in that it comprises:
• rapeseed tobacco, corn, peas, tomatoes, carrots, wheat, barley, apples of earth, soy, sunflower, lettuce, rice; alfalfa, and beet.
• pancreatic lipase and colipase recombinant or derived protein or polypeptide characterized in that it is obtained according to the method of the invention
• the plant extract with enzymatic activity as obtained by implementing a method according to the invention characterized in that it contains recombinant pancreatic lipase and / or recombinant colipase or the proteins or polypeptides derived , - pharmaceutical compositions, characterized in that they comprise an activity of the pancreatic colipase type (for example human pancreatic colipase), or of its derivatives, possibly associated with an activity of the pancreatic lipase type (for example LPH), the case appropriate in combination with a pharmaceutically acceptable vehicle
• the process in particular of enzymatic bioconvei sion, or of biocatalysis, by implementing one or more enzymatic reactions in the industrial, agro-food or agro-industrial field, in particular in the industry of fatty substances, of lipochemistry and the dairy industry, these enzymatic reactions being carried out by means of plants, or parts of plants, in particular leaves and / or fruits and / or seeds and / or cells of plants, genetically transformed, according to the invention, from an activity of the pancreatic colipase type (for example human pancreatic colipase), or of derived polypeptides possibly associated with an activity of the pancreatic lipase type (for example LPH) - the enzymatic preparations for industrial, agro-food or agro-industrial use including a (or more) plant (s) with enzymatic activity according to the invention, and / or a purified or partially purified pancreatic colipase (pa xample human pancreatic colipase),
• pancreatic colipase type for example colipase pancreatic human
• pancreatic lipase type for example LPH
• the pancreatic colipase type for example human pancreatic colipase possibly associated with an activity of the pancreatic lipase type (for example LPH)
• alcohol in particular methanol or ethanol
• LPH pancreatic lipase
• the double constitutive promoter 35S (Pd35S) of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987);
• polyA 35S terminator which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the 35S transcript.
• pBIOCA This binary plasmid derives from pGA492 (An et al, 1986) which contains between the right and left borders; derived from the plasmid pTiT37 of Agi obacterium tumefaciens, on its transfer DNA, the following sequences: the promoter constituting the NOS gene of nopaline synthase, the coding sequence of the nptll gene coding for the neomycin phosphotransferase II deleted from the region of the first 8 codons including the initiator codon methionine ATG and fused to the sequence of the first 14 codons of the coding sequence of the nos gene, the coding sequence of the nos gene devoid
• the plasmid pGA492 was doubly digested with Sac1 (restriction site of the polylinker) and by Scal (restriction site present in the sequence of the cat gene) and then subjected to the action of the T4 DNA polymerase enzyme (New England Biolabs) according to the manufacturer's recommendations.
• Ligation of the modified plasmid (20 ng) was carried out in a 10 ⁇ l reaction medium containing 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham); 2.5 U of T4 DNA ligase enzyme (Amersham) at I 4 ° C for 16 hours.
• the Escherichia co / i DH5 ⁇ bacteria, made previously competent, were transformed (Hanahan et al., 1983).
• the HindIII restriction site of the plasmid DNA of the retained clone was modified into an EcoRI restriction site using a phosphorylated HindIII-EcoRI adapter (Stratagene Cloning Systems).
• 500 ng of plasmid DNA of the retained clone were digested with Hindlll, dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations and coprecipitated in the presence of 1500 ng of DNA adapter HindIII-EcoRI, 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -S0 ° C for 30 min.
• the resulting binary plasmid which only has the last 9 codons of the coding sequence of the cat gene and whose EcoRI site is unique, was called pBIOC4.
• the expression cassette consisting of the Pd35S promoter and the polyA 35S terminator, was isolated from the plasmid p.HT 163 ⁇ .
• the plasmid pJIT163 ⁇ derives from the plasmid pJIT 163 which itself derives from the plasmid pJIT60 (Guerineau and Mullineaux, 1993). Plasmid pJIT 163 has an ATG codon between the sites Hindlll and Sali du polylinker.
• the plasmid DNA pJIT163 was digested twice with Hindlll and SalI, purified by electrophoresis on 0.8% agarose gel, electroeluted, precipitated in the presence of 1/10 of 3M volume.
• the plasmid DNA of the clone pJIT I 63 ⁇ retained was digested with Sacl and Xhol.
• Sacl-Xhol fragment carrying the expression cassette was purified by electrophoresis on 0.8% agarose gel, electroeluted, precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2 , 5 volumes of absolute ethanol at -80 ° C for 30 min., Centrifuged at 12,000 g for 30 min., Washed with 70% ethanol, dried, then subjected to the action of the enzyme Mung Bean Nuclease ( New England Biolabs) according to the manufacturer's recommendations.
• This purified insert (200 ng) was cloned into the plasmid DNA of pBIOC4 (20 ng) digested with EcoRI, treated with the enzyme Mung Bean Nuclease and dephosphorylated by the enzyme alkaline phosphatase from calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
• the ligation reaction was carried out in 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
• LPH Human pancreatic lipase
• the plasmid containing the complete cDNA coding for LPH was digested in order to suppress the sequence coding for the signal peptide of LPH consisting of 16 amino acids. This sequence has been replaced by that coding for the signal peptide PS of sweet potato sporamine A (Murakami et al., 1986; Matsukoa and Nakamura, 1991) of 23 amino acids (A TG AAA GCC TIC ACA CTC GCT CTC
• PCR were purified by electrophoresis on 2% agarose gel, electroeluted, precipitated in the presence of 1/10 of volume of 3M sodium acetate pH 4.8 and
• the plasmid DNA of the clones obtained was extracted according to the alkaline lysis method and analyzed by enzymatic digestion with restriction enzymes.
• the plasmid DNA of certain retained clones was verified by sequencing using the T7 * M sequencing kit marketed by Pharmacia according to the dideoxynucleotide method.
• Mature LPH is Ser-Lys. From the resulting plasmid, the fragment carrying the sequence of PS-LPH was isolated by enzymatic digestion, purified by electrophoresis on 0.8% agarose gel, electroeluted, precipitated with alcohol, dried. Then, this fragment of treated DNA was ligated to the plasmid DNA of pBIOC21 digested, treated and dephosphorylated by the enzyme alkaline phosphatase from calf intestine.
• the plasmid containing the complete cDNA coding for LPH was digested in order to suppress the sequence coding for the signal peptide of LPH consisting of 16 amino acids.
• This sequence has been replaced by that coding for the signal peptide PPS of sweet potato sporamine A (Murakami et al., 1986; Matsukoa and Nakamura, 1991) of 37 amino acids (A TG AAA GCC TIC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TA T CTC CTG CGC AAT CCA GCC CAT TCC AGG TTC AA T CGC A TC CGC CTC CGC ACC ACA CAC GAA CCC GCC) following the PCR amplification protocol described above in paragraph I. Fragments of DNA from PCR amplification were processed in a similar manner to that described in 1. 1. 1. 1.
• the PPS and mature LPH sequences were cloned while keeping their reading phases open.
• the cleavage sequence between the two sequences is Ala-Lys.
• the resulting plasmid is treated as described in 1. 1. 1.
• the binary vector plasmid DNA was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of
• Rabbit gastric lipase is synthesized in the form of a precursor composed of a signal peptide of 22 amino acids, located at the NH 2 ⁇ terminal end and preceding the polypeptide sequence of mature lipase, the clone pJ1010 containing the complete cDNA coding for rabbit gastric lipase is described in European patent application EP542629
• the plasmid containing the complete cDNA coding for LPH was digested in order to suppress the sequence coding for the signal peptide of LPH consisting of 16 amino acids.
• This sequence has been replaced by that coding for the signal peptide of rabbit gastric lipase consisting of the following 22 amino acids: MWVLFMVAALLSALGTTHGLFG (A TG TCC, GTG GIT TTC A TG GTG GCA GCT TTG CTA TCT GCA CTT GGA ACT ACA CA T GGT CTT TTT GGA) by following the PCR amplification protocol described above in paragraph I.
• the DNA fragments resulting from the PCR amplification were treated in a similar manner to that described in 1. 1 1
• the PSLGL and mature LPH sequences were cloned by keeping their reading phases open (i.e., so that they constituted a single open reading phase).
• the cleavage sequence between the PSLGL and mature LPH sequences is Gly-Lys.
• the resulting plasmid is treated as described in LA. at.
• the binary vector plasmid DNA was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978).
• the plasmid containing the complete cDNA coding for the LPH (465 amino acids) comprises the signal peptide PSLPH of 16 amino acids (A TG CTG CCA CTT TGG ACT CTT TCA CTG CTG CTG GGA GCA G TA GCA GGA).
• the DNA fragment carrying the complete cDNA coding for the LPH was treated in a similar manner to that described in 1.1. 1.
• the cleavage sequence between the two sequences coding for PSLPH and LPH is GLy-Lys.
• the binary vector plasmid DNA was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978).
• the binary plasmid pB ⁇ OC21 described in II. is also used.
• Human pancreatic colipase (COLPH) is naturally synthesized in the form of a precursor of 1 12 amino acids
• the mature COLPH protein consists of 95 amino acids.
• Rabbit gastric lipase is synthesized in the form of a precursor composed of a signal peptide of 22 amino acids, located at the NH2-terminal end and preceding the polypeptide sequence of mature lipase, the clone pJO101 containing the complete cDNA coding for rabbit gastric lipase is described in European patent application EP542629
• the plasmid containing the complete cDNA coding for COLPH was digested in order to suppress the sequence coding for the signal peptide of COLPH consisting of 17 amino acids. This sequence has been replaced by that coding for the signal peptide of rabbit gastric lipase consisting of the following 22 amino acids: MWVLFMVAALLSALGTTHGLFG (A TG TGG GTG CTT TTC
• the PSLGL and mature COLPH sequences were cloned by keeping their reading phases open (i.e., so that they constituted a single open reading phase).
• the cleavage sequence between the PSLGL and mature COLPH sequences is Gly-Ala.
• the resulting plasmid is treated as described in 1.1.1.
• the plasmid DNA of the binary vector was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters and a /. (1978).
• the plasmid containing the complete cDNA coding for the COLPH (1 12 amino acids) comprises the signal peptide PSCOLPH of 17 amino acids (A TG GAG AAG ATC CTG A TC CTC CTG CTT GTG GCC CTC TCT GTG GCC TA TGCA).
• the DNA fragment carrying the full cDNA coding for COLPH was treated in a similar manner to that described in 1. 1.1
• the cleavage sequence between the two sequences coding for PSCOLPH and COLPH is Ala-Lys. ⁇
• the binary vector plasmid DNA was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978).
• LPH human pancreatic lipase
• the PCRU promoter corresponding to the 5 ′ non-coding region of the gene for the seed reserve protein, radish CRUCIFERIN A (Depigny-This et al, 1992) and allowing specific expression in the seeds; 2. the transcription terminator sequence, polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA viais of the cauliflower mosaic producing the 35S transcript.
• the fragment "EcoRI treated with Klenow-BamHI", containing the promoter PCRU was isolated from the plasmid pBI221 -CRURSP derived from pBI221 (marketed by Clontech) by replacing the 35S promoter with the PCRU promoter.
• the expression cassette consisting of the PCRU promoter and the polyA 35S terminator, was isolated from pBIOC27 by total Xhol digestion followed by partial EcoRI digestion. It was purified by agarose gel electrophoresis
• the ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of XhoI-EcoRI DNA fragments described above in a 20 ⁇ l reaction medium in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
• Escherichia coli DH5 ⁇ bacteria previously made competent, have been transformed
• the fragment carrying the sequence PSLPH-LPH was ligated to the plasmid DNA of pBIOC28 digested, treated and dephosphorylated by the enzyme alkaline phosphatase of intestine of calf (Boehringer Mannheim) according to the recommendations of the manufacturer.
• the fragment carrying the sequence PSCOLPH-COLPH was ligated to the plasmid DNA of pBIOC28 digested, treated and dephosphorylated by the enzyme phosphatase alkaline calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
• PSCOLPH-COLPH were performed as previously described.
• the binary vector plasmid DNA was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). V. CONSTRUCTION OF CHEMICAL GENES ENCODING THE
• Ad35S 35S constitutive promoter of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987);
• the transcription terminator sequence polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the 35S transcript;
• the transcription terminator sequence polyA NOS terminator, which corresponds to the 3 ′ non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain.
• the plasmid where the sequence coding for PSLPH-LPH is placed under the control of PAR-IAR was obtained by cloning the fragment carrying the sequence coding for PSLPH-LPH in pBSU-PAR-1AR-TNOS.
• the fragment carrying the sequence coding for PSLPH-LPH was isolated by enzymatic digestion, purified by electrophoresis on 0.8% agarose gel, electroeluted, subjected to alcoholic precipitation, dried and then treated.
• the plasmid pBSII-PAR-IAR-tNOS has been digested, purified, treated and dephosphorylated by the enzyme calf alkaline phosphatase (Boehringer Mannheim) according to the manufacturers' recommendations Ligation, transformation of bacteria Escherichia coli DH5 ⁇ made previously competent, analysis of the plasmid DNA of the clones obtained and the nucleotide sequence of the fragment coding for the recombinant protein PSLPH-LPH were carried out as previously
• the plasmid pBSII-PAR-IAR-TNOS results from cloning at the sites "Eco0109I treated with Klenow and Kpnl" of pBSII-TNOS of the SnaBI-Kpnl fi agment carrying the sequence corresponding to "PAR-IAR-start of the coding sequence for the gus "gene isolated from the plasmid pAct I -F4
• the ligation, the transformation of the Escherichia coli DH5 bacteria made previously competent, the analysis of the plasmid DNA of the clones obtained were carried out as described above
• the plasmid pBSII-TNOS was obtained by cloning at the dephosphorylated EcoRV site of pBSIISK + as specialized by Su atagene, from the SacI fragment.
• the plasmid or the coding sequence for PSLPH-LPH is placed under the control of Pd35S was obtained by cloning at the "Kpnl and BamHI" sites of the plasmid pBSII-T35S of the fragment carrying the sequence corresponding to "Pd35S-PSLPH-LPH" isolated.
• the ligation the transfer of Escherichia coli bacteria
• the plasmid pBSI1-T35S was obtained by cloning at the Spel site treated with Klenow, dephosphorylated from the plasmid pBSIISK + as qualified by Stratagene, from the Smal-EcoRV fragment carrying the sequence T35S isolated from pJIT163 (described above) by double digestion enzymatic by Smal and EcoRV, subjected to purification by electrophoresis on agai gel at 2% Ligation, transformation of Eschei iclua coli DH5 ⁇ bacteria made previously competent, analysis of the plasmid DNA of the clones obtained were carried out as previously described
• the plasmid p63 l results from the cloning of P ⁇ zein at the HindIII and Xbal sites of a plasmid pUC18 i enclosing, between its HindIII sites and EcoRI, the expression cassette "P35S-gus-TNOS" from pBI221 marketed by Clontech. It allows an expression in the albumen of corn seeds
• polyA NOS terminator which corresponds to the 3 ′ non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens strain nopaline
• the plasmid where the sequence coding for PSLPH-LPH is placed under the control of P ⁇ zein was obtained by cloning in the plasmid p63 of the fragment carrying the sequence coding for PSLPH-LPH Ligation, transformation of the bacteria Escherichia coli DH5 ⁇ made pi eally competent , the analysis of the plasmid DNA of the clones obtained were carried out as described above
• the fragment carrying the sequence coding for PSCOLPH-COLPH was isolated by enzymatic digestion, purified by electrophoresis on 2% agarose gel, electroeluted, subjected to alcoholic precipitation, dry, then treated
• the plasmid pBSII-PAR-IAR- TNOS was digested, purified, treated and dephosphorylated by the enzyme alkaline phosphatase from calves (Boehringer Mannheim) according to manufacturers' recommendations Ligation, transformation of bacteria Eschei ichia coli DH5 ⁇ rendered competent beforehand, the analysis of the plasmid DNA of the clones obtained and the nucleotide sequence of the fragment coding for the recombinant protein PSCOLPH-COLPH were carried out as described previously
• the plasmid pBSII-PAR-IAR-TNOS used was disappointed in V 1 1
• the plasmid where the sequence coding for PSCOLPH-COLPH is placed under the control of Pd35S was obtained by clon
• the sequence coding for the tetrapeptide KDEL was introduced upstream of the STOP codon to allow adi essage in the endoplasmic reticulum.
• the binary plasmid of coexpi ession denve of pBIOC2 II contains two expression cassettes each consisting of a piotor Pd35S and a polyA 35S terminator but which differs in the polylinker separating the promoter from the terminator.
• One of the expression cassettes is that of pBIOC21 already described in I.
• the other expression cassette was obtained by replacing the HindIII-BamHI-Smal-EcoRI polylinker of pJIT 163D with a HindIII-EcoRI adapter carrying the sites restriction Pacl, Ascl, Mlul and Hpal.
• This adapter was obtained by renaturation of the 2 oligodeoxynucleotides 5 'AGC TGA TTA ATT AAG GCG CGC CAC GCG TTA AC 3' and 5 'AAT TGT TAA CGC GTG GCG CGC CTT AAT TAA TC 3' which are complementary for their 28 3 'nucleotides terminals.
• One hundred microns of each of these two oligodeoxynucleotides were previously phosphorylated by the action of 10 U of T4 polynucleotide kinase enzyme
• the reaction mixture was incubated at 80 ° C for 10 min; then slowly cooled to room temperature.
• the DNA was then precipitated in the presence of 2.5 volumes of absolute ethanol at -80 ° C for 30 min., Centrifuged at 14000g at 4 ° C for 1 hour, washed with 70% ethanol, centrifuged at 14000g at 4 ° C for 10 min., dried, taken up in 10 ⁇ l of H20.
• the Hindi II-EcoRI DNA fragment was then cloned at the HindIII-EcoRI sites of the plasmid DNA pJIT 163D previously dephosphorylated by the enzyme calf intestine alkaline phosphatase (New England Biolabs) according to the manufacturer's recommendations.
• the ligation reaction was carried out in a reaction volume of 20 ⁇ l in the presence of 1 U of T4 DNA ligase (Gibco-BRL) for a total DNA concentration of 8.5 nM with a vector / insert molar ratio of 1 and 4 ⁇ l of T4 DNA ligase x 5 buffer (Gibco-BRL) at 25 ° C for 16 hours.
• the bacteria made previously competent, have been transformed.
• the fiagment coding for the expression cassette consisting of the Pd35S promoter and the poly 35S terminator was isolated by double digestion with Sacl and Xhol II was purified by electrophoresis on agarose gel.
• the ligation was carried out in a reaction volume of 20 ⁇ l in the presence of 1 U of T4 DNA ligase (Gibco-BRL ) for a total DNA concentration of 8.5 nM with a vector / insert molar i of 1 and 4 ⁇ l of T4 DNA ligase x 5 buffer (Gibco-BRL) at 25 ° C for 16 hours
• the resulting plasmid has was called pBIOC75 From pBIOC75, the DNA fi ament carrying the expression cassette consisting of the Pd35S promoter and the tei mmateui polyA 35S was isolated by digestion with Kpnl II was punished by electiophoiesis on agarose gel 0, 75%, then subjected to the action of the "Geneclean II" kit as approved by BIO101 according to the manufacturer's instructions Then, this DNA fi gment was ligated to the plasmid DNA of pBIOC21 digested with Kpnl and dephosphorylated by the enzyme calf intestine alkaline phosphatase (New England Biolabs) according to the manufacturer's recommendations The ligation was carried out in a reaction volume of 20 ⁇ l in the presence of 1 U of T4 DNA ligase (Gibco-BRL) for one c total DNA oncentiation of 8.5 nM with a vector / insei t molar
• the cDNAs coding for the “PSLPH-LPH” and “PSCOLPH-COLPH” sequences were each cloned under the control of the constitutive promoter Pd35S and of the 35S terminator in the coexpression vector pB10C43
• the ligation, the transformation of the Escherichia coli DH5 ⁇ bacteria made previously competent, the analysis of the plasmid DNA of the clones obtained were carried out as described above.
• the spring rapeseeds (Brassica napiis cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a 15% solution of Domestos (registered trademark). After 4 rinses with sterile water, the seeds are put to germinate, at a rate of 20 seeds per pot of 7 cm in diameter and 10 cm high, on mineral medium of Murashige and Skoog (Sigma M 5519) with 30g / l of sucrose and solidified with 5g / l agargel. These pots are placed in a culture chamber at 26 ° C with a photopiod of 16h / 8h and under a light intensity of the order of 80 ⁇ E m-- s "1
• the cotyledons are removed sterile by cutting each petiole about 1 mm above the cotyledon node.
• a preculture of Agrobacterium tumefaciens strain LBA4404, containing the plasmids, namely, the plasmid pGAZE into which was inserted the sequence coding for human pancreatic lipase fused with that coding for an addressing signal (PS, PPS, PSLGL, PSPHP) under the control of the PCRU promoter (or Pd35S), is carried out in a 50 ml Erlenmeyer flask for 36 h at
• This preculture is used to seed at 1% a new bacterial culture carried out under the same conditions. After 14 h the culture is centrifuged for 15 min at 3000 g and the bacteria are taken up in an equivalent volume of liquid germination medium. This suspension is distributed in petri dishes 5 cm in diameter at a rate of 5 ml / box
• the severed end of the petiole is immersed for a few seconds in the agrobacteria solution thus prepared, then the petiole is pressed a few millimeters into the regeneration medium
• This medium has the same basic composition as the germination medium with an additional 4 ⁇ ng / 1 of benzyl-amino-purine (BAP), phytohormone favoring the new formation of buds
• BAP benzyl-amino-purine
• Ten explants (cotyledon with petiole) are cultured per petri dish 9 cm in diameter (Greiner reference 664102).
• the explants are subcultured in phytatray boxes (Sigma, reference PI 552) containing the preceding medium supplemented with a selective agent: 45 mg / l of kanamycin sulfate (Sigma, reference K4000) and a bacteriostatic: mixture of 1/6 (by weight) potassium salt of clavulanic acid and 5/6 sodium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1.
• a selective agent 45 mg / l of kanamycin sulfate
• a bacteriostatic mixture of 1/6 (by weight) potassium salt of clavulanic acid and 5/6 sodium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1.
• the green buds that appear at the end of the second or third transplanting are separated from the explant and cultured individually in transparent pots 5 cm in diameter and 10 cm high containing a medium identical to the previous one but free of BAP.
• the stem of the transformed bud is cut off and the bud is transplanted into a pot of fresh medium.
• the roots are sufficiently developed to allow acclimatization of the seedling in a phytotron.
• Buds that are not green or rooted are removed. These seedlings are then transplanted into 7 cm side pots filled with potting soil (standard NF U44551: 40% brown peat, 30% sifted heather and 30%> sand) saturated with water.
• standard NF U44551 40% brown peat, 30% sifted heather and 30%> sand
• the seedlings are repotted in 12 cm diameter pots filled with the same potting soil enriched with Osmocote late fertilizer (registered trademark), at a rate of 4 g 1 of potting soil and then transported to the greenhouse (class S2 ) regulated at 18 ° C, with two daily waterings of 2 minutes.
• Osmocote late fertilizer registered trademark
• the pods When the pods have reached maturity, they are harvested, dried and then beaten. The seeds obtained are used for the determination of biochemical activity.
• the selection of the transgenic progeny is done by germination on a medium containing kanamycin sulfate at a rate of 100 to 150 mg / 1 (depending on the genotypes).
• the operating conditions are identical to those described above except that the germinations are carried out in glass tubes with a single seed per tube. Only the seedlings developing secondary roots during the first three weeks are acclimatized in a phytotron before being transferred to the greenhouse.
• the tobacco plants used for the transformation experiments are cultivated / ' // in vitro on the base medium of
• thermoperiod 26 ° C during the day, 24 ° C at night.
• the transformation technique used is derived from that of Horsch et al. (1985).
• a preculture of Agrobacterium tumefaciens strain LBA4404 containing the plasmids is carried out for 48 hours at 28 ° C. with stirring, in LB medium supplemented with suitable antibiotics.
• the preculture is then diluted to 50th in the same medium and cultivated under the same conditions. After one night, the culture is centrifuged (10 min., 3000 g), the bacteria are taken up in an equivalent volume of liquid MS30 medium (30 g / 1 sucrose) and this suspension is diluted with 10.
• Explants of approximately 1 cm2 are cut from the leaves of the seedlings described above. They are then brought into contact with the bacterial suspension for 1 h, then dried quickly on filter paper and placed on a coculture medium (solid MS30). After 2 days, the explants are transferred to petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200 mg / 1), an antibiotic, augmentin (400 mg / 1) and the hormones necessary for the induction of buds (BAP, 1 mg / 1 and ANA, 0, 1 mg / 1). The explants are subcultured on the same medium after 2 weeks of culture. After 2 additional weeks, the buds are subcultured in petri dishes on the development medium composed of the MS20 medium supplemented with kanamycin and augmentin.
• Tomato seeds cv. UC82B are sterilized with Domestos 10% 15 min. and rinsed 3 times in sterile water. The last rinse is carried out for 10 min. under agitation.
• MSSV / 2 medium Merashige and Skoog base medium supplemented with Nitsch vitamins (Thomas and
• the transformation technique used is derived from that of Fillatti et al. (1987).
• a preculture of Agrobacterium tumefaciens strain LBA4404 containing the plasmids is carried out for 24 h at 28 ° C. with stirring in LB medium supplemented with suitable antibiotics.
• the preculture is then diluted to 50th in the same medium and cultivated under the same conditions overnight.
• the OD at 600 nm is measured, the agrobacteria are centrifuged (10 min, 3000 g) and taken up in liquid KCMS medium (described in the publication by Fillatti et al, 1987) so as to obtain an OD at 600 nm of 0, 8.
• Technical improvements have been made to certain stages of the Fillatti et al. (1987).
• the explants preculture and the coculture are carried out as described by Fillatti et al. (1987) except that the KCMS medium is supplemented with acetosyringone (200 mM).
• the 2Z washing medium differs by the addition of cefotaxime 500 mg / 1 instead of carbenicillin.
• the development medium used is composed of the basic medium of Murashige and Skoog (Sigma MS6899) added with the vitamins of Nitsch, sucrose at 20 g / 1, kanamycin at 50 mg / 1, augmentin at 200 mg / 1, ANA at 1 mg / 1 and zeatin at 0.5 mg / 1.
• Seedlings are then regenerated from these calluses by modifying the hormonal and osmotic balance of the cells according to the method described by Vain et al. (1989). These plants are then acclimatized in the greenhouse where they can be crossed or self-fertilized.
• the boxes of calluses thus bombarded are then sealed using Scellofrais® and then cultivated in the dark at 27 ° C.
• the first transplanting takes place 24 hours later, then every fortnight for 3 months on medium identical to the medium of initiation with the addition of a selective agent, the nature and concentration of which may vary depending on the gene used (see paragraph 3)
• the selective agents which can be used generally consist of active compounds of certain herbicides (Basta®, Round up®) or certain antibiotics (Hygromyci e , Kanamycin ).
• calluses whose growth is not inhibited by the selection agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene
• the frequency of obtaining such calluses is around 0.8 cal per bombed box
• calluses are identified, individualized, amplified and then cultivated so as to regenerate seedlings. In order to avoid any interference with untransformed cells, all these operations are carried out on culture media containing the selective agent. The plants thus regenerated are acclimatized then cultivated in a greenhouse where they can be crossed or self-fertilized
• XI ANALYSIS OF THE EXPRESSION OF HUMAN PANCREATIC LI PASS (LPH) IN TRANSGENIC TOBACCO PLANTS.
• the lipase extraction protocol from tobacco leaves taken from plants in the greenhouse is as follows: 1 g of leaves (fresh weight) is ground in liquid nitrogen and then at 4 ° C. in 5 ml of Tris- buffer 50mM HCl neutral pH, added with I mM EDTA and ⁇ -mercaptoethanol l OmM, 0.2% Triton X- 100) and 50 mM NaCl. The total ground material is immediately centrifuged at 4 ° C for 15 min at 10,000 g.
• the lipase activity is determined using a pH-STAT by the titrimetric method of Gargouri et al. (1995) in which the substrate used is tributyrin.
• the tributyrin emulsion (1 ml for 30 ml of emulsion) is produced by vortexing in a 2.5 mM Tris-HCl buffer pH 8.0, 25 mM NaCl, and 5 mM CaCl 2.
• the assay consists in neutralizing the butyric acid released under the action of the lipase with a sodium hydroxide solution at a set pH of 8.0 and at 37 ° C.
• One lipase unit corresponds to the quantity of enzyme which causes the release of a micromole of fatty acids in 1 min. at 37 ° C and under optimal pH conditions (8.0).
• the protocol for extracting lipase from leaves and tomato fruits is similar to that described for tobacco leaves, except that 1 g of fresh material is taken up in 4 ml of buffer. Lipase activity is determined as described for tobacco leaves. XII.2. DOSAGE IN TOMATO FRUITS.
• Lipase activity is determined as described for tobacco leaves.
• XIII. ANALYSIS OF HUMAN PANCREATIC COLIPASE EXPRESSION (COLPH) IN TRANSGENIC TOBACCO PLANTS.
• the lipase extraction protocol from tobacco leaves taken from plants in the greenhouse is as follows: 1 g of leaves (fresh weight) is ground in liquid nitrogen and then at 4 ° C. in 5 ml of Tris buffer -HCI 50mM pH7.5, supplemented with EDTA ImM and ⁇ -mercaptoethanol l OmM, Triton X-100 0.2% and NaCl 50mM. The total ground material is immediately centrifuged at 4 ° C for 15 min at 10,000 g.
• the extraction is carried out with 0.1 g of seeds per 4 ml of buffer.
• the lipase activity is determined using a pH-STAT by the method of
• the effect of colipase on the delay in hydrolysis of intralipids is determined as follows: the intralipids (0.5 ml) are diluted in 10 ml of a solution composed of 2mM Tris-HCl pH8.0, 150mM NaCl , 4mM taurodeoxycholate, CaCh I mM, at 40 ° C. The colipase is added, then after
• pancreatic lipase 10 ⁇ 5 M
• the activity of colipase can also be measured according to the method of
• the lipase is denatured at pH 2, which however makes it possible to keep the colipase intact.
• the colipase activity is then evaluated by the ability of the preparation to restore the activity of a colipase-free lipase preparation.
• the test sample is added to the reaction medium, acidified to pH 2 with PHCI IN, and after one minute, brought to pH 9 with 1M NaOH. An aliquot of semi-purified lipase of known potential activity is added. Colipase is measured based on the level of lipase activity restored from the inactive lipase.
• XIV. "WESTERN" IMMUNODETECTION OF RECOMBINANT LPH. XIV.1. LPH EXPRESSED IN SHEETS AND SEEDS
• XIV.1.1 EXPRESSION WITH PEPTIDES PLANT SIGNALS; IMMUNODETECTION IN TRANSFORMED TOBACCO AND CANNED LEAVES AND SEEDS.
• Immunodetection experiments of the “western” type (“western-blots”) of the LPH were carried out on the proteins of tobacco leaves and of tobacco and rapeseed seeds extracted with the buffer (see extraction protocol below). above).
• the proteins extracted (30 ⁇ g total proteins per sample) are firstly separated by size on 12.5% denaturing polyacrylamide gel and then transferred to a nitrocellulose membrane.
• control protein is natural human pancreatic lipase which migrates in the form of a single band with an apparent molecular mass of approximately 50 kDa No band is detected in the protein extracts of tobacco leaves and seeds non-rapeseed rapeseed
• the control protein is human pancreatic lipase which migrates in the form of a single band of appai ent molecular mass of approximately 50 kDa No band is detected in the pi oteic extracts of unprocessed tobacco leaves (T) XIV 1 3 LPH IN AN EXTRACT CONTAINING PROTEINS
• the protocol for extracting pi otemes for deglycosylation experiments is as follows: 0.5 g of leaves (dry weight) are ground in liquid nitrogen and then at 4 ° C. in 1 ml of denatui ation buffer (phosphate buffer 100 mM pH7.5, adding 1% ⁇ -mercaptoethanol, 25 mM EDTA and 1% SDS) The ground material is centrifuged at 4 ° C for 1 5 mm at 10000 g The supernatant is incubated for 5 min at 100 ° C in order to make the protein denatui ation then centrifuged for 2 min at 10,000 g The supernatant is then diluted with l Oeme in the deglycosylation buffer (phosphate buffer l OOmM pH7.5, addition of ⁇ -mercaptoethanol 1%, 25 mM EDTA, SDS 0.1%) and 1% octylglucoside)
• the enzyme (N-glycosidase F, PNGase Boehr
• the recombinant human pancreatic lipase is purified by ion exchange chromatography on a DEAE-Fast Flow column (Pharmacia) balanced in 50 mM Tris-acetate buffer pH 7.0. The column is washed with the same buffer until the absorption at 280 nm is less than OD 0.05.
• the fixed enzymatic activity is eluted using a linear salt gradient in the same buffer (0 to 0.5M NaCl) using 5 column volumes.
• the fractions containing the enzymatic activity are combined. They are subjected to filtration on Sephadex G-100.
• the protein is purified according to conventional methods for purifying proteins well known to those skilled in the art.
• the esterification reactions are carried out at 37 ° C. for 16 hours in hermetically sealed glass bottles placed on a stirring table (250 rpm).
• the organic solvent used is hexane in which the fatty acids are soluble.
• Methanol is added in stoichiometric proportion to the theoretical amount of triacylglycerol contained in rapeseed.
• the major component of rapeseed fatty acids is oleic acid, so the reference control chosen is a methyl ester of oleic acid.
• the synthesis monitoring is done by thin layer chromatography (TLC).
• the migration solvent is a mixture of hexane, diethyl ether, water (70: 10: 1).
• the revelation of the plates is carried out hot after spraying with sulfuric acid (5%) in ethanol.
• pancreatic lipase makes it possible to obtain the expected product which is the methyl ester of oleic acid.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Pharmacology & Pharmacy (AREA)
• Cell Biology (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Enzymes And Modification Thereof (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Fodder In General (AREA)
• Coloring Foods And Improving Nutritive Qualities (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=9496755

Family Applications (1)

Country Status (6)

Families Citing this family (4)

Family Cites Families (12)

• 1996
  • 1996-10-17 FR FR9612665A patent/FR2754827B1/fr not_active Expired - Fee Related
• 1997
  • 1997-10-17 EP EP97911287A patent/EP0941343A1/fr not_active Withdrawn
  • 1997-10-17 AU AU48719/97A patent/AU4871997A/en not_active Abandoned
  • 1997-10-17 CA CA002269025A patent/CA2269025A1/fr not_active Abandoned
  • 1997-10-17 WO PCT/FR1997/001862 patent/WO1998017807A1/fr not_active Application Discontinuation
  • 1997-10-17 JP JP51903198A patent/JP2001507928A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events