U.S. patent application number 10/245405 was filed with the patent office on 2004-04-15 for recombinant preduodenal lipases and polypeptides derivatives produced by plants, processes for obtaining them and their uses.
This patent application is currently assigned to Meristem Therapeutics. Invention is credited to Baudino, Sylvie, Benicourt, Claude, Cudrey, Claire, Gruber, Veronique, Lenee, Philippe, Merot, Bertrand.
Application Number | 20040072317 10/245405 |
Document ID | / |
Family ID | 9478306 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072317 |
Kind Code |
A1 |
Lenee, Philippe ; et
al. |
April 15, 2004 |
Recombinant preduodenal lipases and polypeptides derivatives
produced by plants, processes for obtaining them and their uses
Abstract
The invention concerns the use of recombinant nucleotides
sequences containing cDNA coding for a preduodenal lipase, or any
sequence derived from this cDNA, for transforming plant cells in
order to obtain recombinant preduodenal lipase or polypeptide
derivatives. The invention also concerns the use of genetically
modified plants or parts thereof, or extracts of these plants or
the use of recombinant preduodenal lipase or resultant polypeptide
derivatives in the field of foodstuffs, or for producing
medicaments, or in industry.
Inventors: |
Lenee, Philippe; (Noumea,
FR) ; Gruber, Veronique; (Chamilieres, FR) ;
Baudino, Sylvie; (Lotissement Les Volcans, FR) ;
Merot, Bertrand; (Volvic, FR) ; Benicourt,
Claude; (Houilles, FR) ; Cudrey, Claire;
(Gieres, FR) |
Correspondence
Address: |
PALMER & DODGE, LLP
KATHLEEN M. WILLIAMS
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Assignee: |
Meristem Therapeutics
|
Family ID: |
9478306 |
Appl. No.: |
10/245405 |
Filed: |
September 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10245405 |
Sep 17, 2002 |
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09348930 |
Jul 2, 1999 |
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6573431 |
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09348930 |
Jul 2, 1999 |
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08945930 |
Aug 26, 1997 |
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5859177 |
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Current U.S.
Class: |
435/198 ;
435/320.1; 435/419; 435/69.1 |
Current CPC
Class: |
C12N 9/20 20130101; A61P
1/00 20180101; A61K 38/00 20130101; C12P 7/62 20130101; A61P 1/18
20180101; C12N 15/8247 20130101; C12P 7/6454 20130101; C10L 1/02
20130101; C12P 7/6436 20130101; C12P 7/6418 20130101 |
Class at
Publication: |
435/198 ;
435/069.1; 435/320.1; 435/419 |
International
Class: |
C12N 009/20; C12N
005/04; C12P 021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 1995 |
FR |
FR9504754 |
Claims
1. A recombinant dog gastric lipase comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 4 and
6.
2. A recombinant dog gastric lipase comprising an amino acid
sequence selected from the group of sequences consisting of: SEQ ID
NOs: 4 and 6, wherein said amino acid sequence has its N-terminal
fused to a signal peptide sequence or a prepropeptide sequence.
3. The recombinant dog gastric lipase of claim 2, wherein said
signal peptide sequence or said prepropeptide sequence is derived
from a plant.
4. The recombinant dog gastric lipase of claim 3, wherein said
plant signal peptide sequence is encoded by a nucleotide sequence
of SEQ ID NO: 14.
5. The recombinant dog gastric lipase of claim 3, wherein said
plant prepropeptide sequence is encoded by a nucleotide sequence of
SEQ ID NO: 17.
Description
[0001] 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.
[0002] 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.sub.2-terminal) end and is
secreted by median cells of the mucosa of the fundus of the stomach
of the dog (Carriere F. et al., 1991).
[0003] 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.
[0004] 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).
[0005] 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).
[0006] 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-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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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 in this field was
the production of antibodies in transgenic tobacco plants (Hiatt et
al., 1989).
[0014] 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).
[0015] 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 from DNA
sequences are similar in animal cells and plant cells. However,
some differences exist between plant and animal cells, in
particular in the maturation of polymannoside glycans into complex
glycans, or in the cleavage sites of signal peptides, and it thus
cannot be ensured that active or sufficiently active mammalian
proteins are obtained by transformation of plant cells.
[0016] The inventors have demonstrated that the use of plant cells
transformed by an appropriate recombinant nucleotide sequence
allows recombinant DGL, or recombinant HGL, or polypeptides derived
from these having a sufficient enzymatic activity to be capable of
being developed in an industrial application to be obtained.
[0017] The aim of the present invention is to provide a new process
for the production, by plants, of mammalian recombinant preduodenal
lipases, and more particularly of recombinant DGL or HGL, or
polypeptides derived from these having an enzymatic activity, and
more particularly a lipase activity, such that the said recombinant
lipases or their derived polypeptides can be used industrially.
[0018] Another aim of the present invention is to provide tools for
carrying out such a process, in particular new recombinant
nucleotide sequences, genetically transformed plant cells,
genetically transformed plants or parts of plant (notably leaves,
stems, fruits, seeds or grains, roots) and genetically transformed
fragments of these plants or parts of plants.
[0019] The aim of the invention is also to provide new mammalian
recombinant preduodenal lipase(s), or any derived polypeptide,
which are enzymatically active and are obtained from genetically
transformed plant cells or plants.
[0020] The aim of the invention is also to provide new enzymatic
compositions which can be used in the context of carrying out
enzymatic reactions, in particular on an industrial scale.
[0021] The aim of the invention is also to provide new
pharmaceutical compositions, in particular in the context of
treatment of pathologies associated with a deficit in the
production of lipase in the organism, such as cystic fibrosis.
[0022] Another aim of the present invention is to provide new
fuels, also called biofuels, which have the advantage of being less
polluting than fuels derived from petroleum and of being cheaper to
produce.
[0023] The invention is illustrated below with the aid of the
following figures:
[0024] FIG. 1 shows the nucleotide sequence of the cDNA, of which
the nucleotides situated at positions 1 to 1,137 code for the DGL
shown on FIG. 2, and corresponding to SEQ ID NO 2,
[0025] FIG. 2 shows the amino acid sequence of the DGL, and
corresponding to SEQ ID NO 2,
[0026] FIG. 3 shows a nucleotide sequence derived from the cDNA
shown on FIG. 1, and corresponding to SEQ ID NO 1, the nucleotides
situated in positions 1 to 1,137 of the said derived sequence
coding for the DGL shown on FIG. 2, and corresponding to SEQ ID NO
2,
[0027] FIG. 4 shows the nucleotide sequence of the cDNA of which
the nucleotides situated in positions 47 to 1240 code for the
precursor of the HGL of 398 amino acids, and the nucleotides
situated in positions 104 to 1240 code for the mature HGL of 379
amino acids,
[0028] FIG. 5 shows the amino acid sequence of HGL, the precursor
of HGL being delimited by the amino acids situated at positions 1
and 398, the mature HGL being delimited by the amino acids situated
at positions 20 and 398,
[0029] FIGS. 6, 7 and 8 show the results of immunodetection
experiments of the "western" type on recombinant polypeptides
produced by the leaves and seeds of the tobacco Xanthi, rape seeds
and tomato leaves and fruits, as transformed with the aid of
recombinant sequences according to the invention,
[0030] FIGS. 9 and 10 show respectively the analysis by
electrophoresis over polyacrylamide gel and the result of the
transfer of this gel onto nitrocellulose membrane and revelation
from a dog gastric anti-lipase antibody, of the DGL purified from
tobacco leaves,
[0031] FIG. 11 shows an example of the detection on membrane of
glycanic residues of the recombinant DGL,
[0032] FIG. 12 shows the analysis by electrophoresis on
polyacrylamide gel of the DGL purified from rape seeds,
[0033] FIGS. 13, 14 and 15 illustrate the various tests carried out
in the context of the preparation of methyl oleate from transformed
seeds according to the invention.
[0034] The present invention relates to the use of a recombinant
nucleotide sequence containing, on the one hand, a cDNA which codes
for any mammalian preduodenal lipase, namely the lipases whose
nucleotide sequences which code for these have a percentage
homology of at least about 75%, in particular of about 77% to about
85%, and of which the amino acid sequences have a percentage
homology of at least 70%, in particular of about 80% to about 90%
and having the property of being acid resistant and of being active
at a pH of about 1 to about 5, and more particularly at a pH of
about 1.5 to about 2, in particular a cDNA which codes for any
mammalian gastric lipase, or a cDNA which codes for any polypeptide
derived from the preduodenal lipases mentioned above by the
addition and/or suppression and/or substitution of one (or more
amino acid(s), this derived polypeptide having the properties
described above for preduodenal lipases and, one the other hand,
elements which allow a plant cell to produce the preduodenal lipase
coded by said cDNA, or to produce a derived polypeptide as defined
above, in particular a transcription promoter and a transcription
terminator recognized by the transcription machinery of plant
cells, for the transformation of plant cells with a view to
obtaining, from these cells, or from plants obtained from these, a
mammalian recombinant preduodenal lipase in active enzyme form or
one (or more) polypeptide(s) derived from the latter as defined
above.
[0035] The present invention more particularly relates to the use
of a recombinant nucleotide sequence containing, on the one hand,
the cDNA shown on FIG. 1, which codes for the dog gastric lipase
(DGL) shown on FIG. 2, or a nucleotide sequence derived from this
cDNA, in particular by addition and/or suppression and/or
substitution of one (or more) nucleotide(s), the said derived
sequence being capable of coding for a polypeptide, the amino acid
sequence of which is identical to that of the DGL shown on FIG. 2,
or for a polypeptide derived from the DGL by addition and/or
suppression and/or substitution of one (or more) amino acid(s),
this derived polypeptide having a lipase activity, and, on the
other hand, elements which allow a plant cell to produce the
polypeptide coded by the said cDNA or by an above mentioned derived
sequence, in particular a transcription promoter and a
transcription terminator recognized by the transcription machinery
of plant cells (and more particularly by the RNA polymerases of the
latter), for transformation of plant cells in order to obtain, from
these cells or plants obtained from the latter, recombinant DGL in
active enzyme form or one (or more) polypeptide(s) derived from the
latter as defined above.
[0036] The invention also relates to any recombinant nucleotide
sequence as described above containing, as the cDNA, that shown on
FIG. 1, or a nucleotide sequence derived from the latter as defined
above.
[0037] In this respect, the invention more particularly relates to
any recombinant nucleotide sequence as described above containing
the cDNA shown on FIG. 1, which codes for the dog gastric lipase
(DGL) shown on FIG. 2.
[0038] The invention more particularly also relates to any
recombinant nucleotide sequence as described above containing a
nucleotide sequence derived from the cDNA shown on FIG. 1, the said
derived nucleotide sequence being as defined above and coding for
the dog gastric lipase (DGL) shown in FIG. 2. Such a derived
sequence is advantageously that shown on FIG. 3, and corresponds to
that shown on FIG. 1 in which the nucleotide A in position 12 is
replaced by the nucleotide G, the nucleotide T in position 13 is
replaced by the nucleotide C, and the nucleotide A in position 15
is replaced by the nucleotide T.
[0039] The recombinant nucleotide sequences according to the
invention advantageously contain one (or more) sequence(s) coding
for a peptide which is responsible for directing the recombinant
polypeptides of the invention (that is to say the recombinant DGL
or the above mentioned derived polypeptides) into a specific
compartment of the plant cell, in particular into the endoplasmic
reticulum or into the vacuoles, or even outside the cell, into the
pectocellulosic wall or into the extracellular space, also called
the apoplasm.
[0040] Among the transcription terminators which can be used for
transformation of plant cells in the context of the present
invention there may be mentioned the terminator polyA 35S of the
cauliflower mosaic virus (CaMV) described in the article by Franck
et al., 1980 or the polyA NOS terminator, which corresponds to the
non-coding region 3' of the gene of nopaline synthase of plasmid Ti
of Agrobacterium tumefaciens strain with nopaline (Depicker et al.,
1982).
[0041] In this respect, the invention relates to any recombinant
nucleotide sequence as described above containing, downstream of
the said cDNA or of its derived sequence, the terminator polyA 35S
of CaMV or the terminator polyA NOS of Agrobacterium
tumefaciens.
[0042] Among the transcription promoters which can be used for
transformation of plant cells in the context of the present
invention there may be mentioned:
[0043] the promoter 35S, or advantageously the double-structured
promoter 35S (pd35S) of CaMV, these promoters allowing expression
of recombinant polypeptides according to the invention in the
entire plant obtained from transformed cells according to the
invention, and being described in the article by Kay et al.,
1987,
[0044] the promoter pCRU of the gene of cruciferin of the radish,
which allows expression of the recombinant polypeptides of the
invention solely in the seeds (or grains) of the plant obtained
from transformed cells according to the invention and is described
in the article by Depigny-This et al., 1992.
[0045] the promoters pGEA1 and pGEA6 corresponding to the
non-coding region 5' of the genes of the reserve protein of grains,
GEA1 and GEA6, respectively, of Arabidopsis thaliana (Gaubier et
al. 1993), and allowing a specific expression in grains,
[0046] the chimaeric promoter super-promoter pSP (PCT/US94/12946),
constituted by the fusion of three transcriptional activators of
the promoter of the gene of octopine synthase of Agrobacterium
tumefaciens, of a transcriptional activator element of the promoter
of the gene of mannopine synthase and of the promoter of mannopine
synthase of Agrobacterium tumefaciens,
[0047] the actine promoter of rice followed by the actine intron of
rice (pAR-IAR) contained in the plasmid pAct1-F4 described by
McElroy et al. (1991),
[0048] the promoter of the gene of yzeine of corn (p.gamma.zeine)
contained in the plasmid py63 described in Reina et al. (1990), and
allows expression in the albumen of corn seeds.
[0049] In this respect, the invention relates to any recombinant
nucleotide sequence as described above containing, upstream of the
said cDNA or of its derived sequence, the double-structured
promoter 35S (pd35S) of CaMV or the promoter pCRU of the gene of
cruciferin of the radish or the promoters pGEA1 or pGEA6 of
Arabidopsis thaliana, or the super-promoter pSP of Agrobacterium
tumefaciens, or the promoter pAR-IAR of rice, or the promoter
p.gamma.zeine of corn.
[0050] The sequences which code for a directing peptide used in the
context of the present invention can be of plant, human or animal
origin.
[0051] Among the sequences which code for a directing peptide of
plant origin there may be mentioned:
[0052] the nucleotide sequence of 69 nucleotides (indicated in the
examples which follow) which codes for the prepeptide (signal
peptide) of 23 amino acids of sporamin A in the sweet potato, this
signal peptide allowing entry of recombinant polypeptides of the
invention into the secretion system of transformed plant cells
according to the invention (that is to say chiefly into the
endoplasmic reticulum),
[0053] the nucleotide sequence of 42 nucleotides (indicated in the
examples which follow) which codes for the N-terminal
vacuole-directing propeptide of 14 amino acids of sporamin A in the
sweet potato, allowing the accumulation of recombinant polypeptides
of the invention in the vacuoles of transformed plant cells
according to the invention,
[0054] the nucleotide sequence of 111 nucleotides (indicated in the
examples which follow) which codes for the prepropeptide of 37
amino acids of the sporamin A made up of the N-terminal part to the
C-terminal part of 23 amino acids of the above mentioned signal
peptide followed by the 14 amino acids of the above mentioned
propeptide, this prepropeptide allowing entry of recombinant
polypeptides of the invention into the secretion system, and their
accumulation in the vacuoles, of transformed plant cells according
to the invention,
[0055] the three above mentioned sequences being described in the
articles by Murakami et al., 1986, and Matsuoka and Nakamura,
1991,
[0056] the carboxy-terminal propeptide of the lectin of barley
described, in particular, in the articles by Schroeder et al.,
1993, and Bednarek and Ralkhel, 1991.
[0057] Among the sequences which code for a directing peptide of
human or animal origin, there may be mentioned those which code for
the signal peptide of human gastric lipase (HGL) as described in
European Patent no. 0 191 061, or for that of rabbit gastric lipase
(RGL) as described in European Patent Application no. 0 542 629,
the sequence of which is indicated in the examples which follow, or
for that of human pancreatic lipase (HPL), or also for that of dog
gastric lipase (DGL).
[0058] There can also be mentioned, among the sequences which code
for a directing peptide, that which codes for the tetrapeptide KDEL
and allows directing in the endoplasmic reticulum.
[0059] In this respect, the invention relates to any recombinant
nucleotide sequence as described above containing a sequence which
codes for all or part of a signal peptide, such as that of sporamin
A of the sweet potato or that of HGL or of RGL or of DGL, this
sequence which codes for a signal peptide being situated, in the
said recombinant nucleotide sequence, upstream of the said cDNA or
of its derived sequence and downstream of the promoter used, such
that the last C-terminal amino acid of the signal peptide is bonded
to the first N-terminal amino acid of the polypeptide coded by the
said cDNA or its derived sequence in the protein coded by the said
recombinant nucleotide sequence.
[0060] The invention also relates to any recombinant nucleotide
sequence as described above containing a sequence which codes for
all or part of a vacuole-directing peptide, in particular that of
sporamin A of the sweet potato, this sequence which codes for a
vacuole-directing peptide being situated, in the said recombinant
nucleotide sequence, between the sequence which codes for a signal
peptide and that which codes for the said cDNA or its derived
sequence such that first N-terminal amino acid of the
vacuole-directing peptide is bonded to the last C-terminal amino
acid of the signal peptide, and that the last C-terminal amino acid
of the said directing peptide is bonded to the first N-terminal
amino acid of the polypeptide coded by the said cDNA or its derived
sequence in the protein coded by the said recombinant nucleotide
sequence.
[0061] The invention also relates to any recombinant nucleotide
sequence as described above containing a sequence which codes for
all or part of a vacuole-directing peptide, in particular that of
lectin of barley, this sequence which codes for a vacuole-directing
peptide being situated, in the said recombinant nucleotide
sequence, downstream of the sequence which codes for the said cDNA
or its derived sequence such that the first N-terminal amino acid
of the vacuole-directing peptide is bonded to the last C-terminal
amino acid of the polypeptide coded by the said cDNA or its derived
sequence in the protein coded by the said recombinant nucleotide
sequence.
[0062] The invention more particularly relates to the following
recombinant nucleotide sequences:
[0063] that (called pd35S-PS-DGL) containing, in the direction
5'.fwdarw.3', the promoter pd35S of CaMV, the sequence which codes
for the signal peptide of sporamin A, the latter being immediately
followed by the nucleotide sequence shown on FIG. 3, and then the
terminator polyA 35S of CaMV,
[0064] that (called pd35S-PPS-DGL) containing, in the direction
5'.fwdarw.3', the promoter pd35S of CaMV, the sequence which codes
for the prepropeptide of sporamin A, the latter being immediately
followed by the nucleotide sequence shown on FIG. 3, and then the
terminator polyA 35S of CaMV,
[0065] that (called pd35S-RGLSP-DGL) containing, in the direction
5'.fwdarw.3', the promoter pd35S of CaMV, the sequence which codes
for part of the signal peptide of RGL (that is to say for a
sequence made up of the first 19 amino acids, indicated in the
examples which follow below, the 9 nucleotides which code for the
last 3 C-terminal amino acids being suppressed), the latter being
immediately followed by the cDNA shown on FIG. 1, and then the
terminator polyA 35S of CaMV,
[0066] that (called pCRU-PS-DGL) containing, in the direction
5'.fwdarw.3', the promoter pCRU of cruciferin, the sequence which
codes for the signal peptide of sporamin A, the latter being
immediately followed by the nucleotide sequence shown on FIG. 3,
and then the terminator polyA 35S of CaMV,
[0067] that (called pCRU-PPS-DGL) containing, in the direction
5'.fwdarw.3', the promoter pCRU of cruciferin, the sequence which
codes for the prepropeptide of sporamin A, the latter being
immediately followed by the nucleotide sequence shown on FIG. 3,
and then the terminator polyA 35S of CaMV,
[0068] that (called pCRU-RGLSP-DGL) containing, in the direction
5'.fwdarw.3' the promoter pCRU of cruciferin, the sequence which
codes for a part of the DGL signal peptide (as described above),
the latter being immediately followed by the cDNA as represented in
FIG. 1, or in FIG. 3, and the polyA 35S terminator of CaMV,
[0069] that (called pGEA1-RGLSP-DGL) containing, in the direction
5'.fwdarw.3' the promoter pGEA1 of Arabidopsis thaliana, the
sequence which codes for a part of the signal peptide of RGL (as
described above), the latter being immediately followed by the cDNA
shown on FIG. 1, or on FIG. 3, then the terminator polyA 35S of
CaMV,
[0070] that (called pGEA6-RGLSP-DGL) containing, in the direction
5'.fwdarw.3' the promoter pGEA6 of Arabidopsis thaliana, the
sequence which codes for a part of the signal peptide of RGL (as
described above), the latter being immediately followed by the cDNA
shown on FIG. 1, or on FIG. 3, then the terminator polyA 35S of
CaMV,
[0071] that (called pAR-IAR-RGLSP-DGL) containing, in the direction
5'.fwdarw.3' the promoter pAR-IAR of rice, the sequence which codes
for a part of the signal peptide of RGL (as described above), the
latter being immediately followed by the cDNA shown on FIG. 1, or
on FIG. 3, then the terminator polyA 35S of CaMV, or the terminator
polyA NOS of Agrobacterium tumefaciens,
[0072] that (called p.gamma.zeine-RGLSP-DGL) containing, in the
direction 5'.fwdarw.3' the promoter p.gamma.zeine of corn, the
sequence which codes for a part of the signal peptide of RGL (as
described above), the latter being immediately followed by the cDNA
shown on FIG. 1, or on FIG. 3, then the terminator polyA 35S of
CaMV,
[0073] that (called p.gamma.zeine-RGLSP-DGL-KDEL) containing, in
the direction 5'.fwdarw.3' the promoter p.gamma.zeine of corn, the
sequence which codes for a part of the signal peptide of RGL (as
described above), the latter being immediately followed by the cDNA
shown on FIG. 1, or on FIG. 3, then the sequence which codes for
the tetrapeptide KDEL, then the terminator polyA 35S of CaMV,
[0074] The recombinant nucleotide sequences of the invention
advantageously also contain a nucleotide sequence which can be used
as a marker of the said recombinant sequences, in particular for
differentiation (and thus selection) of those of the plant cells
which are transformed by the said recombinant sequences from those
which are not.
[0075] Such a nucleotide sequence which can be used as a marker of
the said recombinant sequences is preferably chosen from the genes
of resistance to antibiotics, in particular the gene of resistance
to kanamycin.
[0076] The invention also relates to any vector, in particular a
plasmid vector, containing a recombinant nucleotide sequence
according to the invention inserted at a site which is not
essential for its replication.
[0077] The invention also relates to any cell host, in particular
any bacteria, such as Agrobacterium tumefaciens, transformed by a
vector as defined above.
[0078] The present invention also relates to any process for the
preparation of recombinant DGL in active enzyme form and/or of one
(or more) polypeptide(s) derived from the latter, in particular by
addition and/or suppression and/or substitution of one (or more)
amino acid(s), this (or these) derived polypeptide(s) having a
lipase activity, characterized in that it comprises:
[0079] transformation of plant cells such that one (or more)
recombinant nucleotide sequence(s) according to the invention is
(or are) integrated into the genome of these cells,
[0080] where appropriate, production of transformed plants from the
abovementioned transformed cells,
[0081] recovery of the recombinant DGL and/or of the above
mentioned derived polypeptide(s) produced in the said cells or
above mentioned transformed plants, in particular by extraction,
followed, where appropriate, by purification.
[0082] According to one embodiment of the above mentioned process
of the invention, 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 solution of polyethylene glycol (PEG) in the
presence of divalent cations (Ca.sup.2+) in accordance with the
method described in the article by Krens et al., 1982.
[0083] The transformation of plant cells can also be carried out by
electroporation, in particular in accordance with the method
described in the article by Fromm et al., 1986.
[0084] The transformation of the plant cells can also be carried
out by using a gene gun which allows projection, at very high
speed, of metal particles coated with recombinant nucleotide
sequences according to the invention, thus delivering genes inside
the cell nucleus, in particular in accordance with the technique
described in the article by Sanford, 1988.
[0085] Another method of transformation of plant cells is that of
cytoplasmic or nuclear microinjection as described in the article
by De La Penna et al., 1987.
[0086] According to a particularly preferred embodiment of the
abovementioned process of the invention, the plant cells are
transformed by bringing the latter together with a cell host
transformed by a vector according to the invention, as described
above, the said cell host being capable of infecting the said plant
cells and allowing integration, into the genome of the latter, of
recombinant nucleotide sequences of the invention initially
contained in the genome of the abovementioned vector.
[0087] The above mentioned cell host used is advantageously
Agrobacterium tumefaciens, in particular in accordance with the
methods described in the articles by Bevan, 1984 and An et al.,
1986, or also Agrobacterium rhizogenes, in particular in accordance
with the method described in the article by Jouanin et al.,
1987.
[0088] Among the plant cells which can be transformed in the
context of the present invention there may be mentioned those of
rape, tobacco, maize, pea, tomato, carrot, wheat, barley, potato,
soya, sunflower, lettuce, rice and lucerne.
[0089] According to one embodiment of the abovementioned process of
the invention, the transformed plant cells according to the
invention are cultured in vitro, in particular in bioreactors, in
accordance with the method described in the article by Brodelius,
1988, in a liquid medium, or in accordance with the method
described in the article by Brodelius et al., 1979, in immobilized
form, or also in accordance with the method described in the
article by Deno et al., 1987, which is carried out by culture of
transformed roots in vitro.
[0090] The abovementioned in vitro culture media are then recovered
to extract from them, and where appropriate to purify, in
particular by chromatography, the recombinant DGL and/or the
derived polypeptide(s) defined above produced by the said
transformed cells cultured in vitro.
[0091] According to a preferred embodiment of the above-mentioned
process for the preparation of recombinant DGL and/or derived
polypeptide(s) according to the invention, transformation of the
plant cells is followed by a stage of production of transformed
plants by culturing the said transformed cells in a suitable
medium. The recombinant DGL and/or the derived polypeptide(s)
produced in the cells of the whole plants thus obtained are
recovered by extraction, carried out on the whole plants or
fragments of these plants (in particular on the leaves, the stems
or fruits), or on seeds produced by these plants, this extraction
being followed, where appropriate, by a stage of purification of
the recombinant DGL and/or of the derived polypeptide(s).
[0092] The transformed plants used for recovery of the recombinant
DGL and/or of the derived polypeptide(s) in the context of the
abovementioned process are those of generation TO, that is to say
those obtained by culture of transformed cells of the invention on
a suitable medium, or advantageously those of the following
generations (T1, T2 etc.) which are obtained by autofertilization
of plants of the preceding generation and in which the recombinant
nucleotide sequences of the invention are reproduced in accordance
with the laws of Mendel.
[0093] Among the polypeptides derived from the recombinant DGL
which can be obtained in the context of carrying out a process
according to the invention there may be mentioned:
[0094] the polypeptide delimited by the amino acids situated in
positions 55 and 379 of FIG. 2, also called polypeptide
(.DELTA.54), and represented by SEQ ID NO 4, said polypeptide being
encoded by the nucleotide sequence represented by SEQ ID NO 3,
[0095] the polypeptide delimited by the amino acids situated in
positions 5 and 379 in FIG. 2, also called polypeptide (.DELTA.4),
and represented by SEQ ID NO 6, said polypeptide being encoded by
the nucleotide sequence represented by SEQ ID NO 5.
[0096] The invention more particularly relates to any process as
described above for the preparation of the recombinant DGL shown on
FIG. 2 and, where appropriate, the preparation of one (or more)
derived polypeptide(s), in particular the abovementioned
polypeptide (.DELTA.54) and/or polypeptide (.DELTA.4), the said
process being characterized in that the stage of transformation of
plant cells is carried out by integration, into the genome of the
latter, of a recombinant sequence as described above containing, on
the one hand, the cDNA shown on FIG. 1 and, on the other hand, the
sequence which codes for the signal peptide of 22 amino acids of
RGL, advantageously that which codes for the first 19 amino acids
of the signal peptide of RGL.
[0097] The invention more particularly relates to a process for the
preparation, as described above, of the recombinant DGL shown on
FIG. 2, where appropriate in combination with the polypeptide
(.DELTA.54) and/or the polypeptide (.DELTA.4), characterized in
that it comprises:
[0098] transformation of explant cells of the leaves of a plant by
bringing the latter together with a strain of Agrobacterium
tumefaciens transformed by a plasmid as described above containing
the abovementioned recombinant nucleotide sequence pd35S-RGLSP-DGL
on a suitable culture medium,
[0099] selection of the transformed explants on a medium containing
kanamycin,
[0100] production of transformed plants from the abovementioned
transformed explants by culture of the latter on suitable
media,
[0101] extraction of the recombinant DGL and, where appropriate, of
the polypeptide (.DELTA.54) and/or the polypeptide (.DELTA.4), in
particular by grinding the leaves and/or the seeds and/or the
fruits of the abovementioned transformed plants in a suitable
buffer, centrifugation and recovery of the supernatant constituting
the plant extract of enzymatic activity,
[0102] where appropriate, purification of the recombinant DGL from
the extract obtained during the preceding stage, in particular by
chromatography carried out on the supernatant, which leads to the
preparation of the recombinant DGL in an essentially pure form.
[0103] The invention also relates to the use of the abovementioned
process for the preparation of the polypeptide (.DELTA.54) or the
polypeptide (.DELTA.4) in an essentially pure form by purification
of the latter from the extract obtained in the abovementioned
process, in particular by chromatography carried out on the
supernatant of the extraction.
[0104] The invention more particularly relates to the preparation
of the abovementioned polypeptide (.DELTA.4), where appropriate in
an essentially pure form, by implementation of the abovementioned
process in which the cells transformed with the sequence
pd35S-RGLSP-DGL, are explant cells of solanaceae, in particular
tobacco or tomato.
[0105] According to one embodiment of the above-mentioned process,
the polypeptide (.DELTA.4) can be specifically obtained by
extraction from the abovementioned leaves of transformed tobacco
plants, in particular by grinding these leaves in an appropriate
buffer, centrifuging and recovering the supernatant. The
polypeptide (.DELTA.4) can then be purified from the abovementioned
leaf extract, containing said polypeptide (.DELTA.4), in particular
by chromatography carried out on the abovementioned
supernatant.
[0106] The invention more particularly relates to any process as
described above for the preparation of recombinant DGL and/or of
one (or more) polypeptide(s) derived from the latter, as defined
above, characterized in that the stage of transformation of the
plant cells is carried out by integration, into the genome of the
latter, of a sequence containing, on the one hand, the nucleotide
sequence shown on FIG. 3 and, on the other hand, the sequence which
codes for the signal peptide of the sporamin A described above.
[0107] Among the polypeptides derived from the recombinant DGL
which can be obtained in the context of carrying out the process
described above there may be mentioned the abovementioned
polypeptide (.DELTA.54) and/or polypeptide (.DELTA.4).
[0108] The invention more particularly relates to a process for the
preparation of the abovementioned recombinant DGL and/or
polypeptide (.DELTA.54) and/or polypeptide (.DELTA.4),
characterized in that it comprises:
[0109] transformation of explant cells of a plant (in particular
explants of leaves) by bringing the latter together with a strain
of Agrobacterium tumefaciens transformed by a plasmid as described
above containing the recombinant nucleotide sequence pd35S-PS-DGL
and/or the sequence pd35SPPS-DGL,
[0110] selection of the transformed explants on a medium containing
kanamycin,
[0111] production of transformed plants from the abovementioned
transformed explants by culture of the latter on suitable
media,
[0112] extraction of the recombinant DGL and/or the polypeptide
(.DELTA.54) and/or the polypeptide (.DELTA.4), in particular by
grinding the leaves and/or the seeds and/or the fruits of the
abovementioned transformed plants in a suitable buffer,
centrifugation and recovery of the supernatant constituting the
plant extract of enzymatic activity,
[0113] where appropriate, purification of the recombinant DGL
and/or the polypeptide (.DELTA.54) and/or the polypeptide
(.DELTA.4) from the extract obtained during the preceding stage, in
particular by chromatography carried out on the supernatant, which
leads to the preparation of the recombinant DGL and/or the
polypeptide (.DELTA.54) and/or the polypeptide (.DELTA.4) in an
essentially pure form.
[0114] The invention more particularly relates to a process for the
preparation of the abovementioned polypeptide (.DELTA.54) and/or
polypeptide (.DELTA.4) by carrying out the process described above,
in which the transformed cells are explant cells of the leaves of
solanaceae, in particular tobacco or tomato.
[0115] According to a particular embodiment of the abovementioned
process of the invention, the polypeptide (.DELTA.54) can be
obtained specifically by extraction from the abovementioned
transformed tobacco seeds, in particular by grinding these seeds in
a suitable buffer, centrifuging and recovering the supernatant. The
polypeptide (.DELTA.54) can then be purified from the
abovementioned seed extract containing the said polypeptide
(.DELTA.54), in particular by chromatography carried out on the
abovementioned supernatant.
[0116] The invention more particularly relates to a process for the
preparation of recombinant DGL and/or the polypeptide (.DELTA.54)
and/or the polypeptide (.DELTA.4), characterized in that it
comprises:
[0117] transformation of explant cells of a plant by bringing the
latter together with a strain of Agrobacterium tumefaciens
transformed by a plasmid as described above containing the
recombinant nucleotide sequence pCRU-PPS-DGL and/or the sequence
pCRU-PS-DGL and/or the sequence pGEA1-RGLSP-DGL and/or the sequence
pGEA6-RGLSP-DGL and/or the sequence pARIAR-RGLSP-DGL and/or the
sequence p.gamma.zeine-RGLSP-DGL and/or the sequence
p.gamma.zeine-RGLSP-DGL-KDEL,
[0118] selection of the transformed explants on a medium containing
kanamycin,
[0119] production of transformed plants from the abovementioned
transformed explants by culture of the latter on suitable
media,
[0120] extraction of the recombinant DGL and/or the polypeptide
(.DELTA.54) and/or the polypeptide (.DELTA.4), in particular by
grinding seeds produced by the abovementioned transformed plants in
a suitable buffer, centrifuging and recovering the supernatant
containing the plant extract of enzymatic activity,
[0121] where appropriate, purification of the recombinant DGL
and/or the polypeptide (.DELTA.54) and/or the polypeptide
(.DELTA.4) from the extract obtained during the preceding stage, in
particular by chromatography carried out on the supernatant, which
leads to the preparation of the recombinant DGL and/or the
polypeptide (.DELTA.54) and/or the polypeptide (.DELTA.4) in an
essentially pure form.
[0122] The invention more particularly relates to a process for the
preparation of the abovementioned polypeptide (.DELTA.54) by
carrying out the process described above, in which the transformed
cells are explant cells of rape, and tobacco, and the extraction of
the polypeptide (.DELTA.54) is made by grinding the transformed
seeds.
[0123] The transformed plant cells in the processes described above
are advantageously chosen from those of tobacco, rape, maize, pea,
tomato, carrot, wheat, barley, potato, soya, sunflower, lettuce,
rice and lucerne.
[0124] A more particular aim of the invention is a process for the
preparation of recombinant DGL and/or of polypeptide (.DELTA.54)
and/or polypeptide (.DELTA.4), characterized in that it
includes:
[0125] the transformation of corn callus, by bombardment of the
latter using a particle gun, with plasmids containing the
recombinant nucleotide sequence pAR-IAR-RGLSP-DGL and/or the
sequence p.gamma.zeine-RGLSP-DGL and/or the sequence
p.gamma.zeine-RGLSPDGL-KDEL,
[0126] selection of the transformed calluses on a medium containing
a selection agent such as kanamycin,
[0127] production of transformed corn plants from the
abovementioned transformed calluses by culture of the latter on
appropriate media,
[0128] extraction of the recombinant DGL and/or the polypeptide
(.DELTA.54) and/or the polypeptide (.DELTA.4) in particular by
grinding seeds produced by the abovementioned transformed plants in
a suitable buffer, centrifuging and recovering the supernatant
containing the plant extract of enzymatic activity,
[0129] where appropriate, purification of the recombinant DGL
and/or the polypeptide (.DELTA.54) and/or the polypeptide
(.DELTA.4), from the extract obtained during the preceding stage,
in particular by chromatography carried out on the supernatant,
which leads to the obtaining of the recombinant DGL and/or the
polypeptide (.DELTA.54) and/or the polypeptide (.DELTA.4) in an
essentially pure form.
[0130] The invention also relates to any genetically transformed
plant cell containing one (or more) recombinant nucleotide
sequence(s) as described above, according to the invention,
integrated into its genome in a stable manner.
[0131] The invention also relates to any transgenic plant cell as
described above, containing one (or more) recombinant
polypeptide(s) according to the invention, such as the recombinant
DGL and/or the polypeptide (.DELTA.54) and/or the polypeptide
(.DELTA.4), the said plant cell also being called a plant cell of
enzymatic activity, and more particularly of lipase activity as
defined below.
[0132] The invention also relates to genetically transformed seeds
containing one (or more) recombinant nucleotide sequence(s) as
described above, according to the invention, integrated into their
genome in a stable manner.
[0133] The invention also relates to the transgenic seeds described
above which contain one (or more) recombinant polypeptide(s)
according to the invention, such as the recombinant DGL and/or the
polypeptide (.DELTA.54) and/or the polypeptide (.DELTA.4), the said
seeds also being called seeds of enzymatic activity, and more
particularly of lipase activity as defined below.
[0134] The transformed seeds according to the invention are those
harvested from genetically transformed plants according to the
invention, these transformed plants being either those of the
abovementioned generation T0 and produced by culture of transformed
cells according to the invention, or those of the following
generations (T1, T2 etc.) obtained by autofertilization or by
crossing plants of preceding generations (as indicated above).
[0135] The invention also relates to genetically transformed plants
or parts of plants (in particular explants, stems, leaves, roots,
pollen etc.), characterized in that they contain one (or more)
recombinant nucleotide sequence(s) as described above, according to
the invention, integrated into their genome in a stable manner.
[0136] The invention also relates to the transgenic plants or parts
of plants described above containing one (or more) recombinant
polypeptide(s) according to the invention, such as the recombinant
DGL and/or the polypeptide (.DELTA.54) and/or the polypeptide
(.DELTA.4), the said plants or parts of plants also being called
plants or plant fragments of enzymatic activity, and more
particularly of lipase activity as defined below.
[0137] The invention more particularly relates to the
abovementioned transformed plants obtained by culture of cells or
seeds as described above, according to the invention.
[0138] The transformed plants, or parts of plants, according to the
invention are advantageously chosen from rape, tobacco, maize, pea,
tomato, carrot, wheat, barley, potato, soya, sunflower, rice,
lettuce and lucerne, or parts of these plants.
[0139] The present invention relates to any plant extract of
enzymatic activity, and more particularly of lipase activity
defined below, prepared by carrying out one of the processes of the
invention described above, and containing, as active enzymes, one
(or more) recombinant polypeptide(s) according to the invention,
such as the recombinant DGL and/or the polypeptide (.DELTA.54)
and/or the polypeptide (.DELTA.4).
[0140] The lipase (or lipolytic) activity of the plants or parts of
plants and plant extracts of enzymatic activity of the invention,
can be measured, in particular, in accordance with the method of
Gargouri (Gargouri et al., 1986) using a short-chain triglyceride
(such as tributyrin) as the substrate. The enzymatic activity is
stated in units U, one unit U corresponding to the amount of enzyme
required to liberate one .mu.mol of free fatty acids per minute at
37.degree. C. under optimum pH conditions.
[0141] The plant extracts of enzymatic activity of the invention
are advantageously such that the percentage by weight of
enzymatically active recombinant polypeptides is about 0.1% to 20%,
in particular about 1% to about 15%, with respect to the total
weight of proteins present in these extracts, which corresponds to
measures of enzymatic activity from about 0.5 U per g of fresh
weight (FW) of leaves to about 1,000 U/g of FW of leaves, in
particular about 10 U/g of FW to about 300 U/g of FW of leaves, or
from about 1 U/g of FW of seeds to about 5,000 U/g of FW, in
particular to about 10 U/g of FW of seeds to about 1,000 U/g of FW
of seeds.
[0142] The invention more particularly relates to the following
plant extracts of enzymatic activity:
[0143] the extracts of leaves and/or fruits and/or seeds of plants
obtained by transformation of explant cells of these plants with
the sequence pd35S-RGLSP-DGL or the sequence pd35S-PS-DGL or the
sequence pd35S-PPS-DGL, according to one of the processes described
above, and containing the recombinant DGL and/or the polypeptide
(.DELTA.54) and/or the polypeptide (.DELTA.4), in particular:
[0144] the extract of tobacco leaves obtained by transformation of
explant cells of tobacco leaves with the sequence pd35S-PS-DGL or
the sequence pd35S-PPS-DGL, according to the process described
above, and containing the polypeptide (.DELTA.54) in combination
with the polypeptide (.DELTA.4), the percentage by weight by the
mixture of these two polypeptides with respect to the total weight
of proteins present in the said extract being from about 0.1% to
about 20%, the enzymatic activity of the said extract being from
about 100 U/g of FW to about 300 U/g of FW,
[0145] the extract of tomato leaves or fruits obtained by
transformation of explant cells of tomato leaves with the sequence
pd35S-PS-DGL, or the sequence pd35S-PPS-DGL, according to the
process described above, and containing the polypeptide (.DELTA.54)
in combination with the polypeptide (.DELTA.4), the percentage by
weight of this mixture of two polypeptides with respect to the
total weight of proteins present in the said extract being from
about 0.1% to about 20%, the enzymatic activity of the said extract
being from about 100 U/g of FW to about 300 U/g of FW,
[0146] the extract of tobacco leaves obtained by transformation of
explant cells of tobacco leaves with the sequence pd35S-RGLSP-DGL,
according to the process described above, and containing the
polypeptide (.DELTA.4), the percentage by weight of this
polypeptide with respect to the total weight of proteins present in
the said extract being from about 0.1% to about 20%, the enzymatic
activity of the said extract being from about 100 U/g of FW to
about 300 U/g of FW,
[0147] the extract of tobacco seeds obtained by transformation of
explant cells of tobacco leaves with the sequence pd35S-PSDGL or
the sequence pd35S-PPS-DGL, according to the process described
above, and containing the polypeptide (.DELTA.54), the percentage
by weight of the polypeptide (.DELTA.54) with respect to the total
weight of proteins present in the said extract being from about
0.1% to about 1%, the enzymatic activity of the said extract being
from about 10 U/g of FW to about 300 U/g of FW,
[0148] the extracts of plant seeds obtained by transformation of
explant cells of these plants with the sequence pCRU-PS-DGL or the
sequence pCRU-PPS-DGL, or the sequence pGEA1-RGLSP-DGL, or the
sequence pGEA6-RGLSP-DGL, according to one of the processes
described above, and containing the recombinant DGL and/or the
polypeptide (.DELTA.54) and/or the polypeptide (.DELTA.4), in
particular:
[0149] the extract of rape seeds obtained by transformation of
explant cells of rape leaves with the sequence pCRU-PS-DGL or the
sequence pCRU-PPS-DGL, or the sequence pGEA1-RGLSP-DGL, or the
sequence pGEA6-RGLSP-DGL, according to the process described above,
and containing the polypeptide (.DELTA.54), the percentage by
weight of polypeptide (.DELTA.54) with respect to the total weight
of proteins present in the said extract being from about 0.1% to
about 1%, the enzymatic activity of the said extract being from
about 10 U/g of FW to about 1,000 U/g of FW,
[0150] the extracts of plant seeds obtained by transformation of
explant cells of these plants with the sequence pAR-IAR-RGLSP-DGL
and/or the sequence p.gamma.zeine-RGLSP-DGL, and/or the sequence
p.gamma.zeine-RGLSP-DGL-KDEL, according to one of the processes
described above, and containing the recombinant DGL and/or the
polypeptide (.DELTA.54) and/or the polypeptide (.DELTA.4), in
particular:
[0151] the extract of corn seeds obtained by transformation of
explant cells of rape leaves with the sequence pAR-IAR-RGLSP-DGL
and/or the sequence p.gamma.zeine-RGLSP-DGL, and/or the sequence
p.gamma.zeine-RGLSP-DGL-KDEL, according to the process described
above, and containing the polypeptide (.DELTA.54), the percentage
by weight of polypeptide (.DELTA.54) with respect to the total
weight of proteins present in the said extract being from about
0.1% to about 1%, the enzymatic activity of the said extract being
from about 10 U/g of FW to about 1,000 U/g of FW.
[0152] The present invention also relates to any enzymatically
active recombinant DGL, the amino acid sequence of which is that
shown on FIG. 2, or polypeptides derived from the latter, in
particular by addition and/or suppression and/or substitution of
one (or more) amino acid(s), these derived polypeptides having a
lipase activity, such as are obtained in an essentially pure form
by carrying out one of the processes of the invention described
above, these processes comprising a stage of purification of the
recombinant polypeptides of the invention, in particular by
chromatography carried out on the enzymatic extracts described
above.
[0153] As the polypeptides derived from the above-mentioned
recombinant DGL, the invention more particularly relates to the
polypeptides (.DELTA.54) and (.DELTA.4) mentioned above, the
molecular weights of which are, respectively, about 37 kDa and
about 49 kDa.
[0154] Enzymatically active recombinant DGL or derived polypeptides
having a lipase activity, as mentioned above, is understood as
meaning any recombinant polypeptide which is capable of having a
lipase activity as measured in accordance with the method of
Gargouri mentioned above.
[0155] By way of illustration, the recombinant polypeptides
according to the invention have a lipase activity of from about 10
U/mg of recombinant polypeptides to about 1,000 U/mg,
advantageously from about 100 U/mg to about 600 U/mg.
[0156] The invention more particularly relates to the recombinant
DGL obtained by purification of the enzymatic extract of tobacco
leaves or seeds, these leaves or seeds originating from transformed
tobacco plants, themselves obtained from tobacco cells transformed
with the sequence pd35S-RGLSP-DGL according to the process
described above, the said recombinant DGL having a lipase activity
as described above.
[0157] The invention also relates to the polypeptide (.DELTA.54)
and the polypeptide (.DELTA.4) obtained by purification of the
enzymatic extract of plant leaves and/or seeds and/or fruits,
notably solanaceae, such as transformed tobacco or tomato,
themselves obtained from plant cells transformed with the sequence
pd35SPS-DGL or the sequence pd35S-PPS-DGL, or the sequence
pd35SRGLSP-DGL, according to the process described above, the said
recombinant polypeptides (.DELTA.54) and (.DELTA.4) having a lipase
activity as described above.
[0158] The invention also relates to the polypeptide (.DELTA.54)
obtained by purification of the enzymatic extract of tobacco seeds,
or that of rape seeds, these seeds originating, respectively, from
transformed tobacco or rape plants, themselves obtained,
respectively, from tobacco or rape cells transformed with the
sequence pCRU-PS-DGL or the sequence pCRU-PPS-DGL, according to the
processes described above, the recombinant polypeptide (.DELTA.54)
having a lipase activity as described above.
[0159] The invention also relates to the polypeptide (.DELTA.54)
and the polypeptide (.DELTA.4) obtained by purification of the
enzymatic extract of rape seeds, these seeds originating from
transformed rape plants, themselves obtained from rape cells
transformed with the sequence pGEA1-RGLSP-DGL and/or the sequence
pGEA6-RGLSP-DGL, according to the processes described above, the
said recombinant polypeptides (.DELTA.54) and (.DELTA.4) having a
lipase activity as described above.
[0160] The invention also relates to the polypeptide (.DELTA.54)
and the polypeptide (.DELTA.4) obtained by purification of the
enzymatic extract of corn seeds, these seeds originating from
transformed corn plants, themselves obtained from corn cells
transformed with the sequence pAR-IAR-RGLSP-DGL and/or the sequence
p.gamma.zeine-RGLSP-DGL, and/or the sequence
p.gamma.zeine-RGLSPDGL-KDEL, according to the processes described
above, the said recombinant polypeptides (.DELTA.54) and (.DELTA.4)
having a lipase activity as described above.
[0161] The apparent molecular weights of the polypeptides
(.DELTA.54) and (.DELTA.4) according to the invention are,
respectively, 37 kDa and 49 kDa, measured by analysis in
polyacrylamide gel and by immunodetection after electrotransfer on
nitrocellulose (these methods being detailed in the embodiment
examples of the invention which follow).
[0162] The invention relates to antibodies directed against the
recombinant polypeptides of the invention, and more particularly
those directed against the recombinant DGL according to the
invention and/or against the above-mentioned polypeptide
(.DELTA.54) and/or against the above-mentioned polypeptide
(.DELTA.4), which can also recognize HGL.
[0163] Such antibodies can be obtained by immunization of an animal
with these polypeptides, followed by recovery of the antibodies
formed.
[0164] It goes without saying that this production is not limited
to polyclonal antibodies.
[0165] It also applies to any monoclonal antibody produced by any
hybridoma which can be formed by conventional methods from animal
spleen cells, in particular from the mouse or rat, immunized
against one of the purified polypeptides of the invention on the
one hand, and cells of a suitable myeloma on the other hand, and
which can be selected according to its capacity to produce
monoclonal antibodies which recognize the abovementioned
polypeptide initially used for immunization of the animals, as well
as HGL.
[0166] The invention also relates to the use of transformed plants,
plant parts, plant cells or seeds according to the invention for
the preparation of one (or more) recombinant polypeptide(s)
according to the invention, such as recombinant DGL or its derived
polypeptides as defined above, in particular by carrying out one of
the abovementioned processes of the invention, the said recombinant
polypeptides being in an essentially pure form or contained in
plant extracts of enzymatic activity as defined above.
[0167] The invention also relates to the use, in the field of human
or animal foods, plants or parts of plants, of enzymatic activity
according to the invention, or of plant extracts of enzymatic
activity as defined above or of recombinant polypeptides according
to the invention, such as the recombinant DGL or its derived
polypeptides as defined above.
[0168] The invention more particularly relates to the use of plants
or parts of plants, notably leaves, fruits, seeds of enzymatic
activity according to the invention as foods.
[0169] In this respect, the invention more particularly relates to
any food comprising a plant of enzymatic activity as described
above or parts of this plant, notably leaves or fruits, or seeds
produced by the latter, which can be of an edible character to man
or animal.
[0170] The invention also relates to any alimentary composition
comprising one (or more) plant(s) of enzymatic activity as
described above and/or parts of this (these) plant(s) notably
leaves and/or seeds and/or fruits of this (these) plants and/or one
(or more) plant extract(s) of enzymatic activity as described above
and/or one (or more) recombinant polypeptide(s) of the invention,
where appropriate in combination with one (or more) other edible
compound(s).
[0171] The plants or parts of plants contained in the
abovementioned alimentary composition are advantageously in the
form of ground material.
[0172] The foods according to the invention, also called functional
foods, or the alimentary compositions according to the invention
are more particularly intended to facilitate the absorption of
animal or vegetable fats ingested by a healthy individual or an
individual suffering from one or more pathologies which may or may
not affect the level of production of gastric and/or pancreatic
lipase. In this respect, the foods or alimentary compositions of
the invention are advantageously used as nutritional
supplements.
[0173] The invention also relates to the use of plants or parts of
plants, notably leaves and/or fruits and/or seeds, or plant cells
of enzymatic activity according to the invention, or plant extracts
of enzymatic activity as defined above, or recombinant polypeptides
according to the invention, such as the recombinant DGL or its
derived polypeptides as defined above, for the preparation of
medicaments (or pharmaceutical compositions) intended to facilitate
the absorption of animal or vegetable fats ingested by a healthy
individual or an individual suffering from one or more pathologies
which may or may not affect the level of production of gastric
and/or pancreatic lipase.
[0174] In particular, such pharmaceutical compositions are
advantageously used on individuals undergoing medical treatment
which changes the mechanism of absorption of fats, or on elderly
persons.
[0175] The pharmaceutical compositions according to the invention
are also more particularly intended for treatment of pathologies
associated with lipase (in particular gastric and/or pancreatic
lipase) insufficiency in the organism, and more particularly
pathologies such as cystic fibrosis and exocrine pancreatic
insufficiency.
[0176] The invention more particularly relates to any
pharmaceutical composition comprising one (or more) plant
extract(s) of enzymatic activity described above and/or one (or
more) recombinant polypeptide(s) according to the invention, where
appropriate in combination with a pharmaceutically acceptable
vehicle.
[0177] The invention more particularly relates to any
abovementioned pharmaceutical composition comprising the
recombinant DGL and/or the polypeptide (.DELTA.54) and/or the
polypeptide (.DELTA.4) in an essentially pure form or in the form
of enzymatic extracts as described above.
[0178] The pharmaceutical compositions according to the invention
can preferably be administered orally, and are, in particular, in
the form of capsules, tablets or powders for dilution.
[0179] The daily dosage in humans is advantageously from about 200
mg to about 1,000 mg, preferably distributed over the main
mealtimes, if the said pharmaceutical compositions comprise
enzymatic extracts as described above, and from about 100 mg to
about 500 mg if the said pharmaceutical compositions comprise the
recombinant polypeptides according to the invention in an
essentially pure form.
[0180] The invention also relates to the use of plants or parts of
plants, notably leaves and/or fruits and/or seeds, or plant cells
of enzymatic activity according to the invention, or plant extracts
of enzymatic activity as defined above, or recombinant polypeptides
according to the invention, such as the recombinant DGL or its
derived polypeptides as defined above, for carrying out enzymatic
reactions in the industrial, agro-alimentary or agro-industrial
field, in particular in the fats industry, in lipochemistry and in
the milk industry.
[0181] In this respect, the invention relates to any process, in
particular of enzymatic bioconversion or of biocatalysis by
carrying out one or more enzymatic reactions, in the industrial,
agro-alimentary or agro-industrial field, in particular in the fats
industry, in lipochemistry and in the milk industry, these
enzymatic reactions being carried out by means of plants or parts
of plants, notably leaves and/or fruits and/or seeds, or plant
cells of enzymatic activity according to the invention, or plant
extracts of enzymatic activity as defined above, or recombinant
polypeptides according to the invention, such as the recombinant
DGL or its derived polypeptides as defined above.
[0182] The invention more particularly relates to enzymatic
preparations intended for industrial, agro-alimentary or
agro-industrial use which can be used in the context of carrying
out a process as described above and comprise one (or more) plant
extract(s) of enzymatic activity as defined above, and/or one (or
more) recombinant polypeptide(s) according to the invention, in
particular the recombinant DGL and/or the polypeptide (.DELTA.54)
and/or the polypeptide (.DELTA.4), where appropriate in combination
with one (or more) additive(s) or other enzyme(s) which can be used
in the context of the abovementioned industrial use.
[0183] The invention more particularly relates to the use of plants
or parts of plants, notably leaves and/or fruits and/or seeds, or
plant cells of enzymatic activity according to the invention, for
carrying out, on an industrial scale, enzymatic bioconversion
reactions or biocatalysis reactions, such as enzymatic hydrolyses
or trans-esterifications.
[0184] The plants of enzymatic activity or parts of these plants,
notably leaves and/or fruits and/or seeds, or plant cells according
to the invention are advantageously used both as the enzymatic
source and as the reactive substrate.
[0185] The invention also relates to any biocatalysis process which
uses plants or parts of plants, notably leaves and/or fruits and/or
seeds, or plant cells of enzymatic activity according to the
invention, and more particularly plants containing the recombinant
DGL and/or the polypeptide (.DELTA.54) and/or the polypeptide
(.DELTA.4), the said plants or parts of plants, being used both as
the enzymatic source and as the reactive substrate.
[0186] The invention more particularly relates to the use of plants
of enzymatic activity or parts of these plants according to the
invention for the preparation of biofuels.
[0187] In this respect, the present invention relates to any
process for the preparation of a biofuel by addition of alcohol, in
particular methanol or ethanol, to ground material of all or part
of transformed plants according to the invention, advantageously
ground material of transformed rape, sunflower or soya seeds
according to the invention, and recovery of the biofuel, in
particular by filtration.
[0188] The invention also relates to the esters of plant fatty
acids such as are obtained by carrying out the above-mentioned
process, in particular the methyl ester of oleic acid.
[0189] The invention also relates to any biofuel obtained by
carrying out a process as described above, and more particularly
any abovementioned biofuel comprising esters of plant fatty
acids.
[0190] The invention more particularly relates to any biofuel
obtained by carrying out the abovementioned process on rape seeds
and comprising a methyl ester of oleic acid.
[0191] The invention also relates to the use of the abovementioned
antibodies directed against the recombinant polypeptides of the
invention for carrying out a method for detection or assay of DGL
or HGL in a biological sample which may contain it.
[0192] The invention more particularly relates to the use of these
antibodies for carrying out a method for in vitro diagnosis of
pathologies associated with excess production or, as the opposite,
insufficiency, or even the absence of production, of lipase in the
organism.
[0193] This method for in vitro diagnosis carried out on a
biological sample taken from a patient comprises a stage of
bringing this sample together with one or more antibodies according
to the invention, followed by a stage of detection of any
antibody-HGL complexes formed during the preceding stage.
[0194] In this respect, the invention also relates to a kit for
carrying out an abovementioned method of in vitro detection or
diagnosis comprising:
[0195] antibodies as described above, advantageously labelled in a
radioactive or enzymatic manner, and reagents for constitution of a
medium favourable for carrying out the immunological reaction
between these antibodies and the HGL,
[0196] reagents which allow detection of immunological complexes
formed between these antibodies and the HGL.
[0197] The present invention more particularly relates to the use
of a recombinant nucleotide sequence containing on the one hand,
the cDNA shown on FIG. 4 and which codes for the human gastric
lipase (HGL) shown on FIG. 5, or a nucleotide sequence derived from
this cDNA, in particular by addition and/or suppression and/or
substitution of one (or more) nucleotide (s), the said derived
sequence being capable of coding for a polypeptide, the amino acid
sequence of which is identical to that of the HGL shown on FIG. 5,
or for a polypeptide derived from the HGL by addition and/or
suppression and/or substitution of one (or more) amino acid(s),
this derived polypeptide having a lipase activity, and, on the
other hand, elements which allow a plant cell to produce the
polypeptide coded by the said cDNA or by an abovementioned derived
sequence, in particular a transcription promoter and a
transcription terminator recognized by the transcription machinery
of plant cells (and more particularly by the RNA polymerases of the
latter), for transformation of plant cells in order to obtain, from
these cells or plants obtained from the latter, recombinant DGL in
active enzyme form or one (or more) polypeptide(s) derived from the
latter as defined above.
[0198] In this respect, the invention relates to any recombinant
nucleotide sequence as described above in the context of the
transformation of plants with a view to the preparation of
recombinant DGL, in which the nucleotide sequence which codes for
the DGL and shown on FIG. 1 or FIG. 3, is replaced by the
nucleotide sequence which codes for the HGL and shown on FIG.
4.
[0199] The invention more particularly relates to the following
recombinant nucleotide sequences:
[0200] that (called pSP-HGLSP-HGL) containing, in the direction
5'.fwdarw.3', the promoter pSP of Agrobacterium tumefaciens, the
sequence which codes for the signal-peptide of HGL, the latter
being immediately followed by the nucleotide sequence shown on FIG.
4, and then the terminator polyA 35S of CaMV,
[0201] that (called pSP-HPLSP-HGL) containing, in the direction
5'.fwdarw.3', the promoter pSP of Agrobacterium tumefaciens, the
sequence which codes for the signal peptide of HPL, the latter
being immediately followed by the nucleotide sequence shown on FIG.
4, and then the terminator polyA 35S of CaMV,
[0202] that (called pSP-RGLSP-HGL) containing, in the direction
5'.fwdarw.3', the promoter pSP of Agrobacterium tumefaciens, the
sequence which codes for part of the signal peptide of RGL (as
described above), the latter being immediately followed by the
nucleotide sequence shown on FIG. 4, and then the terminator polyA
35S of CaMV.
[0203] The invention also relates to the vectors and cellular hosts
transformed by these vectors, as described above, and containing
the abovementioned recombinant nucleotide sequences which code for
the HGL and/or its derived polypeptides.
[0204] The present invention also relates to any process for the
preparation of recombinant HGL in active enzyme form and/or of one
(or more) polypeptide(s) derived from the latter, in particular by
addition and/or suppression and/or substitution of one (or more)
amino acid(s), this (or these) derived polypeptide(s) having a
lipase activity, characterized in that it comprises:
[0205] transformation of plant cells such that one (or more)
recombinant nucleotide sequence(s) according to the invention is
(or are) integrated into the genome of these cells,
[0206] where appropriate, production of transformed plants from the
abovementioned transformed cells,
[0207] recovery of the recombinant HGL and/or of the abovementioned
derived polypeptide(s) produced in the said cells or abovementioned
transformed plants, in particular by extraction, followed, where
appropriate, by purification.
[0208] The invention more particularly relates to any production
process for recombinant HGL by the implementation of a process as
described above in the context of the production of recombinant DGL
and/or its derived polypeptides using an abovementioned recombinant
sequence containing the sequence shown on FIG. 4.
[0209] Among the polypeptides derived from the recombinant HGL
which can be obtained in the context of carrying out a process
according to the invention there may be mentioned:
[0210] the polypeptide delimited by the amino acids situated in
positions 74 and 398 in FIG. 5, also called polypeptide
(.DELTA.54HGL),
[0211] the polypeptide delimited by the amino acids situated in
positions 24 and 398 in FIG. 5, also called polypeptide
(.DELTA.4HGL).
[0212] The invention more particularly relates to a process for the
preparation, as described above, of the recombinant HGL and/or the
polypeptide (.DELTA.54HGL) and/or the polypeptide (.DELTA.4HGL),
characterized in that it comprises:
[0213] transformation of explant cells of the leaves of a plant by
bringing the latter together with a strain of Agrobacterium
tumefaciens transformed by a plasmid as described above containing
the abovementioned recombinant nucleotide sequence pSP-HGLSP-HGL
and/or pSP-HPLSP-HGL and/or pSP-RGLSP-HGL, on a suitable culture
medium,
[0214] selection of the transformed explants on a medium containing
kanamycin,
[0215] production of transformed plants from the abovementioned
transformed explants by culture of the latter on suitable
media,
[0216] extraction of the recombinant HGL and/or the polypeptide
(.DELTA.54HGL) and/or the polypeptide (.DELTA.4HGL), in particular
by grinding the leaves and/or the seeds and/or the fruits of the
abovementioned transformed plants in a suitable buffer,
centrifuging and recovery of the supernatant constituting the plant
extract of enzymatic activity,
[0217] where appropriate, purification of the recombinant HGL
and/or the polypeptide (.DELTA.54HGL) and/or the polypeptide
(.DELTA.4HGL) from the extract obtained during the preceding stage,
in particular by chromatography carried out on the supernatant,
which leads to the preparation of the recombinant HGL and/or the
polypeptide (.DELTA.54HGL) and/or the polypeptide (.DELTA.4HGL) in
an essentially pure form.
[0218] The invention also relates to any plant or part of this
plant, notably leaves and/or fruits and/or seeds, containing one
(or more) recombinant nucleotide sequence(s) as described above,
according to the invention, integrated into its genome in a stable
manner.
[0219] The present invention relates to any plant extract of
enzymatic activity, and more particularly of lipase activity
defined below, prepared by carrying out one of the processes of the
invention described above, and containing, as active enzymes, one
(or more) recombinant polypeptide(s) according to the invention,
such as the recombinant HGL and/or the polypeptide (.DELTA.54HGL)
and/or the polypeptide (.DELTA.4HGL).
[0220] The invention more particularly relates to extracts of
leaves and/or seeds of plants, as obtained by the transformation of
explant cells of these plants with the sequence pSP-HGLSP-HGL, or
the sequence pSP-HPLSP-HGL, or the sequence pSP-RGLSP-HGL,
according to one of the processes described above, and containing
the recombinant HGL, and/or the polypeptide (.DELTA.54HGL), and/or
the polypeptide (.DELTA.4HGL), in particular:
[0221] the extract of tobacco leaves or seeds as obtained by
transformation of explant cells of tobacco leaves with the sequence
pSP-HGLSP-HGL, or the sequence pSP-HPLSP-HGL, or the sequence
pSP-RGLSP-HGL according to the process described above, and
containing the polypeptide (.DELTA.54HGL), in combination with the
polypeptide (.DELTA.4HGL), the percentage by weight of the mixture
of these two polypeptides with respect to the total weight of
proteins present in the said extract being from about 0.1% to about
20%, the enzymatic activity of the said extract being from about
100 U/g of FW to about 300 U/g of FW,
[0222] the extract of tobacco leaves as obtained by transformation
of explant cells of tobacco leaves with the sequence pSP-RGLSP-HGL,
and containing the polypeptide (.DELTA.4HGL), the percentage by
weight of this polypeptide with respect to the total weight of
proteins present in the said extract being from about 0.1% to about
20%, the enzymatic activity of the said extract being from about
100 U/g of FW to about 300 U/g of FW.
[0223] The present invention also relates to any enzymatically
active recombinant HGL, the amino acid sequence of which is that
shown on FIG. 5, or polypeptides derived from the latter, in
particular by addition and/or suppression and/or substitution of
one (or more) amino acid(s), and more particularly the polypeptides
(.DELTA.54HGL) and (.DELTA.4HGL), these derived polypeptides having
a lipase activity, such as are obtained in an essentially pure form
by carrying out one of the processes of the invention described
above, these processes comprising a stage of purification of the
recombinant polypeptides of the invention, in particular by
chromatography carried out on the enzymatic extracts described
above.
[0224] As has been described previously in the context of the
recombinant DGL, the invention also relates to:
[0225] the polyclonal or monoclonal antibodies directed against the
recombinant HGL or its derived polypeptides according to the
invention, and their uses as described above,
[0226] foods or alimentary compositions or pharmaceutical
compositions, or enzymatic preparations for industrial purposes
based on plants, or parts of plants, notably leaves and/or fruits
and/or seeds, or plant cells or extracts of enzymatic activity as
defined above, or also recombinant HGL or its derived polypeptides
according to the invention,
[0227] any enzymatic bioconversion process or biocatalysis, or
biofuel preparation as described above, carried out from
recombinant HGL or its derived polypeptides according to the
invention, or plants, or parts of plants, notably leaves and/or
fruits and/or seeds and/or plant extracts of enzymatic activity as
defined above.
[0228] The invention will be illustrated further in the detailed
description which follows for the preparation of recombinant
nucleotide sequences as described above and transformed plants
which produce the recombinant polypeptides according to the
invention, and for a process for the preparation of a biofuel.
[0229] 1. Construction of Chimaeric Genes which Code for the
Recombinant Protein of Dog Gastric Lipase and Allow Expression in
the Leaves and Seeds of Solanaceae.
[0230] I-A) Construction of Chimaeric Genes which Code for the
Recombinant DGL and Allow Expression in Tobacco.
[0231] Expression in tobacco leaves and seeds of the gene which
codes for dog gastric lipase (DGL) requires the following regulator
sequences:
[0232] 1. The double-structured promoter 35S (pd35S) of CaMV
(cauliflower mosaic virus).
[0233] This corresponds to duplication of the sequences which
activate transcription and are situated upstream of the TATA
element of the natural promoter 35S (Kay et al., 1987);
[0234] 2. The terminal transcription sequence, terminator polyA
35S, which corresponds to the non-coding region 3' of the sequence
of the cauliflower mosaic virus, of double-stranded circular DNA,
which produces the transcript 35S (Franck et al., 1980).
[0235] The constructions of the various plasmids via the use of
recombinant DNA techniques (Sambrook et al., 1989) are derived from
pBIOC4. This binary plasmid is derived from pGA492 (An, 1986),
which contains, between the right and left borders, the following
sequences originating from the plasmid pTiT37 of Agrobacterium
tumefaciens, on its transfer DNA:
[0236] The structural promoter of the nos gene which codes for
nopaline synthase (Depicker et al., 1982), the sequence which codes
for the nptII gene which codes for neomycin phosphotransferase II
(Berg and Berg, 1983) deleted from the region of the first 8
codons, of which the initiator codon is methionine ATG, and fused
to the sequence of the first 14 codons of the sequence which codes
for the nos gene (Depicker et al., 1982), the sequence which codes
for the nos gene devoid of the region of the first 14 codons, the
nos terminator (Depicker et al., 1982), a region containing
multiple cloning sites (also called polylinker)
(HindIII-XbaISacI-HpaI-KpnI-ClaI-BglII) preceding the cat gene
which codes for chloramphenicol acetyltransferase (Close and
Rodriguez, 1982) and the terminal sequences of the gene 6 of the
plasmid pTiA6 of Agrobacterium tumefaciens (Liu et al., 1993). To
eliminate virtually all the sequence which codes for the cat gene,
the plasmid pGA492 was digested twice by SacI (restriction site of
the polylinker) and by ScaI (restriction site present in the
sequence of the cat gene) and then subjected to the action of the
enzyme T4 DNA polymerase (New England Biolabs) in accordance with
the manufacturer's instructions. The ligation of the modified
plasmid (20 ng) was carried out in a reaction medium of 10 .mu.l
comprising 1 .mu.l of the buffer T4 DNA ligase.times.10 (Amersham);
2.5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5.alpha., rendered
competent beforehand, were transformed (Hanahan, 1983). The plasmid
DNA of the clones obtained, selected on 12 .mu.g/ml of
tetracycline, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The HindIII restriction site of the plasmid DNA of the
clone retained was then modified into an EcoRI restriction site
with the aid of an phosphorylated HindIII-EcoRI adaptor (Stratagene
Cloning Systems). To carry out this modification, 500 ng of plasmid
DNA of the clone retained were digested by HindIII,
dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions and coprecipitated in the presence of
1,500 ng of the HindIII-EcoRI DNA adaptor, 1/10 volume of 3M sodium
acetate, pH 4.8, and 2.5 volumes of absolute ethanol at -80.degree.
C. for 30 min. After centrifugation at 12,000 g for 30 min, the DNA
precipitated was washed with 70% ethanol, dried, taken up in 8
.mu.l of water, kept at 65.degree. C. for 10 min and then ligated
in the presence of 1 .mu.l of the buffer T4 DNA ligase.times.10
(Amersham) and 2.5 U of the enzyme T4 DNA ligase (Amersham) at
14.degree. C. for 16 hours. After inactivation of the T4 DNA ligase
at 65.degree. C. for 10 min, the ligation reaction mixture was
digested by EcoRI, purified by electrophoresis over 0.8% agarose
gel, electroeluted (Sambrook et al., 1989), precipitated in the
presence of {fraction (1/10)} volume of 3M sodium acetate, pH 4.8,
and 2.5 volumes of absolute ethanol at -80.degree. C. for 30 min,
centrifuged at 12,000 g for 30 min, washed with 70% ethanol, dried
and then ligated as described above. The bacteria Escherichia coli
DH5, rendered competent beforehand, were transformed (Hanahan,
1983). The plasmid DNA of the clones obtained, selected on 12
.mu.g/ml of tetracycline, was extracted by the alkaline lysis
method (Birnboim and Doly, 1979) and analysed by enzymatic
digestion by HindIII and EcoRI in particular. The resulting binary
plasmid, which only has the last 9 codons of the sequence which
codes for the cat gene and of which the EcoRI site is unique, was
called pBIOC4.
[0237] The expression cassette made up of the promoter pd35S and
the terminator polyA 35S was isolated using the plasmid
pJIT163.DELTA.. The plasmid pJIT163.DELTA. is derived from the
plasmid pJIT163, which itself is derived from the plasmid pJIT60
(Guerineau and Mullineaux, 1993). The plasmid pJIT163 possesses an
ATG codon between the HindIII and SalI sites of the polylinker. To
suppress this ATG and to obtain the plasmid pJIT163.DELTA., the
plasmid pJIT163 DNA was digested twice by HindIII and SalI,
purified by electrophoresis over 0.8% agarose gel, electroeluted
(Sambrook et al., 1989), precipitated in the presence of {fraction
(1/10)} volume of 3M sodium acetate, pH 4.8, and 2.5 volumes of
absolute ethanol at -80.degree. C. for 30 min, centrifuged at
12,000 g for 30 min, washed with 70% ethanol, dried, subjected to
the action of Klenow enzyme (New England Biolabs) in accordance
with the manufacturer's instructions, deproteinated by extraction
with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) and
then 1 volume of chloroform:isoamyl alcohol (24:1), precipitated in
the presence of {fraction (1/10)} volume of 3M sodium acetate, pH
4.8, and 2.5 volumes of absolute ethanol at -80.degree. C. for 30
min, centrifuged at 12,000 g for 30 min, washed with 70% ethanol,
dried and, finally, ligated in the presence of 1 .mu.l of the
buffer T4 DNA ligase.times.10 (Amersham) and 2.5 U of the enzyme T4
DNA ligase (Amersham) at 14.degree. C. for 16 hours. The bacteria
Escherichia coli DH5.alpha., rendered competent beforehand, were
transformed (Hanahan, 1983). The plasmid DNA of the clones
obtained, selected on 50 .mu.g/ml of ampicillin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes. To isolate the
expression cassette made up of the promoter pd35S and the
terminator polyA 35S (SacI-XhoI fragment), the plasmid DNA of the
clone pJIT163.DELTA.retained was digested by SacI and XhoI. The
SacI-XhoI fragment, carrying the expression cassette, was purified
by electrophoresis over 0.8% agarose gel, electroeluted (Sambrook
et al., 1989), precipitated in the presence of {fraction (1/10)}
volume of 3M sodium acetate, pH 4.8, and 2.5 volumes of absolute
ethanol at -80.degree. C. for 30 min, centrifuged at 12,000 g for
30 min, washed with 70% ethanol, dried and then subjected to the
action of Mung Bean Nuclease enzyme (New England Biolabs) in
accordance with the manufacturer's instructions. This purified
insert (200 ng) was cloned in the plasmid DNA of pBIOC4 (20 ng),
which had been digested by EcoRI, treated with the enzyme Mung Bean
Nuclease and dephosphorylated by the alkaline phosphatase enzyme of
the intestine of the calf (Boehringer Mannheim) in accordance with
the manufacturer's instructions. The ligation reaction was carried
out in 20 .mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5, rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on 12 .mu.l/ml of tetracycline, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes. The
resulting plasmid was called pBIOC21.
[0238] Dog gastric lipase (DGL) is synthesized naturally in the
form of a precursor. The mature DGL protein is made up of 379 amino
acids. The complementary DNA of DGL was cloned at the BglII and
SalI sites of the expression vector pRU303, leading to the vector
pDGL5.303 described in the international application no. WO
94/13816. It was used for construction of the binary plasmids
pBIOC25, containing PS-DGL, and pBIOC26, containing PPS-DGL, where
the sequence which codes for the mature DGL is preceded by that
which codes for a signal peptide (PS) or a prepropeptide (PPS, that
is to say a signal peptide followed by N-terminal vacuole-directing
sequences) of plant origin respectively. The PS and PPS sequences,
made up, respectively, of 23 and 37 amino acids, are those of a
reserve protein of the tuberous roots of the sweet potato: sporamin
A (Murakami et al., 1986; Matsukoa and Nakamura, 1991).
[0239] To simplify fusions between the sequence of mature DGL and
that of the directing signals, PS or PPS, the plasmid pDGL5.303 was
modified by introduction of a supplementary HindIII restriction
site into the fourth and fifth codons of the mature DGL sequence by
mutagenesis directed by PCR using 2 oligodeoxynucleotides, 5'
caggagatc TTG TTT GGA AAG CTT CAT CCC 3' (containing the unique
BglII site in the plasmid and providing the supplementary HindIII
site) and 5'CAT ATT CCT CAG CTG GGT ATC 3' (containing the unique
PvuII site in the plasmid). Amplification of the BglII-PvuII
fragment by PCR was carried out in 100 .mu.l of reaction medium
comprising 10 .mu.l of the buffer Taq DNA polymerase.times.10 (500
mM KCl, 100 mM TrisHCl, pH 9.0, and 1% Triton.times.100), 6 .mu.l
of 25 mM MgCl.sub.2, 3 .mu.l of 10 mM DNTP (DATP, dCTP, dGTP and
dTTP), 100 pM of each of the 2 oligodeoxynucleotides described
above, 5 ng of matrix DNA (expression vector pRU303 including the
complementary DNA of DGL), 2.5 U of Taq DNA polymerase (Promega)
and 2 drops of vaseline oil. The DNA was denatured at 94.degree. C.
for 5 min, subjected to 30 cycles, each of 1 min of denaturation at
94.degree. C., 1 min of hybridization at 50.degree. C. and 1 min of
elongation at 72.degree. C., and then elongation at 72.degree. C.
was continued for 5 min. This PCR reaction was carried out in the
"DNA Thermal Cycler" machine of PERKIN ELMER CETUS. The oil was
removed by extraction with chloroform. The DNA fragments contained
in the reaction medium were then precipitated in the presence of
1/10 volume of 3M sodium acetate, pH 4.8, and 2.5 volumes of
absolute ethanol at -80.degree. C. for 30 min, centrifuged at
12,000 g for 30 min, washed with 70% ethanol, dried and digested by
the 2 restriction enzymes BglII and PvuII. The digested DNA
fragments originating from the PCR were purified by electrophoresis
over 2% agarose gel, electroeluted (Sambrook et al., 1989),
precipitated in the presence of {fraction (1/10)} volume of 3M
sodium acetate, pH 4.8, and 2.5 volumes of absolute ethanol at
-80.degree. C. for 30 min, centrifuged at 12,000 g for 30 min,
washed with 70% ethanol, dried and then ligated to the plasmid DNA
of the vector pDGL5.303, which had been digested twice by BglII and
PvuII, purified by electrophoresis over 0.8% agarose gel,
electroeluted, subjected to precipitation in alcohol, dried and
dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 100
ng of the dephosphorylated vector described above and 50 ng of the
digested DNA fragments, originating from the amplification by PCR,
described above in a reaction medium of 10 .mu.l in the presence of
1 .mu.l of the buffer T4 DNA ligase.times.10 (Amersham) and 2.5 U
of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16
hours. The bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on 50 .mu.g/ml of ampicillin, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes. The
plasmid DNA of some of the clones retained was verified by
sequencing with the aid of the T7.TM. sequencing kit, marketed by
Pharmacia, by the dideoxynucleotide method (Sanger et al., 1977).
Introduction of this HindIII restriction site does not modify the
genetic code of the DGL. In fact, the natural DGL sequence AAA TTA
(Lys-Leu) becomes AAG CTT (Lys-Leu). The resulting plasmid was
called pBIOC22 and includes the sequence of the mature DGL protein
corresponding to:
1 LEU PHE GLY LYS LEU------THR ASP ASN LYS AMB agatcTTG TTT GGA AAG
CTT------ACA GAT AAT AAG TAG TTCTAGA ------ ------- ------ BglII
HindIII XbaI unique restriction site unique restriction site LEU:
first codon of the mature DGL, AMB: Stop codon.
[0240] LEU: first codon of the mature DGL, AMB: Stop codon.
[0241] a. Construction of the Binary Plasmid pBIOC25 Containing
PS-DGL.
[0242] The plasmid pBIOC22 was digested totally by BglII and partly
by HindIII in order to suppress the sequence which codes for the
polypeptide Leu-Phe-Gly-Lys (first 4 amino acids) of the mature DGL
protein. This sequence was replaced by that which codes for the
signal peptide PS of 23 amino acids (ATG AAA GCC TTC ACA CTC GCT
CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC)
fused to that of the first 4 codons of the sequence which codes for
the mature DGL protein ("PS-first 4 codons of mature DGL"). The
sequence "PS-first 4 codons of mature DGL" was amplified by PCR
using the plasmid pMAT103 (Matsuoka and Nakamura, 1991) with the
aid of the 2 following oligodeoxynucleotides 5' caggagatctgATG AAA
GCC TTC ACA CTC GC 3' and 5' G ATG AAG CTT TCC AAA CAA GGA ATG GGC
TGG ATT GGG CAG G 3', in accordance with the protocol of PCR
amplification described above in paragraph I. After double
enzymatic digestion by BGlII and HindIII, the DNA fragments
originating from the PCR amplification were purified by
electrophoresis over 2% agarose gel, electroeluted (Sambrook et
al., 1989), precipitated in the presence of {fraction (1/10)}
volume of 3M sodium acetate, pH 4.8, and 2.5 volumes of absolute
ethanol at -80.degree. C. for 30 min, centrifuged at 12,000 g for
30 min, washed with 70% ethanol, dried and then ligated to the
plasmid DNA of pBIOC22, which had been doubly digested by BglII and
HindIII, purified by electrophoresis over 0.8% agarose gel,
electroeluted (Sambrook et al., 1989), subjected to precipitation
with alcohol, dried and dephosphorylated by the alkaline
phosphatase enzyme of the intestine of the calf (Boehringer
Mannheim) in accordance with the manufacturer's instructions. The
ligation was carried out with 100 ng of the dephosphorylated vector
described above and 50 ng of the digested DNA fragments,
originating from the PCR amplification, described above in a
reaction medium of 10 .mu.l in the presence of 1 ul of the buffer
T4 DNA ligase.times.10 (Amersham) and 2.5 U of the enzyme T4 DNA
ligase (Amersham) at 14.degree. C. for 16 hours. The bacteria
Escherichia coli DH5.alpha., rendered competent beforehand, were
transformed (Hanahan, 1983). The plasmid DNA of the clones
obtained, selected on 50 .mu.g/ml of ampicillin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes. The plasmid DNA of some
of the clones retained was verified by sequencing with the aid of
the T7.TM. sequencing kit, marketed by Pharmacia, by the
dideoxynucleotide method (Sanger et al., 1977). The sequences of
the PS and the mature DGL were cloned, maintaining their
open-reading frames. The cleavage sequence between the sequences of
the PS and the mature DGL is Ser-Leu. The resulting plasmid was
called pBIOC23. Starting from pBIOC23, the BglII-XbaI fragment
carrying the sequence of PS-DGL was isolated by double enzymatic
digestion by BglII and XbaI, purification by electrophoresis over
0.8% agarose gel, electroelution (Sambrook et al., 1989),
precipitation with alcohol and drying. This DNA fragment was then
treated with Klenow enzyme in accordance with the manufacturer's
instructions and ligated to the plasmid DNA of pBIOC21, which had
been digested at the HindIII site, treated with Klenow and
dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 20
ng of the dephosphorylated vector described above and 200 ng of DNA
fragments, containing the PS-DGL, described above in a reaction
medium of 20 .mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5.alpha., rendered
competent beforehand, were transformed (Hanahan, 1983). The plasmid
DNA of the clones obtained, selected on 12 .mu.g/ml of
tetracycline, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The resulting clone was called pBIOC25. The nucleotide
sequence of the fragment which codes for the recombinant protein
PS-DGL was verified by sequencing with the aid of the T7 sequencing
kit, marketed by Pharmacia, by the dideoxynucleotide method (Sanger
et al., 1977). The plasmid DNA of the binary vector pBIOC25 was
introduced by direct transformation into the strain LBA4404 of
Agrobacterium tumefaciens in accordance with the process of
Holsters et al. (1978). The validity of the clone retained was
verified by enzymatic digestion of the plasmid DNA introduced.
[0243] b. Construction of the Binary Plasmid pBIOC26 Containing
PPS-DGL
[0244] The plasmid pBIOC22 was digested totally by BglII and partly
by HindIII in order to suppress the sequence which codes for the
polypeptide Leu-Phe-Gly-Lys of the mature DGL protein. This
sequence was replaced by that which codes for the signal peptide
PPS of 37 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT
CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC AGG TTC AAT CCC ATC
CGC CTC CCC ACC ACA CAC GAA CCC GCC) fused to that of the first 4
codons of the mature DGL protein ("PPS-first 4 codons of mature
DGL"). The sequence "PPS-first 4 codons of mature DGL" was
amplified by PCR using the plasmid pMAT103 (Matsuoka and Nakamura,
1991) with the aid of the 2 following oligodeoxynucleotides 5'
caggagatctgATG AAA GCC TTC ACA CTC GC 3' and 5' G ATG AAG CTT TCC
AAA CAA GGC GGG TTC GTG TGT GGT TG 3', in accordance with the
protocol of PCR amplification described above in paragraph I. After
double enzymatic digestion by BGlII and HindIII, the DNA fragments
originating from the PCR amplification were purified by
electrophoresis over 2% agarose gel, electroeluted (Sambrook et
al., 1989), precipitated in the presence of {fraction (1/10)}
volume of 3M sodium acetate, pH 4.8, and 2.5 volumes of absolute
ethanol at -80.degree. C. for 30 min, centrifuged at 12,000 g for
30 min, washed with 70% ethanol, dried and then ligated to the
plasmid DNA of pBIOC22, which had been doubly digested by BglII and
HindIII, purified by electrophoresis over 0.8% agarose gel,
electroeluted, subjected to precipitation with alcohol, dried and
dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 100
ng of the dephosphorylated vector described above and 50 ng of the
digested DNA fragments, originating from the PCR amplification,
described above in a reaction medium of 10 .mu.l in the presence of
1 .mu.l of the buffer T4 DNA ligase.times.10 (Amersham) and 2.5 U
of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16
hours. The bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on 50 .mu.g/ml of ampicillin, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes.
[0245] The plasmid DNA of some of the clones retained was verified
by sequencing with the aid of the T.sub.7.TM. sequencing kit,
marketed by Pharmacia, by the dideoxynucleotide method (Sanger et
al., 1977). The sequences of the PPS and the mature DGL were
cloned, maintaining their open-reading frames. The cleavage
sequence between the two sequences is Ala-Leu. The resulting
plasmid was called pBIOC24. Starting from pBIOC24, the BglII-XbaI
fragment carrying the sequence of PPS-DGL was isolated by double
enzymatic digestion by BglII and XbaI, purified by electrophoresis
over 0.8% agarose gel, electroeluted (Sambrook et al., 1989),
precipitated with alcohol and dried. This DNA fragment was then
treated with Klenow enzyme in accordance with the manufacturer's
instructions and ligated to the plasmid DNA of pBIOC21, which had
been digested at the HindIII site, treated with Klenow and
dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 20
ng of the dephosphorylated vector described above and 200 ng of DNA
fragments, containing the PPS-DGL, described above in a reaction
medium of 20 .mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.alpha.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5.alpha., rendered
competent beforehand, were transformed (Hanahan, 1983). The plasmid
DNA of the clones obtained, selected on 12 .mu.g/ml of
tetracycline, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The resulting clone was called pBIOC26. The nucleotide
sequence of the fragment which codes for the recombinant protein
PPS-DGL was verified by sequencing with the aid of the T7
sequencing kit, marketed by Pharmacia, by the dideoxynucleotide
method (Sanger et al., 1977). The plasmid DNA of the binary vector
pBIOC26 was introduced by direct transformation into the strain
LBA4404 of Agrobacterium tumefaciens in accordance with the process
of Holsters et al. (1978). The validity of the clone obtained was
verified by enzymatic digestion of the plasmid DNA introduced.
[0246] c. Construction of the Binary Plasmid pBIOC41 Containing
RGLSP-DGL.
[0247] Rabbit gastric lipase is synthesized in the form of a
precursor composed of a signal peptide of 22 amino acids situated
at the NH.sub.2-terminal end and preceding the polypeptide sequence
of the mature lipase. The clone pJ0101 containing the complete cDNA
which codes for rabbit gastric lipase is described in European
Patent Application no. 92 403 055.4 filed on 12.11.92 by "Institut
de Recherche Jouveinal" and entitled "Nucleic acids which code for
rabbit gastric lipase and polypeptide derivatives, their use for
the production of these polypeptides, and pharmaceutical
compositions based on the latter".
[0248] Alignment of the polypeptide sequences of dog gastric lipase
and of the precursor of rabbit gastric lipase has demonstrated that
the sequence LFGK is present in the two proteins. In the
polypeptide sequence of the rabbit lipase determined from the
purified natural protein (Moreau et al., 1988), the first three
residues L, F and G are absent and form part of the signal peptide
of 22 amino acids of RGL. As a result, the signal peptide of rabbit
gastric lipase devoid of these three common amino acids was fused
to the mature protein sequence of dog gastric lipase. Its
polypeptide sequence is made up of the following 19 amino acids:
MWVLFMVAALLSALGTTHG.
[0249] The plasmid pBIOC22 was thus digested totally by BglII and
partly by HindIII in order to suppress the sequence which codes for
the polypeptide Leu-Phe-Gly-Lys (first 4 amino acids) of the mature
DGL protein. This sequence was replaced by that which codes for the
signal peptide RGLSP of rabbit gastric lipase of 19 amino acids
(ATG TGG GTG CTT TTC ATG GTG GCA GCT TTG CTA TCT GCA CTT GGA ACT
ACA CAT GGT) fused to that of the first 4 codons of the mature DGL
protein ("RGLSP-first 4 codons of mature DGL"). The sequence
"RGLSP-first 4 codons of mature DGL" was amplified by PCR using the
plasmid pJO101 with the aid of the 2 following
oligodeoxynucleotides 5' aggagatctcaacaATG TGG GTG CTT TTC ATG GTG
3' and 5' G ATG AAG CTT TCC AAA CAA ACC ATG TGT AGT TCC AAG TG 3',
in accordance with the protocol of PCR amplification described
above in paragraph I. After double enzymatic digestion by BGlII and
HindIII, the DNA fragments originating from the PCR amplification
were purified by electrophoresis over 2% agarose gel, electroeluted
(Sambrook et al., 1989), precipitated in the presence of {fraction
(1/10)} volume of 3M sodium acetate, pH 4.8, and 2.5 volumes of
absolute ethanol at -80.degree. C. for 30 min, centrifuged at
12,000 g for 30 min, washed with 70% ethanol, dried and then
ligated to the plasmid DNA of pBIOC22, which had been doubly
digested by BglII and HindIII, purified by electrophoresis over
0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected
to precipitation with alcohol, dried and dephosphorylated by the
alkaline phosphatase enzyme of the intestine of the calf
(Boehringer Mannheim) in accordance with the manufacturer's
instructions. The ligation was carried out with 100 ng of the
dephosphorylated vector described above and 50 ng of the digested
DNA fragments, originating from the PCR amplification, described
above in a reaction medium of 10 .mu.l in the presence of 1 .mu.l
of the buffer T4 DNA ligase.times.10 (Amersham) and 2.5 U of the
enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16 hours. The
bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on a medium containing 50 .mu.g/ml of
ampicillin, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The plasmid DNA of some of the clones retained was
verified by sequencing with the aid of the T.sub.7.TM. sequencing
kit, marketed by Pharmacia, by the dideoxynucleotide method (Sanger
et al., 1977). The sequences of the RGLSP and the mature DGL were
cloned, maintaining their open reading frames (that is to say such
that they constitute a unique open reading frame). The cleavage
sequence between the sequences of the RGLSP and the mature DGL is
Gly-Leu. The resulting plasmid was called pBIOC40. Starting from
pBIOC40, the BglII-XbaI fragment carrying the sequence of
RGLSPS-DGL was isolated by double enzymatic digestion by BglII and
XbaI, purification by electrophoresis over 0.8% agarose gel,
electroelution (Sambrook et al., 1989), precipitation with alcohol
and drying. This DNA fragment was then treated with Klenow enzyme
in accordance with the manufacturer's instructions and ligated to
the plasmid DNA of pBIOC21, which had been digested at the HindIII
site, treated with Klenow and dephosphorylated by the alkaline
phosphatase enzyme of the intestine of the calf (Boehringer
Mannheim) in accordance with the manufacturer's instructions. The
ligation was carried out with 20 ng of the dephosphorylated vector
described above and 200 ng of DNA fragments, containing the
RGLSP-DGL, described above in a reaction medium of 20 .mu.l in the
presence of 2 .mu.l of the buffer T4 DNA ligase.times.10
(Amersham), 2 .mu.l of 50% polyethylene glycol 8000 and 5 U of the
enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16 hours. The
bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on a medium containing 12 .mu.g/ml of
tetracycline, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The resulting clone was called pBIOC41. The nucleotide
sequence of the fragments which code for the recombinant protein
RGLSP-DGL was verified by sequencing with the aid of the
T.sub.7.TM. sequencing kit, marketed by Pharmacia, by the
dideoxynucleotide method (Sanger et al., 1977). The plasmid DNA of
the binary vector pBIOC41 was introduced by direct transformation
into the strain LBA4404 of Agrobacterium tumefaciens in accordance
with the process of Holsters et al. (1978). The validity of the
clone retained was verified by enzymatic digestion of the plasmid
DNA introduced.
[0250] I-B) Construction of Chimaeric Genes which Code for the
Recombinant DGL and Allow Expression in the Tomato.
[0251] The constructions used are the same as those used for the
genetic transformation of tobacco, namely the plasmids pBIOC25,
pBIOC26 and pBIOC41.
[0252] II. Construction of Chimaeric Genes which Code FOR THE
Recombinant Protein of Dog Gastric Lipase and Allow Expression in
Rape Seeds.
[0253] a) Construction of the Binary Plasmid pBIOC28 Containing the
Promoter pCRU.
[0254] Expression of dog gastric lipase (DGL) in rape seeds
required the insertion of the cDNA which codes for the DGL between
the following regulator sequences:
[0255] 1. The promoter pCRU which corresponds to the non-coding
region 5' of the gene of the reserve protein of the seeds,
CRUCIFERIN A of radish (Depigny-This et al., 1992), and allows
specific expression in the seeds;
[0256] 2. The terminal transcription sequence, terminator polyA
35S, which corresponds to the non-coding region 3' of the sequence
of the cauliflower mosaic virus, of double-stranded circular DNA,
which produces the transcript 35S (Franck et al., 1980).
[0257] To obtain a binary plasmid similar to pBIOC21, but in which
the promoter pd35S was replaced by the promoter PCRU, the fragment
"EcoRI treated with Klenow-BamHI", containing the promoter pCRU,
was isolated using the plasmid pBI221-CRURSP derived from pBI221
(marketed by Depigny-This et al., 1992.
[0258] The plasmid pBI221-CRURSP is derived from pBI221 (marketed
by Clontech) by replacement of the promoter 35S by the promoter
PCRU.
[0259] The fragment "EcoRI treated with Klenow-BamHI" carrying the
promoter pCRU was purified by electrophoresis over 0.8% agarose
gel, electroeluted (Sambrook et al., 1989), subjected to
precipitation with alcohol, dried and ligated to the plasmid DNA of
pJIT163 (described in paragraph I.), digested by KpnI, which had
been treated with T4 DNA Polymerase (New England Biolabs) in
accordance with the manufacturer's instructions and then digested
with BamHI, purified by electrophoresis over 0.8% agarose gel,
electroeluted (Sambrook et al., 1989), subjected to precipitation
with alcohol, dried and dephosphorylated by the alkaline
phosphatase enzyme of the intestine of the calf (Boehringer
Mannheim) in accordance with the manufacturer's instructions. The
ligation was carried out with 20 ng of the dephosphorylated vector
described above and 200 ng of DNA fragments of "EcoRI treated with
Klenow-BamHI" described above in a reaction medium of 20 .mu.l in
the presence of 2 .mu.l of the buffer T4 DNA ligase.times.10
(Amersham), 2 .mu.l of 50% polyethylene glycol 8000 and 5 U of the
enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16 hours. The
bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on a medium containing 50 .mu.g/ml of
ampicillin, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The resulting plasmid was called pBIOC27.
[0260] The expression cassette made up of the promoter PCRU and the
terminator polyA 35S was isolated using pBIOC27 by total digestion
with XhoI followed by partial digestion with EcoRI. It was purified
by electrophoresis over 0.8% agarose gel, electroeluted (Sambrook
et al., 1989), subjected to precipitation with alcohol, dried,
treated with Klenow (New England Biolabs) in accordance with the
manufacturer's instructions and ligated to the plasmid DNA of
pBIOC24 at the EcoRI site treated with Klenow and dephosphorylated
by the alkaline phosphatase enzyme of the intestine of the calf
(Boehringer Mannheim) in accordance with the manufacturer's
instructions. The ligation was carried out with 20 ng of the
dephosphorylated vector described above and 200 ng of the DNA
fragments XhoI-EcoRI described above in a reaction medium of 20
.mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5, rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on a medium containing 12 .mu.g/ml of
tetracycline, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The resulting plasmid was called pBIOC28.
[0261] b. Construction of the Binary Plasmid pBIOC29 Containing
PPS-DGL.
[0262] Isolation of the BglII-XbaI fragment carrying the sequence
PPS-DGL using pBIOC24 has already been described in I-A-b. This
fragment was ligated to the plasmid DNA of pBIOC28 at the EcoRI
site treated with Klenow and dephosphorylated by the alkaline
phosphatase enzyme of the intestine of the calf (Boehringer
Mannheim) in accordance with the manufacturer's instructions. The
ligation was carried out with 20 ng of the dephosphorylated vector
described above and 200 ng of the DNA fragments BglII-XbaI
containing PPS-DGL in a reaction medium of 20 .mu.l in the presence
of the buffer T4 DNA ligase.times.10 (Amersham), 2 .mu.l of 50%
polyethylene glycol 8000 and 5 U of the enzyme T4 DNA ligase
(Amersham) at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on a medium containing 12 .mu.g/ml of tetracycline, was extracted
by the alkaline lysis method (Birnboim and Doly, 1979) and analysed
by enzymatic digestion by restriction enzymes. The resulting
plasmid was called pBIOC29. The nucleotide sequence of the
recombinant protein PPS-DGL was verified by sequencing with the aid
of the T7 sequencing kit, marketed by Pharmacia, by the
dideoxynucleotide method (Sanger et al., 1977). The plasmid DNA of
the binary vector pBIOC29 was introduced by direct transformation
into the strain LBA4404 of Agrobacterium tumefaciens in accordance
with the process of Holsters et al. (1978). The validity of the
clone retained was verified by enzymatic digestion of the plasmid
DNA introduced.
[0263] c) Construction of Binary Plasmids pBIOC90 and pBIOC91
Containing the pGEA1D Promoter.
[0264] Expression of the animal gene which codes for dog gastric
lipase (DGL) in rape seeds required the following regulator
sequences:
[0265] 1. the promoter pGEA1 corresponding to the non-coding region
5' of the gene of the reserve protein of the seeds, GEA1 of
Arabidopsis thaliana (Gaubier et al., 1993), and allows specific
expression in the seeds;
[0266] 2. the terminal transcription sequence, terminator polyA
35S, which corresponds to the non-coding region 3' of the sequence
of the cauliflower mosaic virus, of double-stranded circular DNA,
which produces the transcript 35S (Franck et al., 1980).
[0267] To obtain the binary plasmid pBIOC90 similar to pBIOC21 but
in which the promoter pd35S was replaced by the promoter pGEA1D,
the fragment HindIII-BamHI treated with Klenow, containing the
promoter pGEA1, was isolated using plasmid pGUS2-pGEA1. The clone
pGUS-2-pGEA1 deriving from pBI221 by replacement of the promoter
p35S by the promoter pGEA1, contains 2 ATG in frame: ATG of the
gene GEA1 (Em2) and ATG of the gene gus. The ATG of the gene GEAL
was destroyed. The DNA fragment contained between the SalI site and
the sequences upstream from the ATG of the gene GEAL of the clone
pGUS-2pGEA1 was then amplified by PCR using 2 oligonucleotides: 5'
CAAACGTGTACAATAGCCC 3' and 5'CCCGGGGATCCTTTTTTG 3'. The
hybridization temperature was adjusted. The fragment amplified by
PCR was digested by SalI and BamHI, purified by electrophoresis
over 2% agarose gel, electroeluted (Sambrook et al. 1989),
precipitated in the presence of {fraction (1/10)} volume of 3M
sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at
-80.degree. C. for 30 minutes, centrifuged at 12000 g for 30
minutes, washed with 70% ethanol, then ligated to plasmid DNA of
pGUS-2-GEA1 double digested by SalI and BamHI, purified by
electrophoresis over 0.8% agarose gel, electoeluted (Sambrook et
al., 1989), subjected to precipitation with alcohol, dried.
Ligation was carried out with 100 ng of vector and 50 ng of
digested DNA fragments originating from the PCR amplification
described above, in a reaction medium of 10 .mu.l in the presence
of 1 .mu.l of the buffer T4 DNA ligase.times.10 (Amersham) and 2.5
U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16
hours. The bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on 50 .mu.g/ml ampicillin, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes. Certain
retained clones were verified by sequencing with the aid of the T7
sequencing kit, marketed by Pharmacia, by the dideoxynucleotide
method (Sanger et al., 1977). The resulting clone was called
pGUS-2-pGEA1D.
[0268] The HindIII-BamHI fragment carrying the promoter pGEA1D
isolated from pGUS-2-pGEA1D, treated with Klenow in accordance with
the manufacturer's instructions (Biolabs), was purified by
electrophoresis over 0.8% agarose gel, electreoluted (Sambrook et
al., 1989), subjected to precipitation with alcohol, dried and
ligated to the plasmid DNA of pBIOC21 at KpnI and HINDIII sites
treated with the enzyme T4 DNA polymerase (Biolabs) in accordance
with the manufacturer's instructions. The ligation was carried out
with 20 ng of dephosphorylated pBIOC21 and 200 ng of the XhoI-EcoRI
DNA fragments described above in a reaction medium of 20 .mu.l in
the presence of 2 .mu.l of the buffer T4 DNA ligase.times.10
(Amersham), 2 .mu.l of 50% polyethylene glycol 8000 and 5 U of the
enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16 hours. The
bacteria Escherichia coli DHSA, rendered competent beforehand, were
transformed (Hanahan, 1983). The plasmid DNA of the clones
obtained, selected on 12 .mu.g/ml tetracyclin, was extracted by the
alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes. The resulting plasmid
was called pBIOC90.
[0269] To obtain the binary plasmid pBIOC91, the expression
cassette "pGEA1D-tNOS", isolated from pBSII-pGEA1D, was introduced
into the binary plasmid pSCV1.2 itself obtained by cloning of the
HindIII fragment carrying the expression cassette "p35S-nptII-tNOS"
described by Fromm et al. (1986) at the HindIII site of pSCV1
constructed by Edwards G. A. in 1990 following the usual cloning
procedures.
[0270] The plasmid pBSII-pGEA1D was obtained in two stages:
[0271] on the one hand, the SacI-EcoRI fragment carrying tNOS
(terminator of the gene of nopaline synthase) of Agrobacterium
tumefaciens, treated with the enzyme T4 DNA polymerase (Biolabs) in
accordance with the manufacturer's instructions and purified, was
cloned at the EcoRV site of pBSIISK+ marketed by Stratagene,
dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 20
ng of the dephosphorylated vector and 200 ng of the DNA fragments
containing tNOS described above in a reaction medium of 20 .mu.l in
the presence of 2 .mu.l of the buffer T4 DNA ligase.times.10
(Amersham), 2 .mu.l of 50% polyethylene glycol 8000 and 5 U of the
enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16 hours. The
bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on 50 .mu.g/ml ampicillin, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes. Certain
retained clones were verified by sequencing with the aid of the
T7.TM. sequencing kit, marketed by Pharmacia, by the
dideoxynucleotide method (Sanger et al., 1977). The resulting
plasmid was called pBSII-tNOS.
[0272] on the other hand, the fragment carrying pGEA1D double
digested by HindIII treated with the enzyme Klenow and BamHI,
purified, was cloned at the "XbaI treated with Klenow and BamHI"
sites of the plasmid pBSII-tNOS. The ligation was carried out with
100 ng of the vector described above and 50 ng of the DNA fragments
described above in a reaction medium of 10 .mu.l in the presence of
1 .mu.l of the buffer T4 DNA ligase.times.10 (Amersham) and 2.5 U
of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16
hours. The bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on 50 .mu.g/ml ampicillin, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes. The
resulting plasmid was called pBSII-pGEAID.
[0273] Then, the expression cassette "pGEA1D--tNOS" carried by the
XbaI-HindIII fragment treated with Klenow, was cloned at the SmaI
site of pSCV1.2 dephosphorylated by the alkaline phosphatase enzyme
of the intestine of the calf (Boehringer Mannheim) in accordance
with the manufacturer's instructions. The ligation was carried out
with 20 ng of dephosphorylated pSCV1.2 and 200 ng of the fragments
carrying the expression cassette "pGEA1D-tNOS", in a reaction
medium of 20 .mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5.alpha., rendered
competent beforehand, were transformed (Hanahan, 1983). The plasmid
DNA of the clones obtained, selected on 50 .mu.g/ml ampicillin, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes. The
resulting plasmid was called pBIOC91.
[0274] d) Construction of Binary Plasmid pBIOC92 Containing the
pGEA6D Promoter.
[0275] Expression of the animal gene which codes for dog gastric
lipase (DGL) in rape seeds required the following regulator
sequences:
[0276] 1. the promoter pGEA6 corresponding to the non-coding region
5' of the gene of the reserve protein of the seeds, GEA6 of
Arabidopsis thaliana (Gaubier et al., 1993), and allows specific
expression in the seeds;
[0277] 2. the terminal transcription sequence, terminator polyA
35S, which corresponds to the non-coding region 3' of the sequence
of the cauliflower mosaic virus, of double-stranded circular DNA,
which produces the transcript 35S (Franck et al., 1980).
[0278] To obtain the binary plasmid pBIOC92 similar to pBIOC21 but
in which the promoter pd35S was replaced by the promoter pGEA6D,
the fragment EcoRI-BamHI treated with Klenow, containing the
promoter pGEA6, was isolated using plasmid pGUS2-pGEA6. The clone
pGUS-2-pGEA6 deriving from pUC18, contains 2 ATG in phase: ATG of
the gene GEA6 (Em6) and ATG of the gene gus. The ATG of the gene
GEA6 was destroyed. The DNA fragment contained between the AccI
site and the sequences upstream from the ATG of the gene GEA6 of
the clone pGUS-2-pGEA6 was then amplified by PCR using 2
oligonucleotides: 5' AAGTACGGCCACTACCACG 3' and 5'CCCGGGGATCCTGGCTC
3'. The hybridization temperature was adjusted.
[0279] The fragment amplified by PCR was digested by AccI and
BamHI, purified by electrophoresis over 2% agarose gel,
electroeluted (Sambrook et al. 1989), precipitated in the presence
of {fraction (1/10)} volume of 3M sodium acetate pH 4.8 and 2.5
volumes of absolute ethanol at -80.degree. C. for 30 minutes,
centrifuged at 12000 g for 30 minutes, washed with 70% ethanol,
dried, then ligated to plasmid DNA of pGUS-2-GEA6 double digested
by AccI and BamHI, purified by electrophoresis over 0.8% agarose
gel, electoeluted (Sambrook et al., 1989), subjected to
precipitation with alcohol, dried. Ligation was carried out with
100 ng of the vector described above and 50 ng of digested DNA
fragments originating from the PCR amplification described above,
in a reaction medium of 10 .mu.l in the presence of 1 .mu.l of the
buffer T4 DNA ligase.times.10 (Amersham) and 2.5 U of the enzyme T4
DNA ligase (Amersham) at 14.degree. C. for 16 hours. The bacteria
Escherichia coli DH5.alpha., rendered competent beforehand, were
transformed (Hanahan, 1983). The plasmid DNA of the clones
obtained, selected on 50 .mu.g/ml ampicillin, was extracted by the
alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes. Certain retained clones
were verified by sequencing with the aid of the T.sub.7.TM.
sequencing kit, marketed by Pharmacia, by the dideoxynucleotide
method (Sanger et al., 1977). The resulting clone was called
pGUS-2-pGEA6D.
[0280] The EcoRI-BamHI fragment carrying the promoter pGEA6D
isolated from pGUS-1-pGEA6D, treated with Klenow in accordance with
the manufacturer's instructions (Biolabs), was purified by
electrophoresis over 0.8% agarose gel, electroluted (Sambrook et
al., 1989), subjected to precipitation with alcohol, dried and
ligated to the plasmid DNA of modified pBIOC21 at the XhoI site
treated with Klenow. The modified plasmid pBIOC21 was obtained by
double digestion of pBIOC21 by HindIII treated with Klenow and KpnI
to delete the fragment carrying the promoter pd35S and to replace
it with the KpnIEcoRV fragment carrying the compound polylinker of
sites KpnIXhoI-SalI-ClaL-HindIII-BamHI-SmaI-EcoRI-EcoRV of
pBSIISK+.
[0281] The ligation was carried out with 20 ng of dephosphorylated
modified pBIOC21 and 200 ng of the EcoRI-BamHI DNA fragments
described above in a reaction medium of 20 .mu.l in the presence of
2 .mu.l of the buffer T4 DNA ligase.times.10 (Amersham), 2 .mu.l of
50% polyethylene glycol 8000 and 5 U of the enzyme T4 DNA ligase
(Amersham) at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983) The plasmid DNA of the clones obtained, selected on
12 .mu.g/ml tetracyclin, was extracted by the alkaline lysis method
(Birnboim and Doly, 1979) and analysed by enzymatic digestion by
restriction enzymes. The resulting plasmid was called pBIOC92.
[0282] e) Construction of Binary Plasmids pBIOC93, pBIOC94,
pBIOC95, pBIOC96 and pBIOC97 Containing RGLSP-DGL.
[0283] The plasmid pBIOC40 is described above. This plasmid
contains the fragment BglII-XbaI carrying the sequence
RGLSPDGL.
[0284] This fragment was isolated by double enzymatic digestion by
BglII and XbaI, purified by electrophoresis over 0.8% agarose gel,
electroluted, subjected to precipitation with alcohol, dried,
treated with Klenow and ligated to pBIOC28 (described above)
digested by EcoRI treated with Klenow and dephosphorylated; to
pBIOC90 digested by EcoRI treated with Klenow and dephosphorylated;
to pBIOC91 digested by SmaI and dephosphorylated; to pBIOC92
digested by HindIII treated with Klenow and dephosphorylated to
produce pBIOC93, pBIOC94, pBIOC95 and pBIOC96 respectively.
[0285] The plasmid pBIOC97 results from the cloning of the fragment
KpnI-EcoRV carrying the expression cassette "pGEA6D25
RGLSP-DGL-t35S", treated by the enzyme T4 DNA polymerase, purified
by electrophoresis over 0.8% agarose gel, electroeluted, subjected
to precipitation with alcohol, dried and ligated to pSCV1.2
digested by SmaI and dephosphorylated. The expression cassette
"pGEAGD-RGLSP-DGL-t35S" originates from pBIOC92.
[0286] III. Construction of Chimaeric genes which Code for the
Recombinant Protein of Human Gastric Lipase and Allow Constitutive
Expression, for Example, in Tobacco Leaves and Seeds.
[0287] a) Construction of the Binary Plasmid pBIOC82 Containing the
Chimaeric Promoter SUPER-PROMOTER pSP.
[0288] Expression of the gene which codes for human gastric lipase
(HGL) in tobacco leaves required the following regulator
sequences:
[0289] 1. the chimaeric promoter super-promoter (pSP;
PCT/US94/12946). It is constituted by the fusion of three
transcriptional activator elements of the promoter of the gene of
octopine synthase of Agrobacterium tumefaciens, of a
transcriptional activator element of the promoter of the gene of
mannopine synthase and of the mannopine synthase promoter of
Agrobacterium tumefaciens;
[0290] 2. the terminal transcription sequence, terminator polyA
35S, which corresponds to the non-coding region 3' of the sequence
of the cauliflower mosaic virus of double-stranded circular DNA,
which produces the transcript 35S (Franck et al., 1980).
[0291] To obtain a binary plasmid similar to pBIOC21, but in which
the promoter pd35S was replaced by the promoter pSP, the pvuII-SalI
fragment, subjected to Klenow, containing the promoter pSP, was
isolated using the plasmid pBISNI (PCT/US94/12946), purified by
electrophoresis over 1% agarose gel, electroeluted (Sambrook et
al., 1989), subjected to precipitation with alcohol, dried and
ligated to the plasmid DNA of pBIOC81, doubly digested by KpnI and
EcoRI, subjected to DNA T4 polymerase and dephosphorylated by the
alkaline phosphatase enzyme of the intestine of the calf
(Boehringer Mannheim) in accordance with the manufacturer's
instructions.
[0292] The plasmid pBIOC81 corresponds to pBIOC21 the XbaI site of
which has been deleted. In order to do this, the plasmid pBIOC21
was digested by XbaI, then subjected to the action of the Klenow
enzyme and ligated by the action of T4 DNA ligase.
[0293] The ligation was carried out with 20 ng of the
dephosphorylated vector described above and 200 ng of DNA fragments
carrying pSP described above in a reaction medium of 20 .mu.l in
the presence of 2 .mu.l of the buffer T4 DNA ligase.times.10
(Amersham), 2 .mu.l 50% polyethylene glycol 8000 and 5 U of the
enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16 hours. The
bacteria Escherichia coli DH5, rendered competent beforehand, were
transformed (Hanahan, 1983). The plasmid DNA of the clones
obtained, selected on 12 .mu.g/ml of tetracyclin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes. The resulting plasmid
was called pBIOC82.
[0294] Human gastric lipase (HGL) is synthesized naturally in the
form of a precursor 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. The
restriction site between HGLSP and HGL is Gly-Leu.
[0295] The sequence which codes for the precursor of HGL was used
for the construction of the binary plasmids pBIOC85 containing
HGLSP-HGL, pBIOC87 containing HPLSP-HGL and pBIOC89 containing
RGLSP-HGL where the sequence which codes for HGL is preceded by
those which code for its natural signal peptide HGLSP, the signal
peptide of human pancreatic lipase (HPLSP; Giller et al., 1992) and
the signal peptide of rabbit gastric lipase (RGLSP; already
described previously; European Patent No. 92.403055.4)
respectively.
[0296] The sequence which codes for the precursor of HGL was
isolated by double digestion with PstI and DraI, purified by
electrophoresis over 0.8% agarose gel, electroeluted (Sambrook et
al., 1989), precipitated in the presence of {fraction (1/10)}
volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute
ethanol at minus 80.degree. C. for 30 minutes, centrifuged at 12000
g for 30 minutes, washed with 70% ethanol and dried. Then, it was
cloned at the PstI and SpeI sites (subjected to the action of the
enzyme T4 DNA polymerase (Biolabs) in accordance with the
manufacturer's instructions) of the plasmid pBSIISK+ marketed by
Stratagene. The ligation was carried out with 100 ng of the vector
and 50 ng of the DNA fragments carrying the sequence which codes
for the precursor of HGL described above, in a reaction medium of
10 .mu.l in the presence of 1 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham) and 2.5 U of the enzyme T4 DNA ligase
(Amersham) at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on 50 .mu.g/ml of ampicillin, was extracted by the alkaline lysis
method (Birnboim and Doly, 1979) and analysed by enzymatic
digestion by restriction enzymes. The resulting plasmid was called
pBIOC83.
[0297] The sequence which codes for mature HGL was modified by the
introduction of a BamHI site, which did not exist beforehand, in
the ninth and tenth codons, by directed mutagenesis by PCR using 2
oligodeoxynucleotides, 5' aaactgcaggctcgag TTG TTT GGA AAA TTA CAT
CCT GGA tcc CCT GAA GTG ACT ATG 3' (containing the unique PstI,
XhoI and BamHI sites) and 5' AAT GGT GGT GCC CTG GGA ATG GCC AAC
ATA GTG TAG CTG C 3' (containing the unique MscI site in the
plasmid pBIOC83).
[0298] The PCR amplification of the fragment PstI-MscI was carried
out in 100 pl of reaction medium containing 10 .mu.l of the buffer
Taq DNA polymerase.times.10 (500 mM KCl, 100 mM TrisHCl, pH 9.0 and
1 Triton.times.100), 6 .mu.l of 25 mM MgCl.sub.2, 3 .mu.l of 10 mM
dNTP (DATP, dCTP, dGTP and dTTP), 100 pM of each of the 2
oligodeoxy-nucleotides described above, 5 ng of matrix DNA (vector
pBIOC83), 2.5 U of Tag DNA polymerase (Promega) and 2 drops of
vaseline oil. The DNA was denatured at 94.degree. C. for S minutes,
subjected to 30 cycles each constituted by 1 minute of denaturation
at 94.degree. C., 1 minute of hybridization at 65.degree. C. and 1
minute of elongation at 72.degree. C., then elongation at
72.degree. C. was continued for 5 minutes. This PCR reaction was
carried out in the "DNA Thermal Cycler" machine of PERKIN ELMER
CETUS. The oil was removed by extraction with chloroform. The DNA
fragments contained in the reaction medium were then precipitated
in the presence of {fraction (1/10)} volume of 3M sodium acetate,
pH 4.8, and 2.5 volumes of absolute ethanol at minus 80.degree. C.
for 30 min, centrifuged at 12,000 g for 30 min, washed with 70%
ethanol, dried and digested by the 2 restriction enzymes PstI and
MscI. The digested DNA fragments originating from the PCR
amplification were purified by electrophoresis over 2% agarose gel,
electroeluted (Sambrook et al., 1989), precipitated in the presence
of {fraction (1/10)} volume of 3M sodium acetate, pH 4.8, and 2.5
volumes of absolute ethanol at -80.degree. C. for 30 min,
centrifuged at 12,000 g for 30 min., washed with 70% ethanol, dried
and then ligated to the plasmid DNA of pBIOC83, which had been
digested twice by PstI and MscI, purified by electrophoresis over
0.8% agarose gel, electroeluted, subjected to precipitation with
alcohol, dried. The ligation was carried out with 100 ng of the
vector and 50 ng of the digested DNA fragments, originating from
the amplification by PCR, described above, in a reaction medium of
10 .mu.l in the presence of 1 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham) and 2.5 U of the enzyme T4 DNA ligase
(Amersham) at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on 50 .mu.g/ml of ampicillin, was extracted by the alkaline lysis
method (Birnboim and Doly, 1979) and analysed by enzymatic
digestion by restriction enzymes. Some of the retained clones were
verified by sequencing with the aid of the T7 sequencing kit,
marketed by Pharmacia, by the dideoxynucleotide method (Sanger et
al., 1977). Introduction of the BamHI restriction site does not
modify the genetic code of the HGL. In fact, the natural HGL
sequence GGA AGC (Gly-Ser) becomes GGA TCC (GlySer). The resulting
plasmid was called pBIOC84.
[0299] b) Construction of the Binary Plasmid pBIOC85 Containing
HGLSP-HGL.
[0300] The fragment PstI-XbaI carrying the sequence of HGLSP-HGL
was isolated by double enzymatic digestion by PstI and XbaI from
pBIOC83, purified by electrophoresis over 0.8% agarose gel,
electroeluted (Sambrook et al., 1989), subjected to precipitation
with alcohol and dried. Then, this DNA fragment was treated with
the enzyme T4 DNA polymerase (Biolabs) in accordance with the
manufacturer's instructions and ligated to the plasmid DNA of
pBIOC82 digested at the EcoRI site, treated with Klenow (Biolabs)
and dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 20
ng of the dephosphorylated vector described above and 200 ng of DNA
fragments containing the HGLSP-HGL described above, in a reaction
medium of 20 .mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5.alpha., rendered
competent beforehand, were transformed (Hanahan, 1983). The plasmid
DNA of the clones obtained, selected on 12 .mu.g/ml of
tetracycline, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. The resulting clone was called pBIOC85. The nucleic
sequence of the fragment which codes for the recombinant protein
HGLSP-HGL was verified by sequencing with the aid of the T7
sequencing kit, marketed by Pharmacia, by the dideoxynucleotide
method (Sanger et al., 1977). The restriction sequence between the
HGLSP and mature HGL sequences is Gly-Leu. The plasmid DNA of the
binary vector pBIOC85 was introduced by direct transformation into
the strain LBA4404 of Agrobacterium tumefaciens in accordance with
the method of Holsters et al. (1978). The validity of the retained
clone was verified by enzymatic digestion of the plasmid DNA
introduced.
[0301] c) Construction of the Binary Plasmid pBIOC86 Containing
HPLSP-HGL.
[0302] The plasmid pBIOC84 was digested twice by PstI and BamHI in
order to suppress the sequence which codes for the signal peptide
HGLSP and the first 8 amino acids of the mature HGL protein
(Leu-Phe-Gly-Lys-Leu-His-Pr- o-Gly). This sequence was replaced by
that which codes for the signal peptide HPLSP of 16 amino acids
(ATG CTG CCA CTT TGG ACT CTT TCA CTG CTG CTG GGA GCA GTA GCA GGA)
fused to that which codes for the first 8 codons of the mature HGL
protein ("HPLSP--first 8 codons of mature HGL"). The sequence
"HPLSP--first 8 codons of mature HGL" was amplified by PCR from the
matrix 5' aaactgcaggctcgagaacaATG CTG CCA CTT TGG ACT CTT TCA CTG
CTG CTG GGA GCA GTA GCA GGA TTG TTT GGA AAA TTA CAT CCT GGA tcc CCT
G 3' using the 2 oligodeoxynucleotides, 5' aaactgcaggctcgagaacaATG
C 3' and 5.degree. C. AGG gga TCC AGG ATG TAA TTT TCC 3', following
the PCR amplification protocol described previously (see paragraph
I above). The hybridization temperature was adjusted. After double
enzymatic digestion by PstI and BamHI, the DNA fragments
originating from the PCR amplification were purified by
electrophoresis over 2% agarose gel, electroeluted (Sambrook et al.
1989), precipitated in the presence of {fraction (1/10)} volume of
3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at
-80.degree. C. for 30 minutes, centrifuged at 12000 g for 30
minutes, washed with 70% ethanol, dried, then ligated with plasmid
DNA of pBIOC84 double digested by PstI and BamHI, purified by
electrophoresis over 0.8% agarose gel, electoeluted (Sambrook et
al., 1989), subjected to precipitation with alcohol, dried.
Ligation was carried out with 100 ng of vector and 50 ng of
digested DNA fragments originating from the PCR amplification
described above, in a reaction medium of 10 .mu.l in the presence
of 1 .mu.l of the buffer T4 DNA ligase.times.10 (Amersham) and 2.5
U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16
hours. The bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on 50 .mu.g/ml ampicillin, was
extracted by the alkaline lysis method (Birnboim and Doly, 1979)
and analysed by enzymatic digestion by restriction enzymes. Certain
retained clones were verified by sequencing with the aid of the T7
sequencing kit, marketed by Pharmacia, by the dideoxynucleotide
method (Sanger et al., 1977). The sequences of HPLSP and mature HGL
were cloned while maintaining their open reading frames (that is to
say, such that they constitute a unique open reading frame). The
restriction sequence between the sequences HPLSP and mature HGL is
Gly-Leu. The resulting plasmid was called pBIOC86.
[0303] The fragment PstI-XbaI carrying the sequence HPLSP-HGL was
isolated from BIOC86 by double enzymatic digestion by PstI and
XbaI, purified by electrophoresis over 0.8% agarose gel,
electroeluted (Sambrook et al., 1989), subjected to precipitation
with alcohol and dried. Then, this DNA fragment was treated with
the enzyme T4 DNA polymerase (Biolabs) in accordance with the
manufacturer's instructions and ligated to the plasmid DNA of
pBIOC82 digested at the EcoRI site, treated with Klenow (Biolabs)
and dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 20
ng of the dephosphorylated vector described above and 200 ng of DNA
fragments containing the HPLSP-HGL described above in a reaction
medium of 20 .mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5.alpha., rendered
competent beforehand, were transformed (Hanahan, 1983). The plasmid
DNA of the clones obtained, selected on a medium containing 12
.mu.g/ml of tetracyline, was extracted by the alkaline lysis method
(Birnboim and Doly, 1979) and analysed by enzymatic digestion by
restriction enzymes. The resulting clone was called pBIOC87. The
nucleic sequence of the fragment which codes for the recombinant
protein HPLSP-HGL was verified by sequencing with the aid of the
T7.TM. sequencing kit, marketed by Pharmacia, by the
dideoxynucleotide method (Sanger et al., 1977). The plasmid DNA of
the binary vector pBIOC87 was introduced by direct transformation
in the strain LBA4404 of Agrobacterium tumefaciens in accordance
with the method of Holsters et al. (1978). The validity of the
retained clone was verified by enzymatic digestion of the plasmid
DNA introduced.
[0304] d) Construction of the Binary Plasmid pBIOC89 Containing
RGLSP-HGL.
[0305] The plasmid pBIOC84 was double digested by PstI and BamHI in
order to suppress the sequence which codes for the signal peptide
HGLSP and the first 8 amino acids of the mature HGL protein
(Leu-Phe-Gly-Lys-Leu-His-Pr- o-Gly). This sequence was replaced by
that which codes for the signal peptide RGLSP of 19 amino acids
(ATG TGG GTG CTT TTC ATG GTG GCA GCT TTG CTA TCT GCA CTT GGA ACT
ACA CAT GGT) fused to that which codes for the first 8 codons of
the mature HGL protein ("RGLSP--first 8 codons of mature HGL"). The
sequence "RGLSP--first 8 codons of mature HGL" was amplified by PCR
from the matrix 5' aaactgcaggctcgagaacaATG TGG GTG CTT TTC ATG GTG
GCA GCT TTG CTA TCT GCA CTT GGA ACT ACA CAT GGTTTG TTT GGA AAA TTA
CAT CCT GGA tcc CCT G 3' using the 2 oligodeoxynucleotides, 5'
aaactgcaggctcgagaacaATG TGG 3' and 5.degree. C. AGG gga TCC AGG ATG
TAA TTT TCC 3', following the PCR amplification protocol described
previously (see paragraph I above). The hybridization temperature
was adjusted. After double enzymatic digestion by PstI and BamHI,
the DNA fragments originating from the PCR amplification were
purified by electrophoresis over 2% agarose gel, electroeluted
(Sambrook et al. 1989), precipitated in the presence of {fraction
(1/10)} volume of 3M sodium acetate pH 4.8 and 2.5 volumes of
absolute ethanol at -80.degree. C. for 30 minutes, centrifuged at
12000 g for 30 minutes, washed with 70% ethanol, dried, then
ligated with plasmid DNA of pBIOC84 double digested by PstI and
BamHI, purified by electrophoresis over 0.8% agarose gel,
electoeluted (Sambrook et al., 1989), subjected to precipitation
with alcohol, dried. Ligation was carried out with 100 ng of vector
and 50 ng of digested DNA fragments originating from the PCR
amplification described above, in a reaction medium of 10 .mu.l in
the presence of 1 .mu.l of the buffer T4 DNA ligase.times.10
(Amersham) and 2.5 U of the enzyme T4 DNA ligase (Amersham) at
14.degree. C. for 16 hours. The bacteria Escherichia coli DH5,
rendered competent beforehand, were transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected on 50 .mu.g/ml
ampicillin, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes. Certain retained clones were verified by sequencing with
the aid of the T7 sequencing kit, marketed by Pharmacia, by the
dideoxynucleotide method (Sanger et al., 1977). The sequences of
RGLSP and mature HGL were cloned while maintaining their open
reading frames (that is to say, such that they constitute a unique
open reading frame). The restriction sequence between the sequences
RGLSP and mature HGL is Gly-Leu. The resulting plasmid was called
pBIOC88.
[0306] The fragment PstI-XbaI carrying the sequence RGLSP-HGL was
isolated from pBIOC88 by double enzymatic digestion by PstI and
XbaI, purified by electrophoresis over 0.8% agarose gel,
electroeluted (Sambrook et al., 1989), subjected to precipitation
with alcohol and dried. Then, this DNA fragment was treated with
the enzyme T4 DNA polymerase (Biolabs) in accordance with the
manufacturer's instructions and ligated to the plasmid DNA of
pBIOC82 digested at the XbaI site, treated with Klenow (Biolabs)
and dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 20
ng of the dephosphorylated vector and 200 ng of DNA fragments
containing the RGLSP-HGL described above in a reaction medium of 20
.mu.l in the presence of 2 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham), 2 .mu.l of 50% polyethylene glycol 8000
and 5 U of the enzyme T4 DNA ligase (Amersham) at 14.degree. C. for
16 hours. The bacteria Escherichia coli DH5.alpha., rendered
competent beforehand, were transformed (Hanahan, 1983). The plasmid
DNA of the clones obtained, selected on 12 .mu.g/ml of tetracyline,
was extracted by the alkaline lysis method (Birnboim and Doly,
1979) and analysed by enzymatic digestion by restriction enzymes.
The resulting clone was called pBIOC89. The nucleic sequence of the
fragment which codes for the recombinant protein RGLSP-HGL was
verified by sequencing with the aid of the T7.TM. sequencing kit,
marketed by Pharmacia, by the dideoxynucleotide method (Sanger et
al., 1977). The plasmid DNA of the binary vector pBIOC89 was
introduced by direct transformation in the strain LBA4404 of
Agrobacterium tumefaciens in accordance with the method of Holsters
et al. (1978). The validity of the retained clone was verified by
enzymatic digestion of the plasmid DNA introduced.
[0307] IV. Construction of Chimaeric Genes which Code for the
Recombinant Protein of Dog Gastric Lipase and Allow Expression in
Corn Seeds.
[0308] a) Construction of Plasmids pBIOC98 and pBIOC99 Containing
RGLSP-DGL and Allow Constitutive Expression in Corn Seeds.
[0309] The constitutive expression of the animal gene which codes
for dog gastric lipase (DGL) in corn seeds required the following
regulator sequences:
[0310] 1. one of two promoters which allow constitutive
expression:
[0311] actine promoter of rice followed by the actine intron of
rice (pAR-IAR) contained in the plasmid pAct1-F4 described by
McElroy et al. (1991);
[0312] double constitutive promoter 35S (pd35S) of CaMV
(cauliflower mosaic virus). It corresponds to a duplication of
sequences activating the transcription situated upstream from the
TATA element of the natural 35S promoter (Kay et al., 1987);
[0313] 2. one of two terminators:
[0314] the terminal transcription sequence, terminator polyA 35S,
which corresponds to the non-coding region 3' of the sequence of
the cauliflower mosaic virus, of double-stranded circular DNA,
which produces the transcript 35S (Franck et al., 1980);
[0315] the terminal transcription sequence, terminator polyA NOS,
which corresponds to the non-coding region 3' of the gene of
nopaline synthase of plasmid Ti of Agrobacterium tumefaciens
nopaline strain (Depicker et al., 1982).
[0316] The plasmid pBIOC98 where the sequence which codes for
RGLSP-DGL is placed under the control of pAR-IAR was obtained by
cloning the fragment BglII-XbaI carrying the sequence which codes
for RGLSP-DGL at the sites "NcoI and SalI" of
pBSII-pARIAR-tNOS.
[0317] The fragment BglII-XBaI carrying the sequence which codes
for RGLSP-DGL was isolated from pBIOC40 (described above) by double
enzymatic digestion by BglII and XbaI, purified by electrophoresis
over 0.8% agarose gel, electroeluted, subjected to precipitation
with alcohol, dried, then treated with Klenow enzyme. The plasmid
pBSII-pAR-IAR-tNOS was double digested by SalI and NcoI, purified,
treated with Mung Bean Nuclease enzyme (Biolabs) and
dephosphorylated by the alkaline phosphatase enzyme of the
intestine of the calf (Boehringer Mannheim) in accordance with the
manufacturer's instructions. The ligation was carried out with 20
ng of the dephosphorylated vector and 200 ng of DNA fragments
containing the sequence which codes for RGLSP-DGL described, above
in a reaction medium of 20 .mu.l in the presence of 2 .mu.l of the
buffer T4 DNA ligase.times.10 (Amersham), 2 .mu.l of 50%
polyethylene glycol 8000 and 5 U of the enzyme T4 DNA ligase
(Amersham)-at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on a medium containing 50 .mu.g/ml of ampicillin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes. The resulting plasmid
was called pBIOC98.
[0318] The plasmid pBSII-pAR-IAR-tNOS results from the cloning at
sites "Eco01091 treated with Klenow and KpnI" of pBSII-tNOS of the
fragment SnaBI-KpnI carrying the sequence corresponding to
"pAR-IAR-start of the sequence which codes for the gene gus"
isolated from the plasmid pAct1-F4. The ligation was carried out
with 100 ng of the vector and 50 ng of DNA fragments described
above in a reaction medium of 10 .mu.l in the presence of 1 .mu.l
of the buffer T4 DNA ligase.times.10 (Amersham) and 2.5 U of the
enzyme T4 DNA ligase (Amersham) at 14.degree. C. for 16 hours. The
bacteria Escherichia coli DH5.alpha., rendered competent
beforehand, were transformed (Hanahan, 1983). The plasmid DNA of
the clones obtained, selected on a medium containing 50 .mu.g/ml of
ampicillin, was extracted by the alkaline lysis method (Birnboim
and Doly, 1979) and analysed by enzymatic digestion by restriction
enzymes.
[0319] The plasmid pBSII-tNOS was obtained by cloning at the
dephosphorylated site EcoRV of pBSIISK+ marketed by Stratagene, of
the fragment SacI-EcoRI carrying the sequence tNOS isolated from
pBI121 marketed by Clontech by double enzymatic digestion by SacI
and EcoRV, subjected to purification by electrophoresis over 2%
agarose gel and treated with the enzyme T4 DNA polymerase. The
ligation was carried out with 20 ng of the dephosphorylated vector
and 200 ng of DNA fragments containing the sequence tNOS described
above, in a reaction medium of 20 .mu.l in the presence of 2 .mu.l
of the buffer T4 DNA ligase.times.10 (Amersham), 2 .mu.l of 50%
polyethylene glycol 8000 and 5 U of the enzyme T4 DNA ligase
(Amersham) at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on a medium containing 50 .mu.g/ml of ampicillin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes.
[0320] The plasmid pBIOC99 where the sequence which codes for
RGLSP-DGL is placed under the control of pd35S was obtained by
cloning at sites "KpnI and BamHI" of the plasmid pBSII-t35S, of the
fragment KpnI-BamHI carrying the sequence corresponding to
"pd35S-RGLSP-DGL" isolated from pBIOC41 described above. The
ligation was carried out with 100 ng of the vector and 50 ng of DNA
fragments described above in a reaction medium of 10 .mu.l digested
in the presence of 1 ml of the buffer T4 DNA ligase.times.10
(Amersham) and 2.5 U of the enzyme T4 DNA ligase (Amersham) at
14.degree. C. for 16 hours. The bacteria Escherichia coli
DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on a medium containing 50 .mu.g/ml of ampicillin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes.
[0321] The plasmid pBSII-t35S was obtained by cloning at the
dephosphorylated site SpeI treated with Klenow, of plasmid pBSIISK+
marketed by Stratagene, of the fragment SmaI-EcoRV carrying the
sequence t35S isolated from pJIT163 (described above) by double
enzymatic digestion by SmaI and EcoRV, subjected to purification by
electrophoresis over 2% agarose gel. The ligation was carried out
with 20 ng of the dephosphorylated vector and 200 ng of DNA
fragments containing the sequence t35S described above, in a
reaction medium of 20 .mu.l in the presence of 2 .mu.l of the
buffer T4 DNA ligase.times.10 (Amersham), 2 .mu.l of 50%
polyethylene glycol 8000 and 5 U of the enzyme T4 DNA ligase
(Amersham) at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on a medium containing 50 .mu.g/ml of ampicillin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes.
[0322] b) Construction of Plasmids pBIOC100 and pBIOC101 Containing
RGLSP-DGL and RGLSP-DGL-KDEL Respectively and Allow Expression in
the Albumen of Corn Seeds.
[0323] The expression of the animal gene which codes for dog
gastric lipase (DGL) in the albumen of corn seeds required the
following regulator sequences:
[0324] 1. the gene promoter of yzeine of corn (p.gamma.zeine)
contained in the plasmid py63 described in Reina et al., 1990. The
plasmid py63 results from the cloning of p.gamma.zeine at the
HindIII and XbaI sites of a plasmid pUC18 containing, between its
HindIII and EcoRI sites, the expression cassette "p35S-gustNOS" of
pBI221 marketed by Clontech. It allows expression in the albumen of
corn seeds.
[0325] 2. the terminal transcription sequence, terminator polyA
NOS, which corresponds to the non-coding region 3' of the gene of
nopaline synthase of the plasmid Ti of Agrobacterium tumefaciens
nopaline strain (Depicker et al., 1982).
[0326] The plasmid pBIOC100 where the sequence which codes for
RGLSP-DGL is placed under the control of p.gamma.zeine was obtained
by cloning at the sites "SacI treated by the enzyme T4 DNA
polymerase and BamHI" of the plasmid py63, of the fragment "XbaI
treated with Klenow--BglII" isolated from pBIOC40 (described
above). The ligation was carried out with 100 ng of the vector and
50 ng of DNA fragments described above in a reaction medium of 10
.mu.l in the presence of 1 .mu.l of the buffer T4 DNA
ligase.times.10 (Amersham) and 2.5 U of the enzyme T4 DNA ligase
(Amersham) at 14.degree. C. for 16 hours. The bacteria Escherichia
coli DH5.alpha., rendered competent beforehand, were transformed
(Hanahan, 1983). The plasmid DNA of the clones obtained, selected
on a medium containing 50 .mu.g/ml of ampicillin, was extracted by
the alkaline lysis method (Birnboim and Doly, 1979) and analysed by
enzymatic digestion by restriction enzymes. The resulting clone was
called pBIOC100.
[0327] The plasmid pBIOC101 results from the substitution of
fragment NcoI-AflII of pBIOC100 by the fragment NcoI-AflII carrying
the sequence which codes for the tetrapeptide KDEL (allowing
directing in the endoplasmic reticulum) placed before the stop
codon obtained by PCR amplification according to the processes
described above. The 2 oligodeoxynucleotides used during this
reaction were: 5' AAT CAC TTG GAC TTT ATC TGG Gcc atg gAT GCC 3'
(unique NcoI site) and 5' ATT ctt aag AAA CTT TAT TGC CAA ATG TTT
GAA CGA TCG GGG AAA TTC GAC GCG TCT AGA ACT ATA GCT CAT CCT TAT TAT
CTG TTC CCA TCA TGG 3' (the sequence which codes for KDEL and a
unique AflII site). The hybridization temperature was 70.degree. C.
The cloning was carried out as described previously.
[0328] V. Example of the Production of Transgenic Rape Plants
[0329] Seeds of spring rape (Brassica napus cv WESTAR or Limagrain
stock) are disinfected for 40 minutes in a 15% solution of
Domestos. After rinsing 4 times with sterile water, the seeds are
germinated in an amount of 20 seeds per pot of 7 cm diameter and 10
cm height on a mineral medium of Murashige and Skoog (Sigma M 5519)
with 30 g/l of sucrose, solidified with 5 g/l of agar gel. These
pots are placed in a growing room at 26.degree. C. with a
photoperiod of 16 h/8 h under a luminous intensity of the order of
80 .mu.m.sup.-2 s.sup.-1.
[0330] After 5 days of germination, the cotyledons are removed
under sterile conditions by cutting each petiole about 1 mm above
the cotyledonary node.
[0331] In parallel, Agrobacterium tumefaciens, strain LBA4404,
containing the plasmid pBIOC29 (or pBIOC25), that is to say the
plasmid pGAZE into which has been inserted the sequence which codes
for dog gastric lipase fused to that which codes for a directing
signal PPS (or PS), under control of the promoter pCRU (or pd35S),
is precultured in a conical flask of 50 ml for 36 h at 28.degree.
C. in 10 ml of bacterial medium 2YT (Sambrook et al., 1989),
supplemented with the antibiotics which can be used for selection
of the strain used.
[0332] This preculture is used to seed, in an amount of 1%, a new
bacterial culture prepared under the same conditions. After 14 h,
the culture is centrifuged for 15 min at 3,000 g and the bacteria
are taken up in an equivalent volume of the liquid germination
medium. This suspension is divided among Petri dishes of 5 cm
diameter in an amount of 5 ml/dish.
[0333] The cut end of the petiole is immersed for a few seconds in
the agrobacterial solution thus prepared and the petiole is then
pushed a few millimeters into the regeneration medium. This medium
has the same base composition as the germination medium, with an
addition 4 mg/l of benzyl-amino-purine (BAP), a phytohormone which
promotes neoformation of buds. Ten explants (cotyledon with
petiole) are grown per Petri dish of 9 cm diameter (Greiner
reference 664102).
[0334] After 2 days of coculture under the same environmental
conditions as the germination, the explants are planted out into
Phytatray boxes (Sigma, reference P1552) containing the previous
medium supplemented with a selective agent: 45 mg/l of kanamycin
sulphate (Sigma, reference K4000) and a bacteriostatic: mixture of
1/6 (by weight) of the potassium salt of clavulanic acid and 5/6 of
the sodium salt of amoxicillin (Augmentin, injectable) in an amount
of 600 mg/l.
[0335] The explants are planted out under sterile conditions on new
medium under the same conditions on two subsequent occasions, at an
interval of 3 weeks.
[0336] The green buds which have appeared at the end of the second
or third planting out are separated from the explant and grown
individually in transparent pots of 5 cm diameter and 10 cm height
containing a medium identical to the previous but devoid of BAP.
After growing for 3 weeks, the stem of the transformed bud is cut
and the bud is planted out in a pot of fresh medium. At the end of
three to four weeks, the roots are sufficiently developed to allow
acclimatization of the plantlet in a phytotron. The buds which are
not green or have not taken root are removed. These plantlets are
then transplanted into bowls with 7 cm sides filled with soil
(standard NF U44551: 40% brown peat, 30% sifted heath and 30% sand)
saturated with water. After two weeks of acclimatization in the
phytotron (temperature 21.degree. C., photoperiod 16 h/8 h and 84%
relative humidity), the plantlets are repotted in pots of 12 cm
diameter filled with the same soil enriched with slowacting
fertilizer (Osmocote, in an amount of 4 g/l of soil) and then
transferred to a greenhouse (class S2) regulated at 18.degree. C.
with two daily waterings with water for 2 minutes.
[0337] When the flowers appear, these are placed in bags (Crispac,
reference SM 570.times.300 mm*700 mm) in a manner such that
cross-fertilization is prevented.
[0338] When the pods have reached maturity, these are harvested,
dried and then crushed. The seeds obtained are used for analysis of
the biochemical activity. The transgenic descendants are selected
by germination on a medium containing kanamycin sulphate in an
amount of 100 to 150 mg/l (depending on the genotypes). The working
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 plantlets which develop secondary roots in the first
three weeks are acclimatized in the phytotron before being passed
to the greenhouse.
[0339] VI. Example of the Production of Transgenic Solanaceae
Plants.
[0340] a) Production of Transgenic Tobacco Plants
[0341] The tobacco plants used for the transformation experiments
(Nicotiana tabacum var. Xanthi NC and PBD6) are cultivated in vitro
on the base medium of Murashige and Skoog (1962), to which are
added vitamins according to Gamborg et al. (1968, Sigma reference
M0404), sucrose in an amount of 20 g/l and agar (Merck) in an
amount of 8 g/l. The pH of the medium is adjusted to 5.8 with a
solution of potash, before autoclaving at 120.degree. C. for 20
min. The tobacco plantlets are planted out by taking cuttings at
the internodes every 30 days on this multiplication medium
MS20.
[0342] All the in vitro cultures are carried out in a climatically
controlled chamber under the conditions defined below:
[0343] luminous intensity of 30 .mu.E.m.sup.-2.S.sup.-1;
photoperiod of 16 h;
[0344] thermal period of 26.degree. C. during the day, 24.degree.
C. at night.
[0345] The transformation technique used is derived from that of
Horsch et al. (1985).
[0346] Agrobacterium tumefaciens, strain LBA4404, containing the
plasmids pBIOC29 or pBIOC26 or pBIOC25 is precultured for 48 h at
28.degree. C., under agitation, in medium LB (Sambrook et al.,
1989), to which adequate antibiotics (rifampicin and tetracycline)
have been added. The preculture is then diluted 50-fold in the same
medium and cultured under the same conditions. After one night, the
culture is centrifuged (10 min, 3,000 g), the bacteria are taken up
in an equivalent volume of liquid medium MS30 (30 9/1 of sucrose)
and this suspension is diluted 10-fold.
[0347] Explants of about 1 cm.sup.2 are cut from the leaves of the
plantlets described above. They are subsequently brought into
contact with the bacterial suspension for 1 h and then dried
rapidly on filter paper and placed on a coculture medium (solid
MS30).
[0348] After 2 days, the explants are transferred to Petri dishes
on regeneration medium MS30 containing a selective agent, kanamycin
(200 mg/l), a bacteriostatic, Augmentin (400 mg/l), and the
hormones necessary for induction of buds (BAP, 1 mg/l, and ANA, 0.1
mg/l). The explants are planted out on the same medium after
growing for 2 weeks. After two additional weeks, the buds are
planted out in Petri dishes on the development medium, composed of
medium MS20 to which kanamycin and Augmentin have been added. After
15 days, half the buds are planted out. Rooting takes about 20
days, at the end of which the plantlets can be cloned by cutting at
the internodes or put out in a greenhouse.
[0349] b) Production of Transgenic Tomato Plants.
[0350] The tomato seeds cv. UC82B are sterilized with 10% Domestos
for 15 minutes and rinsed 3 times with sterile water. The last
rinsing is carried out for 10 minutes under agitation.
[0351] The seeds thus sterilized are germinated on medium MSSV/2
(base medium of Murashige and Skoog (1962, Sigma reference M6899)/2
to which are added vitamins according to Nitsch (Thomas and Pratt,
1981), saccharose at 30 g/l, agar (Merck) at 8 g/l, pH 5.9, for 7
or 8 days in a climatically-controlled chamber (luminous intensity
of 30 .mu.E.m.sup.-2, S.sup.-, photoperiod of 16 h/8 h, 26.degree.
C.).
[0352] The transformation technique used is derived from that of
Fillatti et al. (1987).
[0353] Agrobacterium tumefaciens, strain LBA4404, containing the
plasmids pBIOC25 or pBIOC26 is precultured for 24 hours at
28.degree. C. under agitation in medium LB to which adequate
antibiotics (rifampicin and tetracycline) have been added. The
preculture is then diluted 50-fold in the same medium and cultured
under the same conditions for one night. The OD is measured at 600
nm, the agrobacteria are centrifuged (10 minutes, 3000 g) and taken
up in a liquid medium KCMS (described in the publication by
Fillatti et al., 1987) so as to obtain an OD of 0.8 at 600 nm.
[0354] Technical improvements were used in some stages of the
protocol described by Fillatti et al. (1987).
[0355] The preculture of the explants and the coculture were
carried out as described by Fillatti et al. (1987) except that the
medium KCMS was supplemented by acetosyringone (200 mM).
[0356] The washing medium 2Z differs by the addition of cefotaxime
at 500 mg/l instead of carbenicillin. The development medium used
is composed of base medium of Murashige and Skoog (Sigma MS6899) to
which are added vitamins according to Nitsch, saccharose at 20 g/l,
kanamycin at 50 mg/l, augmentin at 200 mg/l, ANA at 1 mg/l and
zeatin at 0.5 mg/l.
[0357] VII. Production of Transgenic Corn Plants
[0358] a) Production and Use of Corn Callus as a Target for Genetic
Transformation.
[0359] The genetic transformation of corn, whatever the method
employed (electroporation; Agrobacterium, microfibres, particle
gun), generally requires the use of rapid division undifferentiated
cells which have retained the ability to regenerate entire plants.
This type of cell includes the embryogenic friable callus (called
type II) of corn.
[0360] These calluses are obtained from immature embryos of
genotype Hl II or (A188 x B73) according to the method and on the
media described by Armstrong (Malze Handbook; (1994) M. Freeling,
V. Walbot Eds.; pp. 665-671). The calluses obtained in this way are
multiplied and maintained by successive plantings every fifteen
days on the initiation medium.
[0361] Plantlets 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. (Plant Cell Tissue and Organ
Culture (1989), 18:143-151). These plants are then acclimatized in
a greenhouse where they can be crossed or self-fertilized.
[0362] b) Use of a Particle Gun for the Genetic Transformation of
Corn
[0363] The preceding paragraph describes the production and the
regeneration of cell lines necessary for the transformation; a
genetic transformation method is described here which leads to a
stable integration of the modified genes in the genome of the
plant. This method depends on the use of a particle gun identical
to that described by J. Finer (Plant Cell Report (1992) 11:
323-328); the target cells are callus fragments described in
paragraph 1. These fragments with a surface area of 10 to 20
mm.sup.2 were arranged, 4 hours before bombardment, at the rate of
16 fragments per dish in the centre of a Petri dish containing a
culture medium identical to the initiation medium, to which is
added 0.2 M of mannitol+0.2M of sorbitol. The plasmids carrying the
genes to be introduced are purified on a Qiagen.RTM. column,
following the manufacturer's instructions. They are then
precipitated on particles of tungsten (M10) following the protocol
described by Klein (Nature (1987) 327:70-73). The particles thus
coated are fired towards the target cells using the gun and in
accordance with the protocol described by J. Finer (Plant Cell
Report(1992) 11:323-328).
[0364] The dishes of calluses thus bombarded are then sealed using
Scellofrais.RTM. then cultured in the dark at 27.degree. C. The
first plantings take place 24 hours later, then every fifteen days
for 3 months on a medium identical to the initiation medium with a
selection agent added to it the type and concentration of which can
vary according to-the gene used (see paragraph 3). The selection
agents which can be used generally consist of the active compounds
of certain herbicides (Basta.RTM., Round up.RTM.) or certain
antibiotics (Hygromycin, Kanamycin . . . ).
[0365] After 3 months or sometimes earlier, calluses are obtained
whose growth is not inhibited by the selection agent, usually and
for the most part is composed of cells resulting from the division
of a cell having integrated into its gene pool one or more copies
of the selection gene. The frequency with which such calluses are
obtained is about 0.8 callus per bombarded dish.
[0366] These calluses are identified, individualized, amplified
then cultured so as to regenerate the plantlets (cf. paragraph a).
In order to avoid any interference with the non-transformed cells
all these operations are carried out on culture media containing
the selection agent.
[0367] The plants thus regenerated are acclimatized then cultivated
in a greenhouse where they can be crossed or self-fertilized.
[0368] VIII. Analysis of the Expression of Dog Gastric Lipase in
Transgenic Tobacco Plants.
[0369] a) Protocol
[0370] The protocol for extraction of the lipase from tobacco
leaves taken from the plants in the greenhouse is as follows: 1 g
of leaves (fresh weight) is ground in liquid nitrogen and then at
4.degree. C. in 5 ml of the buffer 25 mM Tris-HCl buffer, pH 7.5,
to which 1 mM EDTA and 10 mM mercaptoethanol have been added
(buffer A), or 25 mM glycine-HCl buffer, pH 3, to which 1 mM EDTA,
10 mM .beta.-mercaptoethanol, 0.2% Triton X-100 and 250 mM NaCl
have been added (buffer B). The total ground material is
immediately centrifuged at 4.degree. C. for 15 min at 10,000 g.
[0371] For tobacco seeds, the extraction is carried out in buffer B
in an amount of 0.1 g of seeds per 4 ml of buffer.
[0372] The lipase activity is determined with the aid of a pHSTAT
by the titrimetric method of Gargouri et al. (1986), in which the
substrate used is tributyrin. The emulsion of tributyrin (4 ml per
30 ml of emulsion) is prepared in a vortex in the presence of bile
salts (1.04 g/l), bovine albumin (0.1 g/l) and NaCl (9 g/l). The
analysis comprises neutralization of the butyric acid liberated
under the action of the lipase by a solution of sodium carbonate at
a pH regulated at 5.5 and at 37.degree. C. One unit of lipase
corresponds to the amount of enzyme which causes the liberation of
one micromole of fatty acids in 1 min at 37.degree. C. under
optimum pH conditions (5.5). Natural (purified) dog gastric lipase
has a specific activity of 570 units/mg of protein. The lipase
activity can be measured on the total ground material, on the
sediment or on the centrifugation supernatant.
[0373] Analysis of the total soluble proteins on the centrifugation
supernatant (buffer A) is carried out by the method of Bradford
(1976).
[0374] A sandwich ELISA test (Carrire et al., 1993) is also carried
out on the centrifugation supernatant with 2 populations of natural
anti-DGL polyclonal antibodies. The first population includes the
antibodies which react with human gastric lipase and have been
purified by affinity using total antiserum on human gastric lipase
grafted on a column of Affigel 10 (Aoubala et al., (1993). These
antibodies are used to coat the wells of the ELISA plates in a
concentration of 1 .mu.g/ml. The 2nd population comprises
antibodies which do not recognize human lipase. They are
subsequently purified on a column of Sepharose, bound to protein A
and then bound to biotin. Fixing of the antibody is detected by the
indirect means of a streptavidin/peroxidase conjugate, the
enzymatic activity of which is demonstrated by means of the
substrate o-phenylenediamine. The results obtained are of
qualitative value and are recorded with the aid of the symbols +and
-. The extraction yield can be improved by adding to the extraction
buffer a detergent of CHAPS type (3-(3cholamidopropyl)dime-
thyl-ammonio-1-propane sulphonate) (SIGMA). In fact, in this case,
the extraction yields in the supernatant increase by about 100%
(cf. Table 1).
2TABLE 1 Lipase activity (U/gFW) in the extracts obtained from 4
tobacco plants originating from the genetic transformation with
pBIOC25, in the presence of 1% CHAPS. NaCl NaCl 0.2 M/EDTA 0.2
M/EDTA 1 mM 1 mM pH 3.0 + Plants pH 3.0 1% CHAPS 1 80 112 2 40 104
3 43 109 4 54 92 b) Expression with plant signal peptides; analyses
on tobacco leaves and seeds transformed with pBIOC25 and pBIOC26;
ELISA test.
[0375] The results obtained on 98 T0 plants of the genotype Xanthi
(15 to 20 leaf stage) are shown in Table 2. The analyses were all
carried out on the ground mixture of leaves or seeds before
centrifugation. For each construct, the activities measured show a
wide variability according to the transformants. About 20% of the
plants have no activity or an activity which is too weak for
detection. The mean activities and the maximum activities are given
in Table 2.
[0376] The amounts of total proteins are similar for the 3
constructs, with means of 7 and 8 mg/g of FW in the leaves and 34,
31 and 38 mg/g of FW in the seeds.
[0377] The mean lipase activity in the transformed leaves with the
construct pBIOC26 is 34 U/g of FW, that is to say 0.8% expression
with respect to the total proteins. In the seeds, the mean activity
is 36 U/g of FW, that is to say 0.2%. The maximum activity obtained
on one of the transformants is 146 U/g of FW (leaves; 2.5%
expression) and 148 U/g of FW (seeds; 0.7% expression).
[0378] The enzymatic activities analyzed on the plants transformed
with the construct pBIOC25 are similar. The mean activity in the
leaves is 34 U/g of FW (0.8% expression) and the maximum activity
is 134 U/g of FW (3% of the total proteins). In the seeds, the mean
activity is 42 U/g of FW (0.3%) and the maximum activity is 159 U/g
of FW (1%).
[0379] For plants transformed with the construct pBIOC29, no
activity is detected in the leaves (seed-specific promoter). In the
seeds, the mean activity is 12 U/g of FW (0.1% expression) and the
maximum activity is 137 U/g of FW (0.7%).
[0380] The expression in the leaves and seeds of the same
transformation event generally correlates well.
[0381] The results of the ELISA tests also agree with those of the
analyses of the enzymatic activity, that is to say a plant having a
lipase activity gives a positive result in the ELISA test.
[0382] Results obtained subsequently on these same plants show that
the lipase activity can vary in the course of development of the
plant. In fact, a plant of which the expression, with respect to
the total proteins, was 3% at the 12-15 leaf stage gave an
expression of 6% during a subsequent analysis (older plant having
flowered).
3TABLE 2 EXPRESSION OF LIPASE IN THE LEAVES AND SEEDS OF XANTHI
TOBACCO. LIPASE ACTIVITY TOTAL Expression PROTEINS (% of total mg/g
of FW U/g of FW proteins) min-max min-max min-max CONSTRUCT ORGAN
(mean) (mean) (mean) pBIOC26 Leaves 3-15 0-145.6 0-2.5 (n = 34) (7)
(34.4) (0.8) Seeds 24-43 0-147.6 0-0.7 (n = 34) (34) (36.3) (0.2)
pBIOC25 Leaves 3-18 0-133.5 0-3 (n = 35) (8) (34) (0.8) Seeds 17-35
0-158.5 0-1 (n = 35) (31) (41.9) (0.3) pBIOC29 Seeds 29-55 0-137.4
0-0.7 (n = 29) (38) (11.8) (0.1) FW, fresh weight; min, value
obtained for the transformation event expressing the least; max,
value obtained for the transformation event expressing the most; n,
number of transformation events analyzed. b) Expression with the
signal peptide of RGL; analyses of the lipase activity on tobacco
leaves.
[0383] The results obtained on 44 T0 plants of genotypes Xanthi and
pBD6 (15 to 20 leaf stage) are as follows:
[0384] The analyses were all carried out on the ground mixture of
leaves before centrifugation. The activities analyzed show a wide
variability according to the transformants. About 20% of the plants
have no activity or an activity which is too weak for detection.
The mean lipase activities in the leaves transformed with the
construct pBIOC41 is 38 U/g FW for the genotype Xanthi and 48 U/g
FW for the genotype PBD6. The maximum activities are 152 and 226
U/g FW respectively.
[0385] IX. Analysis of the Expression of Dog Gastric Lipase in
Transgenic Tomato Plants.
[0386] a) Protocol
[0387] The protocol for the extraction of the lipase from the
tomato leaves and fruits is similar to that described for the
tobacco leaves, except that 1 g of fresh material is taken up in 4
ml of buffer B. The lipase activity is determined as described for
the tobacco leaves.
[0388] b) Analysis in Tomato Fruits.
[0389] The fruits of thirty primary transformants were
analyzed.
[0390] The lipase activity analyzed in the fruits is variable
depending on the fruits for the same transformant. It is an average
of 5 U/g FW for ripe fruit and 35 U/g FW for unripe fruit,
independent of the construct tested (pBIOC25 or pBIOC26).
[0391] It should be noted that during the ripening of fruit the
activity reduces. For example, for a given primary transformant,
the lipase activity is 132 U/g FW for a green fruit with a diameter
of 10 mm, 44 U/g FW for a red-green fruit with a diameter of 33 mm
and 36 U/g FW for a red fruit with a diameter of 45 mm.
[0392] X. Immunodetection of the "Western" Type of the Recombinant
DGL.
[0393] a) DGL Expressed in Transgenic Tobacco and Rape Leaves and
Seeds.
[0394] a.1) Expression with Plant Signal Peptides; Immunodetection
in Tobacco and Rape Leaves and Seeds Transformed by pBIOC25,
pBIOC26 and pBIOC29.
[0395] Immunodetection experiments of the "western" type ("western
blots") (Renart and Sandoval, 1984) on the dog gastric lipase were
carried out on the proteins of tobacco leaves and tobacco and rape
seeds extracted with buffers A and B (see the extraction protocol
above). To carry out these experiments, the proteins extracted (30
.mu.g of total proteins per sample) are first separated over 12.5%
of denaturing polyacrylamide gel in accordance with the technique
of Laemmli U.K. (1970) and are then transferred on to a
nitrocellulose membrane. An anti-dog lipase polyclonal antibody
obtained in the guinea-pig is used as the probe and the detection
is carried out by means of an anti-IgG antibody of the guinea-pig
labelled with alkaline phosphatase.
[0396] The control protein is natural dog gastric lipase (LC. FIG.
6), which migrates in the form of a single band at an apparent
molecular weight of about 50 kDa. The migration of the dog gastric
lipase is slightly retarded in the presence of the extract of
non-transformed tobacco leaves (FIG. 6, LC+T).
[0397] No band is detected in the protein extracts of
non-transformed leaves and seeds of tobacco and rape. The lipase
produced in the tobacco leaves is in the form of 2 bands. The band
which is the largest quantitatively has an apparent molecular
weight of about 37 kDa and corresponds to the abovementioned
polypeptide (.DELTA.54). The minor band has an apparent molecular
weight of about 49 kDa and corresponds to the abovementioned
polypeptide (.DELTA.4) (FIG. 6, F). In the protein extracts of
seeds, only the band of lower molecular weight is visible (FIG. 6,
GT and GC).
[0398] a.2) Expression with the Signal Peptide of RGL;
Immunodetection in Tobacco Leaves and Seeds Transformed with
pBIOC41.
[0399] Western blots (Renart and Sandoval, 1984) were carried out
on the proteins of tobacco leaves and seeds extracted with buffer B
(see the extraction protocol above). The proteins extracted are
first separated over denaturing polyacrylamide gel (SDS-PAGE) in
accordance with the technique of Laemmli (1970) and are then
transferred on to a nitrocellulose membrane. An anti-dog lipase
polyclonal antibody obtained in the guinea-pig is used as the probe
and the detection is carried out by means of an anti-guinea pig
antibody labelled with alkaline phosphatase.
[0400] An example of a western blot of tobacco leaves is shown in
FIG. 7. The control protein is dog gastric lipase, which migrates
in the form of a single band at an apparent molecular weight of
about 50 kDa. No band is detected in the protein extracts of
non-transformed leaves of tobacco (T). The lipase produced in the
leaf extracts is in the form of a major band at an apparent
molecular weight of about 48-49 kDa and corresponds to the
abovementioned polypeptide (D4).
[0401] In tobacco seeds transformed with the construct pBIOC41, the
recombinant lipase is in the same form as in the leaves.
[0402] b) DGL in a Protein Extract of Transformed Tobacco Leaves:
Deglycosylation Experiment.
[0403] The protocol for extraction of the proteins for the
deglycosylation experiments is as follows: 0.5 g of leaves (fresh
weight) is ground in liquid nitrogen and then at 4.degree. C. in 1
ml of denaturation buffer (100 mM phosphate buffer, pH 7.5, to
which 1% .beta.-mercaptoethanol, 25 mM EDTA and 1% SDS have been
added). The ground material is centrifuged at 4.degree. C. for 15
min at 10,000 g. The supernatant is incubated for 5 min at
100.degree. C. in order to denature the proteins and then
centrifuged for 2 min at 10,000 g. The supernatant is then diluted
10-fold in the deglycosylation buffer (100 mM phosphate buffer, pH
7.5, to which 1% .beta.-mercaptoethanol, 25 mM EDTA, 0.1% SDS and
1% octyl glucoside have been added). The enzyme (N-glycosidase F,
PNGase Boehringer) is added in an amount of 1 U per 100 .mu.l of
supernatant. A control without enzyme is carried out for each
sample. The deglycosylation of the control protein (dog or rabbit
gastric lipase) takes place under the same conditions. The various
protein samples are incubated at room temperature for 8 hours. The
proteins are then separated by electrophoresis over polyacrylamide
gel and transferred on a nitrocellulose membrane as described in
the preceding paragraph.
[0404] According to the "western blots" results, the gastric lipase
used as the control has an apparent molecular weight of about 50
kDa. After deglycosylation, its apparent molecular weight is only
about 43 kDa.
[0405] The lipase produced in the tobacco leaves, after incubation
without PNGase under the conditions described above, appears in the
form of 3 bands of apparent molecular weights 49 (polypeptide
(.DELTA.4)), 37 (polypeptide (.DELTA.54)) and 28 kDa. The band of
molecular weight 28 kDa is without doubt the result of a
proteolysis which has taken place during the incubation for 8 hours
at room temperature. After deglycosylation, the molecular weights
of the 3 bands are reduced by about 1 to 2 kDa, which shows that
the proteins produced in the tobacco leaves are glycosylated.
[0406] c) DGL Expressed in Transgenic Tomato Leaves and Fruits.
[0407] Immunodetection experiments of the "western" type on the dog
gastric lipase were carried out on the proteins of tomato leaves
and fruits extracted with buffer B (see the extraction protocol
above). To carry out these experiments, the proteins extracted (15
.mu.g and 6 .mu.g of total soluble proteins for the leaves and
fruits respectively) are separated over denaturing polyacrylamide
gel as described in paragraph X.a).
[0408] The control protein is natural gastric lipase (track 4, FIG.
8), which migrates in the form of a single band at an apparent
weight of about 50 kDa.
[0409] No band is detected in the protein extracts of
non-transformed tomato leaves and fruits.
[0410] The lipase produced in the tomato leaves and fruits is in
the form of 2 bands, whichever construct is used (pBIOC25 or
pBIOC26). The band which is the largest quantitatively has an
apparent weight of about 37 kDa and corresponds to the
abovementioned polypeptide (.DELTA.54). The minor band has an
apparent molecular weight of about 49 kDa and corresponds to the
abovementioned polypeptide (.DELTA.4) (FIG. 8).
[0411] XI. Purification of the Dog Gastric Lipase from Plants.
[0412] a) Purification of DGL from Tobacco Leaves.
[0413] The activity of the dog gastric lipase produced in the
tobacco leaves is determined by a titrimetric method, the regulated
pH being kept at 5.5 and the temperature at 37.degree. C. with the
aid of a pH-stat (Mettler-Toledo-DL25), using tributyrin as the
substrate: 1 ml of tributyrin is emulsified in 29 ml of an aqueous
solution of 0.15 M NaCl in a vortex. The analysis comprises
neutralizing the butyric acid liberated under the action of the
lipase by addition of 0.02 N sodium carbonate, while the emulsion
is maintained by vigorous mechanical agitation. One lipase unit
corresponds to 1 micromole of fatty acid liberated per minute under
these conditions of pH and temperature.
[0414] After the first chromatography stage, the lipase activity is
demonstrated with an analytical sample of 0.5 ml on an emulsion of
1 ml of tributyrin in 29 ml of a solution of 0.15 M NaCl, 2 mM
sodium taurodeoxycholate and 1.5 .mu.M bovine serum albumin in
accordance with the analysis described by Gargouri et al.
(1986).
[0415] 1 gram of lyophilized leaves is ground at 4.degree. C. in 30
ml of 20 mM glycine buffer, pH 2.5, and the mixture is stirred
gently for 15 minutes. During the steeping, the pH is kept at 2.5
by addition of 1N HCl. The product of the steeping is centrifuged
at 15,000 g for 5 minutes. The pH of the supernatant is adjusted to
4 by addition of 1N NaOH. After filtration over MIRACLOTH
(Calbiochem), all the supernatant is applied to a cation exchange
resin column (S-Sepharose Fast Flow resin--Pharmacia) of 10 ml
(diameter 1.6 cm) equilibrated in a buffer of 20 mM sodium acetate,
pH 4.0, and 20 mM NaCl at a flow rate of 1 ml per minute. Fractions
of 2 ml are collected. After passage of the supernatant, the column
is washed with 40 ml of the equilibration buffer. The proteins
retained on the column are eluted in accordance with the following
protocol:
[0416] linear gradient in 20 mM sodium acetate buffer, pH 4.0, of
20 mM to 210 mM NaCl in the course of 30 minutes for elution of a
first set of peaks of proteins which do not contain lipase
activity, the test being carried out on analytical samples of 1
ml,
[0417] plateau at 210 mM NaCl for 20 minutes,
[0418] linear gradient in 20 mM sodium acetate buffer, pH 4.0, of
210 mM to 500 mM NaCl in the course of 30 minutes for elution of a
second set of peaks. The lipase activity-measured on analytical
samples of 0.5 ml is eluted during this second gradient at an ionic
strength of between 300 and 400 mM.
[0419] The active fractions are collected and concentrated with the
aid of an OMEGACELL concentration cell of molecular weight limit 30
kDa (Filtron Technology Corporation).
[0420] The concentrate is dialysed for 12 hours against a buffer of
10 mM Tris-HCl, pH 8, and then applied to an anion exchange resin
column (MonoQ HR 5/5 of diameter 0.5 cm and height 5 cm--Pharmacia)
equilibrated in 10 mM Tris-HCl buffer, pH 8. The lipase activity is
measured on an analytical sample of 0.5 ml. The flow rate is kept
at 1 ml per minute, that is to say a pressure of 2.0 mPa. After
elution of the fraction which is not retained and washing of the
column with 10 ml of 10 mM Tris-HCl buffer, pH 8, a linear gradient
of ionic strength from 0 to 400 mM NaCl is applied in the course of
60 minutes. The lipase activity is eluted for an ionic strength
between 100 mM and 200 mM.
[0421] The active fractions are collected and concentrated with the
aid of an OMEGACELL concentration cell of molecular weight limit 30
kDa. The concentrate constitutes the purified form of the dog
gastric lipase extracted from tobacco leaves.
[0422] The concentration of proteins is determined in accordance
with the method of M. Bradford (1976) on the concentration
supernatants and in accordance with the method of O. H. Lowry
(1951) for the solutions after the first ion exchange
chromatography.
[0423] The various stages of the purification are analyzed by
electrophoresis over denaturing polyacrylamide gel (SDS-PAGE 12.5%)
in accordance with the technique of U. K. Laemmli (1970). The
proteins separated in this way on the polyacrylamide gel are, on
the one hand, detected by staining with Coomassie blue and, on the
other hand, transferred on a nitrocellulose membrane by the
technique of semi-dry electrotransfer (Transblot SD, BIORAD) in a
buffer of 20 mM Tris base, 150 mM glycine and 20% ethanol at 2.3 mA
per cm.sup.2 of membrane. The recombinant dog gastric lipase
transferred on the nitrocellulose membrane is detected by
immunodetection in accordance with the following protocol:
[0424] labelling of the target protein by an dog gastric
anti-lipase polyclonal antibody obtained in the guinea-pig and
diluted to 1/5,000 in PBS buffer, to which lipid-free milk powder
in an amount of 5% and Tween 20 in an amount of 0.1% have been
added, for 1 hour at room temperature,
[0425] rinsing of the membrane in three successive baths of PBS
buffer, to which lipid-free milk powder in an amount of 1% and
Tween 20 in an amount of 0.1% have been added, for 10 minutes for
each bath,
[0426] labelling with an anti-guinea-pig antibody bound to
peroxidase (Sigma) diluted to {fraction (1/2,000)} in PBS buffer,
to which lipid-free milk powder in an amount of 1% and Tween 20 in
an amount of 0.1% have been added, for 1 hour at room
temperature,
[0427] rinsing of the membrane in three successive baths of PBS
buffer, to which lipid-free milk powder in an amount of 1% and
Tween 20 in an amount of 0.1% have been added, for 10 min for each
bath,
[0428] detection by the action of the peroxidase on
4-chloro-1-naphthol (Sigma) in the presence of H.sub.2O.sub.2 to
produce a blue coloration which is stable in the course of
time.
[0429] The lipase produced in the leaves is in the form of two
bands of about 49 (polypeptide (.DELTA.4)) kDa and 37 (polypeptide
(.DELTA.54)) kDa.
[0430] b) Variant for the Purification of DGL from Tobacco
Leaves.
[0431] The activity of the dog gastric lipase produced in the
tobacco leaves is determined by the titrimetric methods described
by Gargouri et al. (1986) with the aid of a pH-stat
(METTLER-TOLEDO-DL25).
[0432] The analysis on short-chain triglycerides is carried out
using tributyrin as the substrate: 1 ml of tributyrin is emulsified
in 29 ml of an aqueous solution of 0.15 M NaCl, 1.5 .mu.M of Bovine
Serum Albumin (BSA) and 2 mM of sodium taurodeoxycholate (NaTDC).
The regulated pH is kept at 5.0 and the temperature at 37.degree.
C.
[0433] The analysis comprises neutralizing the butyric acid
liberated under the action of the lipase by addition of 0.02 N
sodium carbonate, while the emulsion is maintained by vigorous
mechanical agitation.
[0434] The analysis on long-chain triglycerides is carried out
using an aqueous emulsion with 30% purified soya bean oil, 1.2% of
purified egg phospholipids, 1.67% of anhydrous glycerol
(Intralipid.TM. 30% PHARMACIA AB Stockholm, Sweden) as the
substrate. Ten ml of this suspension is emulsified in 20 ml of an
aqueous solution of 0.15 M NaCl, 30 .mu.M of BSA and 3.5 mM of
CaCl.sub.2. The regulated pH is kept at 4.0 and the temperature at
37.degree. C.
[0435] The analysis consists of neutralizing the fatty acids
liberated under the action of the lipase by addition of 0.2 N
sodium carbonate, after a jump in the pH from 4 to 9 while the
emulsion is maintained by vigorous mechanical agitation.
[0436] One lipase unit corresponds to 1 micromole of fatty acid
liberated per minute under the defined conditions of pH and
temperature for each of the substrates. 2 grams of lyophilized
leaves is ground up at 4.degree. C. in 60 ml of 0.2 M NaCl, pH 3,
and the mixture is gently agitated for 15 minutes at 4.degree. C.
During this steeping, the pH is kept at 3 by the addition of 1N
HCl. The product of the steeping (homogenate) is centrifuged at
10,000 g for 10 minutes. After filtration over MIRACLOTH
(Calbiochem) and a 0.45.mu. MILLIPORE filter, all the supernatant
is injected into a cation exchange resin column (RESOURCE S 6
ml--Pharmacia--16 mm i.d..times.30 mm) equilibrated in a buffer of
20 mM sodium acetate, 0.2 M NaCl pH 3 at a flow rate of 8 ml/minute
(240 cm h-1).
[0437] After passage of the non-retained fraction, the column is
washed with 30 times its volume of the equilibration buffer. The
proteins retained on the column are eluted with a linear gradient
in 20 mM sodium acetate buffer, pH 3, of 0.2 M NaCl to 0.5 M NaCl
in 7 column volumes. Fractions of 4 ml are collected. The lipase
activity measured on analytical samples of 0.5 ml is eluted at an
ionic strength of 0.35 M NaCl.
[0438] The active fractions are collected and concentrated with the
aid of an OMEGACELL concentration cell (Filtron Technology
Corporation) with a molecular weight limit of 30 kDa.
[0439] Determination of the protein concentration is carried out in
accordance with the method of O. H. Lowry (1951).
[0440] An example of polyacrylamide gel and the result of the
transfer of this gel onto a nitrocellulose membrane and detection
from a dog gastric anti-lipase antibody are shown (FIGS. 9 and 10),
the detection was carried out by the action of peroxydase on
Luminol in the presence of activator (ECL western
blotting--AMERSHAM LIFE SCIENCE) and recording on a photographic
film.
[0441] The presence of glycanic residues on the protein was
determined according to the following protocol:
[0442] immobilization of the protein on a microcellulose
membrane
[0443] treatment with periodate
[0444] specific reaction with streptavidin coupled with alkaline
phosphatase
[0445] coloured reaction with alkaline phosphate (GLYCOTRACK OXFORD
GLYLOSYSTEM).
[0446] An example of membrane detection of glycanic residues is
shown (FIG. 11).
[0447] The purity of the fractions is ensured by high performance
liquid chromatography.
[0448] Chromatograph WATERS 625 LC
[0449] Diode array detector WATERS 991
[0450] Column VYDAC C4
[0451] Elution conditions:
4 Time Flow rate (min) (ml/min) % A % B 0 1 100 0 35 1 40 60 40 1
40 60 45 1 20 80 50 1 100 0
[0452] Buffer A: 89.9% H2O 10% Acetonitrile, 0.1%
[0453] trifluoroacetic acid
[0454] Buffer B: 100% Acetonitrile.
5 Purification table: recombinant DGL Total mg of Specific
Purification units* proteins activity factor Yield Homogenate 3200
/ / / / Super- 2800 230 13 1 88% natant Outlet 1600 6 250 20 50%
Resource S *Tributyrin units
[0455] Comparative Specific Activities of Natural Dog Gastric
Lipase (n-DGL) and Recombinant Dog Gastric Lipase (r-DGL)
6 r-DGL extract from n-DGL* tobacco leaves Tributyrin 570 U/mg 250
U/mg Intralipid 30% 1000 U/mg 950 U/mg *ref: Carrire et al (1991)
Eur. J. Biochem, 202, 75-83. c) Purification of DGL from rape
seeds
[0456] The activity of the recombinant DGL produced in rape seeds
is determined in the same way as in the case of extraction from
leaves.
[0457] Ten grams of rape seeds are ground up in liquid nitrogen.
The flour obtained is delipidated with hexane by a first steeping
at 4.degree. C. under gentle agitation for 12 hours. The whole is
decanted, the hexane is eliminated. The flour is rinsed twice with
100 ml of hexane under gentle agitation for 1/2 hour at 4.degree.
C. for each rinsing. The hexane is eliminated by decanting. The
flour is dried in a rotary evaporator (HEIDOLPH 94200). The
delipidated flour is stored at -20.degree. C.
[0458] Extraction of the DGL is carried out from the delipidated
flour by steeping at 4.degree. C. in a 0.2 M aqueous solution of
NaCl pH 3 (1N HCl) for 30 minutes at the rate of 2 ml of aqueous
solution per 0.1 g of flour. The resultant material from the
steeping is centrifuged at 10,000 g for 10 minutes at 4.degree.
C.
[0459] The pellet is eliminated. The supernatant constitutes the
seed extract.
[0460] The seed extract is dialyzed against a buffer of 10 mM of
sodium acetate, pH 4, 140 mM of NaCl, 3 mM of KCl then applied to
an immunoaffinity column constituted by dog gastric antilipase
polyclonal antibodies obtained from guinea-pigs coupled to a resin
(hydrazide Avidgel--BIOPROBE INTERNATIONAL, Inc.).
[0461] The resin/seed extract contact is for 30 minutes at
4.degree. C. under gentle agitation. The resin is then rinsed with
10 column volumes of buffer, 10 mM sodium acetate, pH 4, 150 mM
NaCl, 3 mM KCl. The DGL is eluted with 5 column volumes of buffer,
0.2 M glycine, pH 2.8, 150 mM NaCl. The collected fractions have a
volume equal to 1 column volume and contain {fraction (1/20)}th V/V
of 1M Tris buffer, pH 9. Analysis by electrophoresis over
polyacrylamide gel in denaturing medium (cf. FIG. 12) shows a
protein of molecular weight of about 37 kDa.
[0462] XII. Synthesis of Fatty Acid Esters.
[0463] The tests were carried out with non-transformed rape seeds,
the lipase being provided:
[0464] either in the form of an immobilized enzymatic preparation
(lipozyme (NOVO)) or in the free form (rabbit gastric lipase (JO
4002)),
[0465] or in the form of tobacco seeds transformed with the gene of
dog gastric lipase (tobacco T14-44 0.85% expression).
[0466] The esterification reactions are carried out at 37.degree.
C. for 16 hours in hermetically stoppered glass bottles placed on
an agitation bench (250 rpm). The organic solvent used is hexane,
in which the fatty acids are soluble. The methanol is added in a
stoichiometric amount with respect to the theoretical amount of
triacylglycerol contained in the rape seeds.
[0467] The major component of the fatty acids of rape is oleic
acid, and the reference control chosen is also a methyl ester of
oleic acid. The synthesis is monitored by thin layer chromatography
(TLC). The migration solvent is a mixture of hexane, diethyl ether
and water (70:10:1). Detection on the plates is carried out under
hot conditions after spraying with sulphuric acid (5%) in
ethanol.
[0468] In a first test, 27 .mu.l of methanol (0.66 mmol) and 0.02 g
of lipozyme are added to 0.2 g of rape oil (0.22 mmol): no spot
appears at the level of the reference methyl oleate (FIG. 13,
column 2).
[0469] In the second test, the rape oil is replaced by 0.5 g of
rape seeds ground in the dry state, and 1 ml of hexane is added: a
methyl ester is synthesized (FIG. 13, column 5).
[0470] In the following tests, the conditions above are repeated
with the following modifications: the amount of lipozyme is reduced
to 0.006 g and the lipozyme is replaced by rabbit gastric lipase
(0.007 g of JO 4002). Methyl oleate is synthesized in the presence
of the lipozyme but not in the presence of JO 4002 (FIG. 14, column
2).
[0471] Finally, in the last tests, the lipase is provided by
transformed tobacco seeds (1 g of tobacco seeds). A characteristic
spot of a methyl ester appears (FIG. 15, column 3).
[0472] In conclusion, the results described above demonstrate that
if extracts of rape seeds, alcohol and recombinant dog gastric
lipase produced by transgenic tobacco are brought together, an
esterification reaction which leads to the synthesis of a methyl
ester which can be used as a biofuel is obtained.
[0473] Legend to the figures:
[0474] FIG. 1: Nucleotide sequence of the cDNA which codes for the
DGL,
[0475] FIG. 2: Amino acid sequence of the DGL,
[0476] FIG. 3: Nucleotide sequence derived from the cDNA which
codes for the DGL, coding for the DGL shown on FIG. 2,
[0477] FIG. 4: Nucleotide sequence of the cDNA which codes for HGL
and the amino acid sequence of HGL,
[0478] FIG. 5: Amino acid sequence of HGL,
[0479] FIG. 6: Immunodetection of recombinant polypeptides produced
by Xanthi tobacco leaves and seeds and rape-seeds transformed with
the constructs pBIOC26, pBIOC25 and pBIOC29; E, molecular weight
range; LC, dog gastric lipase; F, tobacco leaves and GT, tobacco
seeds transformed with the construct pBIOC25; GC, rape seeds
transformed with the construct pBIOC29; T, non-transformed tobacco
leaves,
[0480] FIG. 7: Immunodetection of recombinant polypeptides produced
by tobacco leaves transformed with the constructs pBIOC25 and
pBIOC41; E, molecular weight range; LC, dog gastric lipase; 1 and
3, tobacco leaves transformed with the construct pBIOC41; 2,
tobacco leaves transformed with the construct pBIOC25; T,
non-transformed tobacco leaves,
[0481] FIG. 8: Immunodetection of recombinant polypeptides produced
by tomato leaves and fruits transformed with the constructs pBIOC25
and pBIOC26:
[0482] T1: non-transformed tomato fruit
[0483] 1 and 3: transformed tomato fruits
[0484] 2: transformed tomato leaf
[0485] R: molecular weight range
[0486] LC: dog gastric lipase
[0487] T2: non-transformed tomato leaf.
[0488] FIG. 9: Analysis by electrophoresis over polyacrylamide gel
in denaturing medium (SDS-PAGE).
[0489] 1: Rape seed extract
[0490] 2: Recombinant dog gastric lipase produced from tobacco
leaves
[0491] 3: Natural dog gastric lipase
[0492] 4: molecular weight range.
[0493] FIG. 10: Immunological detection of the preceding analysis
after transfer on nitrocellulose membrane
[0494] 1: Natural dog gastric lipase
[0495] 2: Recombinant dog gastric lipase produced from tobacco
leaves
[0496] 3: Rape seed extract.
[0497] FIG. 11: Detection of the presence of glycanic residues from
a polyacrylamide gel after transfer on nitrocellulose membrane
[0498] 1: Rape seed extract
[0499] 2: Recombinant dog gastric lipase produced from tobacco
leaves
[0500] 3: Natural dog gastric lipase.
[0501] FIG. 12: SDS-PAGE analysis of the immunopurification of
r-DGL produced in rape seeds
[0502] 1: Natural dog gastric lipase
[0503] 2: Recombinant dog gastric lipase
[0504] 3 to 6: Non-retained fractions eluted from the
immunopurification column.
[0505] FIG. 13: Detection on a TLC plate; 1: rape oil, no enzyme;
2: rape oil+lipozyme; 3: methyl ester of oleic acid; 4: rape seeds,
no enzyme; 5: rape seeds+lipozyme,
[0506] FIG. 14: Detection of a TLC plate; 1 and 4: rape seeds
without enzyme; 2: rape seeds+J04002; 3: methyl ester of oleic
acid; 5: rape seeds+lipozyme,
[0507] FIG. 15: Detection on a TLC plate; 1: monoolein, diolein,
triolein; 2: methyl ester of oleic acid; 3: rape seeds+transformed
tobacco seeds.
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Sequence CWU 1
1
37 1 1528 DNA Canis familiaris CDS (1)..(1137) 1 ttg ttt gga aag
ctt cat ccc aca aac cct gaa gtg acc atg aat ata 48 Leu Phe Gly Lys
Leu His Pro Thr Asn Pro Glu Val Thr Met Asn Ile 1 5 10 15 agt cag
atg atc acc tac tgg gga tac cca gct gag gaa tat gaa gtt 96 Ser Gln
Met Ile Thr Tyr Trp Gly Tyr Pro Ala Glu Glu Tyr Glu Val 20 25 30
gtg acc gaa gac ggt tat atc ctt ggg atc gac aga att cct tat ggg 144
Val Thr Glu Asp Gly Tyr Ile Leu Gly Ile Asp Arg Ile Pro Tyr Gly 35
40 45 agg aaa aat tca gag aat ata ggc cgg aga cct gtt gca ttt ttg
caa 192 Arg Lys Asn Ser Glu Asn Ile Gly Arg Arg Pro Val Ala Phe Leu
Gln 50 55 60 cac ggt ttg ctc gca tca gcc aca aac tgg atc tcc aac
ctg ccc aac 240 His Gly Leu Leu Ala Ser Ala Thr Asn Trp Ile Ser Asn
Leu Pro Asn 65 70 75 80 aac agc ctg gcc ttc atc ctg gcc gac gcc ggg
tac gac gtg tgg ctg 288 Asn Ser Leu Ala Phe Ile Leu Ala Asp Ala Gly
Tyr Asp Val Trp Leu 85 90 95 ggg aac agc agg ggc aac acc tgg gcc
agg agg aat ctg tac tac tcg 336 Gly Asn Ser Arg Gly Asn Thr Trp Ala
Arg Arg Asn Leu Tyr Tyr Ser 100 105 110 ccc gac tcc gtc gaa ttc tgg
gct ttc agc ttt gac gag atg gct aaa 384 Pro Asp Ser Val Glu Phe Trp
Ala Phe Ser Phe Asp Glu Met Ala Lys 115 120 125 tat gac ctt ccc gcc
acc att gac ttc atc ttg aag aaa acg gga cag 432 Tyr Asp Leu Pro Ala
Thr Ile Asp Phe Ile Leu Lys Lys Thr Gly Gln 130 135 140 gac aag cta
cac tac gtt ggc cat tcc cag ggc acc acc att ggt ttc 480 Asp Lys Leu
His Tyr Val Gly His Ser Gln Gly Thr Thr Ile Gly Phe 145 150 155 160
atc gcc ttt tcc acc aat ccc aag ctg gcg aaa cgg atc aaa acc ttc 528
Ile Ala Phe Ser Thr Asn Pro Lys Leu Ala Lys Arg Ile Lys Thr Phe 165
170 175 tat gca tta gct ccc gtt gcc acc gtg aag tac acc gaa acc ctg
tta 576 Tyr Ala Leu Ala Pro Val Ala Thr Val Lys Tyr Thr Glu Thr Leu
Leu 180 185 190 aac aaa ctc atg ctc gtc cct tcg ttc ctc ttc aag ctt
ata ttt gga 624 Asn Lys Leu Met Leu Val Pro Ser Phe Leu Phe Lys Leu
Ile Phe Gly 195 200 205 aac aaa ata ttc tac cca cac cac ttc ttt gat
caa ttt ctc gcc acc 672 Asn Lys Ile Phe Tyr Pro His His Phe Phe Asp
Gln Phe Leu Ala Thr 210 215 220 gag gta tgc tcc cgc gag acg gtg gat
ctc ctc tgc agc aac gcc ctg 720 Glu Val Cys Ser Arg Glu Thr Val Asp
Leu Leu Cys Ser Asn Ala Leu 225 230 235 240 ttt atc att tgt gga ttt
gac act atg aac ttg aac atg agt cgc ttg 768 Phe Ile Ile Cys Gly Phe
Asp Thr Met Asn Leu Asn Met Ser Arg Leu 245 250 255 gat gtg tat ctg
tca cat aat cca gca gga aca tcg gtt cag aac gtg 816 Asp Val Tyr Leu
Ser His Asn Pro Ala Gly Thr Ser Val Gln Asn Val 260 265 270 ctc cac
tgg tcc cag gct gtt aag tct ggg aag ttc caa gct ttt gac 864 Leu His
Trp Ser Gln Ala Val Lys Ser Gly Lys Phe Gln Ala Phe Asp 275 280 285
tgg gga agc cca gtt cag aac atg atg cac tat cat cag agc atg cct 912
Trp Gly Ser Pro Val Gln Asn Met Met His Tyr His Gln Ser Met Pro 290
295 300 ccc tac tac aac ctg aca gac atg cat gtg cca atc gca gtg tgg
aac 960 Pro Tyr Tyr Asn Leu Thr Asp Met His Val Pro Ile Ala Val Trp
Asn 305 310 315 320 ggt ggc aac gac ttg ctg gcc gac cct cac gat gtt
gac ctt ttg ctt 1008 Gly Gly Asn Asp Leu Leu Ala Asp Pro His Asp
Val Asp Leu Leu Leu 325 330 335 tcc aag ctc ccc aat ctc att tac cac
agg aag att cct cct tac aat 1056 Ser Lys Leu Pro Asn Leu Ile Tyr
His Arg Lys Ile Pro Pro Tyr Asn 340 345 350 cac ttg gac ttt atc tgg
gcc atg gat gcc cct caa gcg gtt tac aat 1104 His Leu Asp Phe Ile
Trp Ala Met Asp Ala Pro Gln Ala Val Tyr Asn 355 360 365 gaa att gtt
tcc atg atg gga aca gat aat aag tagttctaga tttaaggaat 1157 Glu Ile
Val Ser Met Met Gly Thr Asp Asn Lys 370 375 tattctttta ttgttccaaa
atacgttctt ctctcacacg tggttttcta tcatgtttga 1217 gacacggtga
ttgttcccat ggttttgatt tcagaaatgt gttagcatca acaatctttc 1277
cattggtaat ttttgaattt aaaatgattt ttaaatttgg ggcatctggg tggctcagtt
1337 ggctaagtcg tctgccttgg cttaagtcat gatctcgggg tcctaggatg
gagccttgtg 1397 tctgggctcc tgccggggcg ggggtctgct tctcctcctg
ctgctccccc ctgctgctgt 1457 gtgcacacac gctctctctc tctcaaataa
ataaataaat aaatacttaa taaaataaaa 1517 aaaaaaaaaa a 1528 2 379 PRT
Canis familiaris 2 Leu Phe Gly Lys Leu His Pro Thr Asn Pro Glu Val
Thr Met Asn Ile 1 5 10 15 Ser Gln Met Ile Thr Tyr Trp Gly Tyr Pro
Ala Glu Glu Tyr Glu Val 20 25 30 Val Thr Glu Asp Gly Tyr Ile Leu
Gly Ile Asp Arg Ile Pro Tyr Gly 35 40 45 Arg Lys Asn Ser Glu Asn
Ile Gly Arg Arg Pro Val Ala Phe Leu Gln 50 55 60 His Gly Leu Leu
Ala Ser Ala Thr Asn Trp Ile Ser Asn Leu Pro Asn 65 70 75 80 Asn Ser
Leu Ala Phe Ile Leu Ala Asp Ala Gly Tyr Asp Val Trp Leu 85 90 95
Gly Asn Ser Arg Gly Asn Thr Trp Ala Arg Arg Asn Leu Tyr Tyr Ser 100
105 110 Pro Asp Ser Val Glu Phe Trp Ala Phe Ser Phe Asp Glu Met Ala
Lys 115 120 125 Tyr Asp Leu Pro Ala Thr Ile Asp Phe Ile Leu Lys Lys
Thr Gly Gln 130 135 140 Asp Lys Leu His Tyr Val Gly His Ser Gln Gly
Thr Thr Ile Gly Phe 145 150 155 160 Ile Ala Phe Ser Thr Asn Pro Lys
Leu Ala Lys Arg Ile Lys Thr Phe 165 170 175 Tyr Ala Leu Ala Pro Val
Ala Thr Val Lys Tyr Thr Glu Thr Leu Leu 180 185 190 Asn Lys Leu Met
Leu Val Pro Ser Phe Leu Phe Lys Leu Ile Phe Gly 195 200 205 Asn Lys
Ile Phe Tyr Pro His His Phe Phe Asp Gln Phe Leu Ala Thr 210 215 220
Glu Val Cys Ser Arg Glu Thr Val Asp Leu Leu Cys Ser Asn Ala Leu 225
230 235 240 Phe Ile Ile Cys Gly Phe Asp Thr Met Asn Leu Asn Met Ser
Arg Leu 245 250 255 Asp Val Tyr Leu Ser His Asn Pro Ala Gly Thr Ser
Val Gln Asn Val 260 265 270 Leu His Trp Ser Gln Ala Val Lys Ser Gly
Lys Phe Gln Ala Phe Asp 275 280 285 Trp Gly Ser Pro Val Gln Asn Met
Met His Tyr His Gln Ser Met Pro 290 295 300 Pro Tyr Tyr Asn Leu Thr
Asp Met His Val Pro Ile Ala Val Trp Asn 305 310 315 320 Gly Gly Asn
Asp Leu Leu Ala Asp Pro His Asp Val Asp Leu Leu Leu 325 330 335 Ser
Lys Leu Pro Asn Leu Ile Tyr His Arg Lys Ile Pro Pro Tyr Asn 340 345
350 His Leu Asp Phe Ile Trp Ala Met Asp Ala Pro Gln Ala Val Tyr Asn
355 360 365 Glu Ile Val Ser Met Met Gly Thr Asp Asn Lys 370 375 3
1048 DNA Canis familiaris CDS (1)..(975) 3 ata ggc cgg aga cct gtt
gca ttt ttg caa cac ggt ttg ctc gca tca 48 Ile Gly Arg Arg Pro Val
Ala Phe Leu Gln His Gly Leu Leu Ala Ser 1 5 10 15 gcc aca aac tgg
atc tcc aac ctg ccc aac aac agc ctg gcc ttc atc 96 Ala Thr Asn Trp
Ile Ser Asn Leu Pro Asn Asn Ser Leu Ala Phe Ile 20 25 30 ctg gcc
gac gcc ggg tac gac gtg tgg ctg ggg aac agc agg ggc aac 144 Leu Ala
Asp Ala Gly Tyr Asp Val Trp Leu Gly Asn Ser Arg Gly Asn 35 40 45
acc tgg gcc agg agg aat ctg tac tac tcg ccc gac tcc gtc gaa ttc 192
Thr Trp Ala Arg Arg Asn Leu Tyr Tyr Ser Pro Asp Ser Val Glu Phe 50
55 60 tgg gct ttc agc ttt gac gag atg gct aaa tat gac ctt ccc gcc
acc 240 Trp Ala Phe Ser Phe Asp Glu Met Ala Lys Tyr Asp Leu Pro Ala
Thr 65 70 75 80 att gac ttc atc ttg aag aaa acg gga cag gac aag cta
cac tac gtt 288 Ile Asp Phe Ile Leu Lys Lys Thr Gly Gln Asp Lys Leu
His Tyr Val 85 90 95 ggc cat tcc cag ggc acc acc att ggt ttc atc
gcc ttt tcc acc aat 336 Gly His Ser Gln Gly Thr Thr Ile Gly Phe Ile
Ala Phe Ser Thr Asn 100 105 110 ccc aag ctg gcg aaa cgg atc aaa acc
ttc tat gca tta gct ccc gtt 384 Pro Lys Leu Ala Lys Arg Ile Lys Thr
Phe Tyr Ala Leu Ala Pro Val 115 120 125 gcc acc gtg aag tac acc gaa
acc ctg tta aac aaa ctc atg ctc gtc 432 Ala Thr Val Lys Tyr Thr Glu
Thr Leu Leu Asn Lys Leu Met Leu Val 130 135 140 cct tcg ttc ctc ttc
aag ctt ata ttt gga aac aaa ata ttc tac cca 480 Pro Ser Phe Leu Phe
Lys Leu Ile Phe Gly Asn Lys Ile Phe Tyr Pro 145 150 155 160 cac cac
ttc ttt gat caa ttt ctc gcc acc gag gta tgc tcc cgc gag 528 His His
Phe Phe Asp Gln Phe Leu Ala Thr Glu Val Cys Ser Arg Glu 165 170 175
acg gtg gat ctc ctc tgc agc aac gcc ctg ttt atc att tgt gga ttt 576
Thr Val Asp Leu Leu Cys Ser Asn Ala Leu Phe Ile Ile Cys Gly Phe 180
185 190 gac act atg aac ttg aac atg agt cgc ttg gat gtg tat ctg tca
cat 624 Asp Thr Met Asn Leu Asn Met Ser Arg Leu Asp Val Tyr Leu Ser
His 195 200 205 aat cca gca gga aca tcg gtt cag aac gtg ctc cac tgg
tcc cag gct 672 Asn Pro Ala Gly Thr Ser Val Gln Asn Val Leu His Trp
Ser Gln Ala 210 215 220 gtt aag tct ggg aag ttc caa gct ttt gac tgg
gga agc cca gtt cag 720 Val Lys Ser Gly Lys Phe Gln Ala Phe Asp Trp
Gly Ser Pro Val Gln 225 230 235 240 aac atg atg cac tat cat cag agc
atg cct ccc tac tac aac ctg aca 768 Asn Met Met His Tyr His Gln Ser
Met Pro Pro Tyr Tyr Asn Leu Thr 245 250 255 gac atg cat gtg cca atc
gca gtg tgg aac ggt ggc aac gac ttg ctg 816 Asp Met His Val Pro Ile
Ala Val Trp Asn Gly Gly Asn Asp Leu Leu 260 265 270 gcc gac cct cac
gat gtt gac ctt ttg ctt tcc aag ctc ccc aat ctc 864 Ala Asp Pro His
Asp Val Asp Leu Leu Leu Ser Lys Leu Pro Asn Leu 275 280 285 att tac
cac agg aag att cct cct tac aat cac ttg gac ttt atc tgg 912 Ile Tyr
His Arg Lys Ile Pro Pro Tyr Asn His Leu Asp Phe Ile Trp 290 295 300
gcc atg gat gcc cct caa gcg gtt tac aat gaa att gtt tcc atg atg 960
Ala Met Asp Ala Pro Gln Ala Val Tyr Asn Glu Ile Val Ser Met Met 305
310 315 320 gga aca gat aat aag tagttctaga tttaaggaat tattctttta
ttgttccaaa 1015 Gly Thr Asp Asn Lys 325 atacgttctt ctctcacacg
tggttttcta tca 1048 4 325 PRT Canis familiaris 4 Ile Gly Arg Arg
Pro Val Ala Phe Leu Gln His Gly Leu Leu Ala Ser 1 5 10 15 Ala Thr
Asn Trp Ile Ser Asn Leu Pro Asn Asn Ser Leu Ala Phe Ile 20 25 30
Leu Ala Asp Ala Gly Tyr Asp Val Trp Leu Gly Asn Ser Arg Gly Asn 35
40 45 Thr Trp Ala Arg Arg Asn Leu Tyr Tyr Ser Pro Asp Ser Val Glu
Phe 50 55 60 Trp Ala Phe Ser Phe Asp Glu Met Ala Lys Tyr Asp Leu
Pro Ala Thr 65 70 75 80 Ile Asp Phe Ile Leu Lys Lys Thr Gly Gln Asp
Lys Leu His Tyr Val 85 90 95 Gly His Ser Gln Gly Thr Thr Ile Gly
Phe Ile Ala Phe Ser Thr Asn 100 105 110 Pro Lys Leu Ala Lys Arg Ile
Lys Thr Phe Tyr Ala Leu Ala Pro Val 115 120 125 Ala Thr Val Lys Tyr
Thr Glu Thr Leu Leu Asn Lys Leu Met Leu Val 130 135 140 Pro Ser Phe
Leu Phe Lys Leu Ile Phe Gly Asn Lys Ile Phe Tyr Pro 145 150 155 160
His His Phe Phe Asp Gln Phe Leu Ala Thr Glu Val Cys Ser Arg Glu 165
170 175 Thr Val Asp Leu Leu Cys Ser Asn Ala Leu Phe Ile Ile Cys Gly
Phe 180 185 190 Asp Thr Met Asn Leu Asn Met Ser Arg Leu Asp Val Tyr
Leu Ser His 195 200 205 Asn Pro Ala Gly Thr Ser Val Gln Asn Val Leu
His Trp Ser Gln Ala 210 215 220 Val Lys Ser Gly Lys Phe Gln Ala Phe
Asp Trp Gly Ser Pro Val Gln 225 230 235 240 Asn Met Met His Tyr His
Gln Ser Met Pro Pro Tyr Tyr Asn Leu Thr 245 250 255 Asp Met His Val
Pro Ile Ala Val Trp Asn Gly Gly Asn Asp Leu Leu 260 265 270 Ala Asp
Pro His Asp Val Asp Leu Leu Leu Ser Lys Leu Pro Asn Leu 275 280 285
Ile Tyr His Arg Lys Ile Pro Pro Tyr Asn His Leu Asp Phe Ile Trp 290
295 300 Ala Met Asp Ala Pro Gln Ala Val Tyr Asn Glu Ile Val Ser Met
Met 305 310 315 320 Gly Thr Asp Asn Lys 325 5 1198 DNA Canis
familiaris CDS (1)..(1125) 5 ctt cat ccc aca aac cct gaa gtg acc
atg aat ata agt cag atg atc 48 Leu His Pro Thr Asn Pro Glu Val Thr
Met Asn Ile Ser Gln Met Ile 1 5 10 15 acc tac tgg gga tac cca gct
gag gaa tat gaa gtt gtg acc gaa gac 96 Thr Tyr Trp Gly Tyr Pro Ala
Glu Glu Tyr Glu Val Val Thr Glu Asp 20 25 30 ggt tat atc ctt ggg
atc gac aga att cct tat ggg agg aaa aat tca 144 Gly Tyr Ile Leu Gly
Ile Asp Arg Ile Pro Tyr Gly Arg Lys Asn Ser 35 40 45 gag aat ata
ggc cgg aga cct gtt gca ttt ttg caa cac ggt ttg ctc 192 Glu Asn Ile
Gly Arg Arg Pro Val Ala Phe Leu Gln His Gly Leu Leu 50 55 60 gca
tca gcc aca aac tgg atc tcc aac ctg ccc aac aac agc ctg gcc 240 Ala
Ser Ala Thr Asn Trp Ile Ser Asn Leu Pro Asn Asn Ser Leu Ala 65 70
75 80 ttc atc ctg gcc gac gcc ggg tac gac gtg tgg ctg ggg aac agc
agg 288 Phe Ile Leu Ala Asp Ala Gly Tyr Asp Val Trp Leu Gly Asn Ser
Arg 85 90 95 ggc aac acc tgg gcc agg agg aat ctg tac tac tcg ccc
gac tcc gtc 336 Gly Asn Thr Trp Ala Arg Arg Asn Leu Tyr Tyr Ser Pro
Asp Ser Val 100 105 110 gaa ttc tgg gct ttc agc ttt gac gag atg gct
aaa tat gac ctt ccc 384 Glu Phe Trp Ala Phe Ser Phe Asp Glu Met Ala
Lys Tyr Asp Leu Pro 115 120 125 gcc acc att gac ttc atc ttg aag aaa
acg gga cag gac aag cta cac 432 Ala Thr Ile Asp Phe Ile Leu Lys Lys
Thr Gly Gln Asp Lys Leu His 130 135 140 tac gtt ggc cat tcc cag ggc
acc acc att ggt ttc atc gcc ttt tcc 480 Tyr Val Gly His Ser Gln Gly
Thr Thr Ile Gly Phe Ile Ala Phe Ser 145 150 155 160 acc aat ccc aag
ctg gcg aaa cgg atc aaa acc ttc tat gca tta gct 528 Thr Asn Pro Lys
Leu Ala Lys Arg Ile Lys Thr Phe Tyr Ala Leu Ala 165 170 175 ccc gtt
gcc acc gtg aag tac acc gaa acc ctg tta aac aaa ctc atg 576 Pro Val
Ala Thr Val Lys Tyr Thr Glu Thr Leu Leu Asn Lys Leu Met 180 185 190
ctc gtc cct tcg ttc ctc ttc aag ctt ata ttt gga aac aaa ata ttc 624
Leu Val Pro Ser Phe Leu Phe Lys Leu Ile Phe Gly Asn Lys Ile Phe 195
200 205 tac cca cac cac ttc ttt gat caa ttt ctc gcc acc gag gta tgc
tcc 672 Tyr Pro His His Phe Phe Asp Gln Phe Leu Ala Thr Glu Val Cys
Ser 210 215 220 cgc gag acg gtg gat ctc ctc tgc agc aac gcc ctg ttt
atc att tgt 720 Arg Glu Thr Val Asp Leu Leu Cys Ser Asn Ala Leu Phe
Ile Ile Cys 225 230 235 240 gga ttt gac act atg aac ttg aac atg agt
cgc ttg gat gtg tat ctg 768 Gly Phe Asp Thr Met Asn Leu Asn Met Ser
Arg Leu Asp Val Tyr Leu 245 250 255 tca cat aat cca gca gga aca tcg
gtt cag aac gtg ctc cac tgg tcc 816 Ser His Asn Pro Ala Gly Thr Ser
Val Gln Asn Val Leu His Trp Ser 260 265 270 cag gct gtt aag tct ggg
aag ttc caa gct ttt gac tgg gga agc cca 864 Gln Ala Val Lys Ser Gly
Lys Phe Gln Ala Phe Asp Trp Gly Ser Pro 275 280 285 gtt cag aac atg
atg cac tat cat cag agc atg cct ccc tac tac aac 912 Val Gln Asn Met
Met His Tyr His Gln Ser Met Pro Pro Tyr Tyr Asn 290 295 300 ctg aca
gac atg cat gtg cca atc gca gtg tgg aac ggt ggc aac gac 960 Leu Thr
Asp Met His Val Pro Ile Ala
Val Trp Asn Gly Gly Asn Asp 305 310 315 320 ttg ctg gcc gac cct cac
gat gtt gac ctt ttg ctt tcc aag ctc ccc 1008 Leu Leu Ala Asp Pro
His Asp Val Asp Leu Leu Leu Ser Lys Leu Pro 325 330 335 aat ctc att
tac cac agg aag att cct cct tac aat cac ttg gac ttt 1056 Asn Leu
Ile Tyr His Arg Lys Ile Pro Pro Tyr Asn His Leu Asp Phe 340 345 350
atc tgg gcc atg gat gcc cct caa gcg gtt tac aat gaa att gtt tcc
1104 Ile Trp Ala Met Asp Ala Pro Gln Ala Val Tyr Asn Glu Ile Val
Ser 355 360 365 atg atg gga aca gat aat aag tagttctaga tttaaggaat
tattctttta 1155 Met Met Gly Thr Asp Asn Lys 370 375 ttgttccaaa
atacgttctt ctctcacacg tggttttcta tca 1198 6 375 PRT Canis
familiaris 6 Leu His Pro Thr Asn Pro Glu Val Thr Met Asn Ile Ser
Gln Met Ile 1 5 10 15 Thr Tyr Trp Gly Tyr Pro Ala Glu Glu Tyr Glu
Val Val Thr Glu Asp 20 25 30 Gly Tyr Ile Leu Gly Ile Asp Arg Ile
Pro Tyr Gly Arg Lys Asn Ser 35 40 45 Glu Asn Ile Gly Arg Arg Pro
Val Ala Phe Leu Gln His Gly Leu Leu 50 55 60 Ala Ser Ala Thr Asn
Trp Ile Ser Asn Leu Pro Asn Asn Ser Leu Ala 65 70 75 80 Phe Ile Leu
Ala Asp Ala Gly Tyr Asp Val Trp Leu Gly Asn Ser Arg 85 90 95 Gly
Asn Thr Trp Ala Arg Arg Asn Leu Tyr Tyr Ser Pro Asp Ser Val 100 105
110 Glu Phe Trp Ala Phe Ser Phe Asp Glu Met Ala Lys Tyr Asp Leu Pro
115 120 125 Ala Thr Ile Asp Phe Ile Leu Lys Lys Thr Gly Gln Asp Lys
Leu His 130 135 140 Tyr Val Gly His Ser Gln Gly Thr Thr Ile Gly Phe
Ile Ala Phe Ser 145 150 155 160 Thr Asn Pro Lys Leu Ala Lys Arg Ile
Lys Thr Phe Tyr Ala Leu Ala 165 170 175 Pro Val Ala Thr Val Lys Tyr
Thr Glu Thr Leu Leu Asn Lys Leu Met 180 185 190 Leu Val Pro Ser Phe
Leu Phe Lys Leu Ile Phe Gly Asn Lys Ile Phe 195 200 205 Tyr Pro His
His Phe Phe Asp Gln Phe Leu Ala Thr Glu Val Cys Ser 210 215 220 Arg
Glu Thr Val Asp Leu Leu Cys Ser Asn Ala Leu Phe Ile Ile Cys 225 230
235 240 Gly Phe Asp Thr Met Asn Leu Asn Met Ser Arg Leu Asp Val Tyr
Leu 245 250 255 Ser His Asn Pro Ala Gly Thr Ser Val Gln Asn Val Leu
His Trp Ser 260 265 270 Gln Ala Val Lys Ser Gly Lys Phe Gln Ala Phe
Asp Trp Gly Ser Pro 275 280 285 Val Gln Asn Met Met His Tyr His Gln
Ser Met Pro Pro Tyr Tyr Asn 290 295 300 Leu Thr Asp Met His Val Pro
Ile Ala Val Trp Asn Gly Gly Asn Asp 305 310 315 320 Leu Leu Ala Asp
Pro His Asp Val Asp Leu Leu Leu Ser Lys Leu Pro 325 330 335 Asn Leu
Ile Tyr His Arg Lys Ile Pro Pro Tyr Asn His Leu Asp Phe 340 345 350
Ile Trp Ala Met Asp Ala Pro Gln Ala Val Tyr Asn Glu Ile Val Ser 355
360 365 Met Met Gly Thr Asp Asn Lys 370 375 7 1528 DNA Canis
familiaris 7 ttgtttggaa aattacatcc cacaaaccct gaagtgacca tgaatataag
tcagatgatc 60 acctactggg gatacccagc tgaggaatat gaagttgtga
ccgaagacgg ttatatcctt 120 gggatcgaca gaattcctta tgggaggaaa
aattcagaga atataggccg gagacctgtt 180 gcatttttgc aacacggttt
gctcgcatca gccacaaact ggatctccaa cctgcccaac 240 aacagcctgg
ccttcatcct ggccgacgcc gggtacgacg tgtggctggg gaacagcagg 300
ggcaacacct gggccaggag gaatctgtac tactcgcccg actccgtcga attctgggct
360 ttcagctttg acgagatggc taaatatgac cttcccgcca ccattgactt
catcttgaag 420 aaaacgggac aggacaagct acactacgtt ggccattccc
agggcaccac cattggtttc 480 atcgcctttt ccaccaatcc caagctggcg
aaacggatca aaaccttcta tgcattagct 540 cccgttgcca ccgtgaagta
caccgaaacc ctgttaaaca aactcatgct cgtcccttcg 600 ttcctcttca
agcttatatt tggaaacaaa atattctacc cacaccactt ctttgatcaa 660
tttctcgcca ccgaggtatg ctcccgcgag acggtggatc tcctctgcag caacgccctg
720 tttatcattt gtggatttga cactatgaac ttgaacatga gtcgcttgga
tgtgtatctg 780 tcacataatc cagcaggaac atcggttcag aacgtgctcc
actggtccca ggctgttaag 840 tctgggaagt tccaagcttt tgactgggga
agcccagttc agaacatgat gcactatcat 900 cagagcatgc ctccctacta
caacctgaca gacatgcatg tgccaatcgc agtgtggaac 960 ggtggcaacg
acttgctggc cgaccctcac gatgttgacc ttttgctttc caagctcccc 1020
aatctcattt accacaggaa gattcctcct tacaatcact tggactttat ctgggccatg
1080 gatgcccctc aagcggttta caatgaaatt gtttccatga tgggaacaga
taataagtag 1140 ttctagattt aaggaattat tcttttattg ttccaaaata
cgttcttctc tcacacgtgg 1200 ttttctatca tgtttgagac acggtgattg
ttcccatggt tttgatttca gaaatgtgtt 1260 agcatcaaca atctttccat
tggtaatttt tgaatttaaa atgattttta aatttggggc 1320 atctgggtgg
ctcagttggc taagtcgtct gccttggctt aagtcatgat ctcggggtcc 1380
taggatggag ccttgtgtct gggctcctgc cggggcgggg gtctgcttct cctcctgctg
1440 ctcccccctg ctgctgtgtg cacacacgct ctctctctct caaataaata
aataaataaa 1500 tacttaataa aataaaaaaa aaaaaaaa 1528 8 1367 DNA Homo
sapiens CDS (47)..(1240) 8 agagaaacag aatcctaact atttctgagg
aaactgcagg tccaaa atg tgg ctg 55 Met Trp Leu 1 ctt tta aca atg gca
agt ttg ata tct gta ctg ggg act aca cat ggt 103 Leu Leu Thr Met Ala
Ser Leu Ile Ser Val Leu Gly Thr Thr His Gly 5 10 15 ttg ttt gga aaa
tta cat cct gga agc cct gaa gtg act atg aac att 151 Leu Phe Gly Lys
Leu His Pro Gly Ser Pro Glu Val Thr Met Asn Ile 20 25 30 35 agt cag
atg att act tat tgg gga tac cca aat gaa gaa tat gaa gtt 199 Ser Gln
Met Ile Thr Tyr Trp Gly Tyr Pro Asn Glu Glu Tyr Glu Val 40 45 50
gtg act gaa gat ggt tat att ctt gaa gtc aat aga att cct tat ggg 247
Val Thr Glu Asp Gly Tyr Ile Leu Glu Val Asn Arg Ile Pro Tyr Gly 55
60 65 aag aaa aat tca ggg aat aca ggc cag aga cct gtt gtg ttt ttg
cag 295 Lys Lys Asn Ser Gly Asn Thr Gly Gln Arg Pro Val Val Phe Leu
Gln 70 75 80 cat ggt ttg ctt gca tca gcc aca aac tgg att tcc aac
ctg ccg aac 343 His Gly Leu Leu Ala Ser Ala Thr Asn Trp Ile Ser Asn
Leu Pro Asn 85 90 95 aac agc ctt gcc ttc att ctg gca gat gct ggt
tat gat gtg tgg ctg 391 Asn Ser Leu Ala Phe Ile Leu Ala Asp Ala Gly
Tyr Asp Val Trp Leu 100 105 110 115 ggc aac agc aga gga aac acc tgg
gcc aga aga aac ttg tac tat tca 439 Gly Asn Ser Arg Gly Asn Thr Trp
Ala Arg Arg Asn Leu Tyr Tyr Ser 120 125 130 cca gat tca gtt gaa ttc
tgg gct ttc agc ttt gat gaa atg gct aaa 487 Pro Asp Ser Val Glu Phe
Trp Ala Phe Ser Phe Asp Glu Met Ala Lys 135 140 145 tat gac ctt cca
gcc aca atc gac ttc att gta aag aaa act gga cag 535 Tyr Asp Leu Pro
Ala Thr Ile Asp Phe Ile Val Lys Lys Thr Gly Gln 150 155 160 aag cag
cta cac tat gtt ggc cat tcc cag ggc acc acc att ggt ttt 583 Lys Gln
Leu His Tyr Val Gly His Ser Gln Gly Thr Thr Ile Gly Phe 165 170 175
att gcc ttt tcc acc aat ccc agc ctg gct aaa aga atc aaa acc ttc 631
Ile Ala Phe Ser Thr Asn Pro Ser Leu Ala Lys Arg Ile Lys Thr Phe 180
185 190 195 tat gct cta gct cct gtt gcc act gtg aag tat aca aaa agc
ctt ata 679 Tyr Ala Leu Ala Pro Val Ala Thr Val Lys Tyr Thr Lys Ser
Leu Ile 200 205 210 aac aaa ctt aga ttt gtt cct caa tcc ctc ttc aag
ttt ata ttt ggt 727 Asn Lys Leu Arg Phe Val Pro Gln Ser Leu Phe Lys
Phe Ile Phe Gly 215 220 225 gac aaa ata ttc tac cca cac aac ttc ttt
gat caa ttt ctt gct act 775 Asp Lys Ile Phe Tyr Pro His Asn Phe Phe
Asp Gln Phe Leu Ala Thr 230 235 240 gaa gtg tgc tcc cgt gag atg ctg
aat ctc ctt tgc agc aat gcc tta 823 Glu Val Cys Ser Arg Glu Met Leu
Asn Leu Leu Cys Ser Asn Ala Leu 245 250 255 ttt ata att tgt gga ttt
gac agt aag aac ttt aac acg agt cgc ttg 871 Phe Ile Ile Cys Gly Phe
Asp Ser Lys Asn Phe Asn Thr Ser Arg Leu 260 265 270 275 gat gtg tat
cta tca cat aat cca gca gga act tct gtt caa aac atg 919 Asp Val Tyr
Leu Ser His Asn Pro Ala Gly Thr Ser Val Gln Asn Met 280 285 290 ttc
cat tgg acc cag gct gtt aag tct ggg aaa ttc caa gct tat gac 967 Phe
His Trp Thr Gln Ala Val Lys Ser Gly Lys Phe Gln Ala Tyr Asp 295 300
305 tgg gga agc cca gtt cag aat agg atg cac tat gat cag tcc caa cct
1015 Trp Gly Ser Pro Val Gln Asn Arg Met His Tyr Asp Gln Ser Gln
Pro 310 315 320 ccc tac tac aat gtg aca gcc atg aat gta cca att gca
gtg tgg aac 1063 Pro Tyr Tyr Asn Val Thr Ala Met Asn Val Pro Ile
Ala Val Trp Asn 325 330 335 ggt ggc aag gac ctg ttg gct gac ccc caa
gat gtt ggc ctt ttg ctt 1111 Gly Gly Lys Asp Leu Leu Ala Asp Pro
Gln Asp Val Gly Leu Leu Leu 340 345 350 355 cca aaa ctc ccc aat ctt
att tac cac aag gag att cct ttt tac aat 1159 Pro Lys Leu Pro Asn
Leu Ile Tyr His Lys Glu Ile Pro Phe Tyr Asn 360 365 370 cac ttg gac
ttt atc tgg gca atg gat gcc cct caa gaa gtt tac aat 1207 His Leu
Asp Phe Ile Trp Ala Met Asp Ala Pro Gln Glu Val Tyr Asn 375 380 385
gac att gtt tct atg ata tca gaa gat aaa aag tagttctgga tttaaagaat
1260 Asp Ile Val Ser Met Ile Ser Glu Asp Lys Lys 390 395 tatccgtttg
tttttccaaa atactttatt ctctcataca tagtattttc ataatgtttg 1320
acatgcagtg cttctttctg taattttgac tttagaaata tattggc 1367 9 398 PRT
Homo sapiens 9 Met Trp Leu Leu Leu Thr Met Ala Ser Leu Ile Ser Val
Leu Gly Thr 1 5 10 15 Thr His Gly Leu Phe Gly Lys Leu His Pro Gly
Ser Pro Glu Val Thr 20 25 30 Met Asn Ile Ser Gln Met Ile Thr Tyr
Trp Gly Tyr Pro Asn Glu Glu 35 40 45 Tyr Glu Val Val Thr Glu Asp
Gly Tyr Ile Leu Glu Val Asn Arg Ile 50 55 60 Pro Tyr Gly Lys Lys
Asn Ser Gly Asn Thr Gly Gln Arg Pro Val Val 65 70 75 80 Phe Leu Gln
His Gly Leu Leu Ala Ser Ala Thr Asn Trp Ile Ser Asn 85 90 95 Leu
Pro Asn Asn Ser Leu Ala Phe Ile Leu Ala Asp Ala Gly Tyr Asp 100 105
110 Val Trp Leu Gly Asn Ser Arg Gly Asn Thr Trp Ala Arg Arg Asn Leu
115 120 125 Tyr Tyr Ser Pro Asp Ser Val Glu Phe Trp Ala Phe Ser Phe
Asp Glu 130 135 140 Met Ala Lys Tyr Asp Leu Pro Ala Thr Ile Asp Phe
Ile Val Lys Lys 145 150 155 160 Thr Gly Gln Lys Gln Leu His Tyr Val
Gly His Ser Gln Gly Thr Thr 165 170 175 Ile Gly Phe Ile Ala Phe Ser
Thr Asn Pro Ser Leu Ala Lys Arg Ile 180 185 190 Lys Thr Phe Tyr Ala
Leu Ala Pro Val Ala Thr Val Lys Tyr Thr Lys 195 200 205 Ser Leu Ile
Asn Lys Leu Arg Phe Val Pro Gln Ser Leu Phe Lys Phe 210 215 220 Ile
Phe Gly Asp Lys Ile Phe Tyr Pro His Asn Phe Phe Asp Gln Phe 225 230
235 240 Leu Ala Thr Glu Val Cys Ser Arg Glu Met Leu Asn Leu Leu Cys
Ser 245 250 255 Asn Ala Leu Phe Ile Ile Cys Gly Phe Asp Ser Lys Asn
Phe Asn Thr 260 265 270 Ser Arg Leu Asp Val Tyr Leu Ser His Asn Pro
Ala Gly Thr Ser Val 275 280 285 Gln Asn Met Phe His Trp Thr Gln Ala
Val Lys Ser Gly Lys Phe Gln 290 295 300 Ala Tyr Asp Trp Gly Ser Pro
Val Gln Asn Arg Met His Tyr Asp Gln 305 310 315 320 Ser Gln Pro Pro
Tyr Tyr Asn Val Thr Ala Met Asn Val Pro Ile Ala 325 330 335 Val Trp
Asn Gly Gly Lys Asp Leu Leu Ala Asp Pro Gln Asp Val Gly 340 345 350
Leu Leu Leu Pro Lys Leu Pro Asn Leu Ile Tyr His Lys Glu Ile Pro 355
360 365 Phe Tyr Asn His Leu Asp Phe Ile Trp Ala Met Asp Ala Pro Gln
Glu 370 375 380 Val Tyr Asn Asp Ile Val Ser Met Ile Ser Glu Asp Lys
Lys 385 390 395 10 29 DNA Artificial sequence primer 10 caggagatct
tgttggaaag cttcatccc 29 11 21 DNA Artificial sequence PCR
mutagenesis primer 11 catattcctc agctgggtat c 21 12 9 PRT Canis
familiaris 12 Leu Phe Gly Lys Leu Thr Asp Asn Lys 1 5 13 42 DNA
Canis familiaris 13 agatcttgtt tggaaagctt acagataata agtagttcta ga
42 14 69 DNA Artificial sequence Sequence encoding signal peptide
of sporamin A 14 atgaaagcct tcacactcgc tctcttctta gctctttccc
tctatctcct gcccaatcca 60 gcccattcc 69 15 31 DNA Artificial sequence
PCR primer 15 caggagatct gatgaaagcc ttcacactcg c 31 16 40 DNA
Artificial sequence PCR primer 16 atgaagcttt ccaaacaagg aatgggctgg
attgggcagg 40 17 111 DNA Artificial sequence Sequence encoding
signal prepropeptide of sporamin A 17 atgaaagcct tcacactcgc
tctcttctta gctctttccc tctatctcct gcccaatcca 60 gcccattcca
ggttcaatcc catccgcctc cccaccacac acgaacccgc c 111 18 38 DNA
Artificial sequence primer 18 atgaagcttt ccaaacaagg cgggttcgtg
tgtggttg 38 19 19 PRT Rabbit 19 Met Trp Val Leu Phe Met Val Ala Ala
Leu Leu Ser Ala Leu Gly Thr 1 5 10 15 Thr His Gly 20 57 DNA Rabbit
20 atgtgggtgc ttttcatggt ggcagctttg ctatctgcac ttggaactac acatggt
57 21 35 DNA Artificial sequence PCR primer 21 aggagatctc
aacaatgtgg gtgcttttca tggtg 35 22 38 DNA Artificial sequence PCR
primer 22 atgaagcttt ccaaacaaac catgtgtagt tccaagtg 38 23 19 DNA
Artificial sequence PCR primer 23 caaacgtgta caatagccc 19 24 18 DNA
Artificial sequence PCR primer 24 cccggggatc cttttttg 18 25 19 DNA
Artificial sequence PCR primer 25 aagtacggcc actaccacg 19 26 17 DNA
Artificial sequence PCR primer 26 cccggggatc ctggctc 17 27 42 DNA
Unknown PCR primer 27 ttgtttggaa aattacatcc tggatcccct gaagtgacta
tg 42 28 40 DNA Artificial sequence PCR primer 28 aatggtggtg
ccctgggaat ggccaacata gtgtagctgc 40 29 48 DNA Homo sapiens 29
atgctgccac tttggactct ttcactgctg ctgggagcag tagcagga 48 30 99 DNA
Homo sapiens 30 aaactgcagg ctcgagaaca atgctgccac tttggactct
ttcactgctg ctgggagcag 60 tagcaggatt gtttggaaaa ttacatcctg gatcccctg
99 31 24 DNA Artificial sequence PCR primer 31 aaactgcagg
ctcgagaaca atgc 24 32 24 DNA Unknown PCR primer 32 aggggatcca
ggatgtaatt ttcc 24 33 57 DNA Rabbit 33 atgtgggtgc ttttcatggt
ggcagctttg ctatctgcac ttggaactac acatggt 57 34 108 DNA Artificial
sequence PCR template 34 aaactgcagg ctcgagaaca atgtgggtgc
ttttcatggt ggcagctttg ctatctgcac 60 ttggaactac acatggtttg
tttggaaaat tacatcctgg atcccctg 108 35 26 DNA Unknown PCR primer 35
aaactgcagg ctcgagaaca atgtgg 26 36 33 DNA Artificial sequence PCR
primer 36 aatcacttgg actttatctg ggccatggat gcc 33 37 97 DNA
Artificial sequence PCR primer 37 attcttaaga aactttattg ccaaatgttt
gaacgatcgg ggaaattcga ctgcgtctag 60 aactatagct catccttatt
atctgttccc atcatgg 97
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