U.S. patent application number 12/819068 was filed with the patent office on 2012-03-08 for methods for the production of apolipoproteins in transgenic plants.
This patent application is currently assigned to SemBioSys Genetics Inc.. Invention is credited to Maurice M. MOLONEY, Alexandra Reid.
Application Number | 20120059150 12/819068 |
Document ID | / |
Family ID | 34594972 |
Filed Date | 2012-03-08 |
United States Patent
Application |
20120059150 |
Kind Code |
A1 |
MOLONEY; Maurice M. ; et
al. |
March 8, 2012 |
METHODS FOR THE PRODUCTION OF APOLIPOPROTEINS IN TRANSGENIC
PLANTS
Abstract
Methods for the production of an apolipoprotein in plants are
described. In one embodiment, the present invention provides a
method for the expression of apolipoprotein in plants comprising:
(a) providing a chimeric nucleic acid construct comprising in the
5' to 3' direction of transcription as operably linked components:
(i) a nucleic acid sequence capable of controlling expression in
plant cells; and (ii) a nucleic acid sequence encoding an
apolipoprotein polypeptide; (b) introducing the chimeric nucleic
acid construct into a plant cell; and growing the plant cell into a
mature plant capable of setting seed wherein the seed expresses
apolipoprotein.
Inventors: |
MOLONEY; Maurice M.;
(Calgary, CA) ; Reid; Alexandra; (Grenoble,
FR) |
Assignee: |
SemBioSys Genetics Inc.
|
Family ID: |
34594972 |
Appl. No.: |
12/819068 |
Filed: |
June 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10987454 |
Nov 15, 2004 |
7786352 |
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12819068 |
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60519606 |
Nov 14, 2003 |
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60579733 |
Jun 16, 2004 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/69.1; 536/23.6 |
Current CPC
Class: |
C12N 15/8257 20130101;
A61P 3/06 20180101; C07K 14/775 20130101 |
Class at
Publication: |
530/350 ;
536/23.6; 435/320.1; 435/69.1 |
International
Class: |
C07K 14/775 20060101
C07K014/775; C12N 15/63 20060101 C12N015/63; C12P 21/02 20060101
C12P021/02; C12N 15/29 20060101 C12N015/29 |
Claims
1-38. (canceled)
39. A composition comprising substantially pure oil bodies
comprising apolipoprotein obtained from plants.
40. A nucleic acid sequence encoding apolipoprotein linked to
nucleic acid sequence comprising a nucleic acid capable of
controlling expression in a plant cell.
41. A nucleic acid sequence according to claim 40 wherein said
plant cell is a seed cell.
42. A nucleic acid sequence according to claim 41 wherein said
nucleic acid sequence capable of controlling expression in a plant
cell is a seed-preferred promoter.
43. A nucleic acid according to claim 42 wherein said
seed-preferred promoter is the phaseolin promoter.
44. A nucleic acid sequence according to claim 41 wherein said
nucleic acid sequence capable of controlling expression in plant
seeds is a constitutive promoter.
45. A nucleic acid sequence according to claim 44 wherein said
promoter is the ubiquitin promoter.
46. A recombinant expression vector suitable for expression in a
plant cell comprising a nucleic acid sequence encoding an
apolipoprotein polypeptide.
47. A method for preparing substantially pure apolipoprotein
comprising: (a) providing a chimeric nucleic acid construct
comprising in the 5' to 3' direction of transcription as operably
linked components: (i) a nucleic acid sequence capable of
controlling expression in plant seed cells; and (ii) a nucleic acid
sequence encoding an apolipoprotein polypeptide; (b) introducing
the chimeric nucleic acid construct into a plant cell; (c) growing
the plant cell into a mature plant; and (d) obtaining seed from
said plant wherein the seed comprises apolipoprotein; and (e)
separating apolipoprotein from the plant seed constituents to
obtain substantially pure apolipoprotein.
48. (canceled)
49. A composition comprising substantially pure apolipoprotein
obtained from a plant.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to plant genetic engineering
methods and to the production of apolipoproteins. More
specifically, the present invention relates to methods for the
production of recombinant apolipoproteins in transgenic plants.
BACKGROUND OF THE INVENTION
[0002] In a healthy human body, there is a balance between the
delivery and removal of cholesterol. When people have a high level
of low-density lipoprotein (LDL) and low level of high-density
lipoprotein (HDL), the imbalance results in more cholesterol being
deposited in the arteries than that being removed (van Dam, M. J.
et al. 2002, Lancet 359: 37-42)). Atherosclerosis, the narrowing or
blocking of arteries, is a consequence of the repeated deposit of
cholesterol, termed plaque (Major, A. S. et al. 2001, Arterioscler.
Thromb. Vasc. Biol. 21: 1790-1795)).
[0003] Lipoproteins can be separated into atherogenetic and
vasoprotective lipoproteins. Atherogenetic lipoproteins are
generally all apolipoprotein (Apo) B-containing lipoprotein such as
very-low-density lipoprotein (VLDL), intermediate (IDL), low (LDL)
or lipoprotein (Lp(a)), whereas vasoprotective lipoproteins are Apo
AI containing, such as HDL.
[0004] Apo AI, the major protein constituent of HDL, plays a
critical role in cholesterol homeostasis. Clinical and
population-based studies have demonstrated a remarkable inverse
correlation between cardiovascular disease and plasma HDL levels,
suggesting Apo AI and HDL help to serve a protective role against
atherogenesis (Rubins, H. B. et al. 1993, Am. J. Cartiol. 71:
45-52)). Studies of transgenic mice (Rubin, E. M. et al. 1991,
Nature 353: 265-267)) and rabbits (Duverger, N. et al. 1996,
Circulation 94: 713-717)) susceptible to atherosclerosis have shown
that expression of human Apo AI inhibits the development of
atherosclerosis. This effect may be related to its efficient
promotion of cholesterol efflux from cells (Castro, G. et al. 1997,
Biochemistry 36: 2243-2249)), the first step in the process of
`reverse cholesterol transport` (RCT) (Glomset, J. A. 1968, J Lipid
Res. 9: 155-167)). Apo AI modulates this process by being a
preferential acceptor of cellular cholesterol (Rothblat, G. H. et
al. 1999, J Lipid Res. 40: 781-796)), increasing the activity of
lecithin-cholesterol-acyl-transferase (LCAT) esterification of
HDL-associated cholesterol several-fold (Jonas, A. 1991, Biochim.
Biophys. Acta 1084: 205-220; Mahley, R. W. et al. 1984, J Lipid
Res. 25: 1277-1294)), and transporting LCAT-derived cholesteryl
esters to the liver (Morrison, J. R. et al. 1992, J Biol. Chem.
267: 13205-13209)).
[0005] Unlike synthetic antihyperlipidemics, such as LIPITOR.RTM.
(atorvastatin calcium) that act to lower lipid levels in the body
by inhibiting the synthesis of cholesterol (Alaupovic, P. et al.
1997, Atherosclerosis 133: 123-133)), an infusion of purified Apo
AI stimulates cholesterol efflux from tissues into plasma (Navab,
M. et al. 2002, Circulation 105: 290-292)). This suggests that Apo
AI could stimulate cholesterol efflux from foam cells in the
arterial wall and induce regression of atherosclerotic plaque,
effectively `cleaning out` the arteries.
[0006] In humans, Apo AI is synthesized in liver and intestinal
cells as a non-glycosylated pre-pro-protein (Gordon, J. I. et al.
1983, J. Biol. Chem. 258: 4037-4044)). The 18 amino acid
pre-segment is removed before the protein leaves the cell and the 6
amino acid pro-segment is cleaved post secretion by an unknown
protease in the plasma, leaving the mature 243 amino acid protein
(Saku, K. et al. 1999, Eur. J. Clin. Invest. 29: 196-203)).
[0007] The Apolipoprotein A-I.sub.Milano (Apo AI-M) is the first
described molecular variant of human Apo AI (Franceschini, G. et
al. 1980, J. Clin. Invest. 66: 892-900)). It is characterized by
the substitution of Arg 173 with Cys (Weisgraber, K. H. et al.
1983, J. Biol. Chem. 258: 2508-2513)). The mutant apolipoprotein is
transmitted as an autosomal dominant trait and 8 generations of
carriers have been identified (Gerli, G. C. et al. 1984, Hum.
Hered. 34: 133-140)).
[0008] The status of the Apo AI-M carrier individual is
characterized by a remarkable reduction in HDL-cholesterol level.
In spite of this, the affected subjects do not apparently show any
increased risk of arterial disease; indeed, by examination of the
genealogic tree it appears that these subjects may be "protected"
from atherosclerosis.
[0009] The mechanism of the possible protective effect of Apo AI-M
in the carriers seems to be linked to a modification in the
structure of the mutant apolipoprotein, with the loss of one
alpha-helix and an increased exposure of hydrophobic residues
(Franceschini, G. et al. 1985, J. Biol. Chem. 260: 16321-16325).
The loss of the tight structure of the multiple alpha-helices leads
to an increased flexibility of the molecule, which associates more
readily with lipids, compared to normal A-I. Moreover,
apolipoprotein/lipid complexes are more susceptible to
denaturation, thus suggesting that lipid delivery is also improved
in the case of the mutant.
[0010] The therapeutic use of Apo AI and the Apo AI-M mutant is
presently limited by the lack of a method allowing the preparation
of said apolipoproteins in sufficient amount and in a suitable
form. In particular, the recombinant production of Apo AI has been
shown to be very difficult due to its amphiphilic character,
autoaggregation, and degradation (Schmidt, H. H. et al. 1997,
Protein Expr. Purif. 10: 226-236). At the time of the present
invention, recombinant human Apo AI has been expressed in vitro in
two eukaryotic systems: Baculovirus transfected Spodoptera
frugiperda (Sf9) cells (Sorci-Thomas, M. G. et al. 1996, J. Lipid
Res. 37: 673-683) and stably transfected Chinese hamster ovary
(CHO) cells (Forte, T. M. et al. 1990, Biochim. Biophys. Acta 1047:
11-18; Mallory, J. B. et al. 1987, J. Biol. Chem. 262: 4241-4247).
In the baculovirus system, once the cells are successfully
transfected, there is an in-depth screening process before cells
with the correct construct can be used for expression. Similarly,
CHO cell colonies must undergo a screening process to find stably
transfected, high expressing colonies. Additionally, both of these
cell types require a relatively long period of time before
significant expression is achieved and a much higher level of
maintenance than bacteria.
[0011] Recombinant expression of proteins in bacterial systems is
generally attractive because it can produce large amounts of pure
protein quickly and economically There are several reports of Apo
AI expression in transformed Escherichia coli (E. coli); however,
while certain recent improvements in expression levels have been
made (Ryan, R. O. et al. 2003, Protein Expr. Purif. 27: 98-103) in
general, these methods provide relatively low yields or the
undesirable presence of extraneous affinity tags or secretion
signals (Bergeron, J. et al. 1997, Biochim. Biophys. Acta 1344:
139-152; Li, H. H. et al. 2001, J. Lipid Res. 42: 2084-2091;
McGuire, K. A. et al. 1996, J. Lipid Res. 37: 1519-1528). Moreover,
E. coli endotoxins are known to form particularly strong complexes
with apolipoproteins (Emancipator et al. (1992) Infect. Immun. 60:
596-601). Reduction or elimination of the toxicity associated with
these E. coli endotoxins in pharmaceutical preparations of
apoliproteins is highly desirable. The removal of these endotoxins,
while technically feasible, involves complex and expensive protein
purification methodologies (U.S. Pat. No. 6,506,879) without fully
eliminating the human health risk.
[0012] The use of plants as bioreactors for the large scale
production of recombinant proteins is known to the art, and
numerous proteins, including human therapeutic proteins, have been
produced. For example, U.S. Pat. Nos. 4,956,282, 5,550,038 and
5,629,175 disclose the production of .gamma.-interferon in plants;
U.S. Pat. Nos. 5,650,307, 5,716,802 and 5,763,748 detail the
production of human serum albumin in plants and U.S. Pat. Nos.
5,202,422, 5,639,947 and 5,959,177 relate to the production of
antibodies in plants. One of the significant advantages offered by
plant-based recombinant protein production systems is that by
increasing the acreage of plants grown, protein production can be
inexpensively scaled up to provide for large quantities of protein.
By contrast, fermentation and cell culture systems have large
space, equipment and energy requirements, rendering scale-up of
production costly. However, despite the fact that the use of plants
as bioreactors is amply documented, and despite the above mentioned
therapeutic applications of apolipoproteins, the prior art provides
no methods for the production of apolipoproteins in plants.
[0013] In order to offer a practical alternative to the
fermentation and cell culture based systems, it is important that
plants remain healthy and that apolipoproteins accumulate to
significant levels in the plants. In view of the inherent property
of apolipoproteins to associate with lipids, recombinantly
expressed apolipoproteins may associate with the endogenous plant
lipids and thereby interfere with the plant's lipid metabolism.
Thus recombinant expression of apolipoproteins may affect the
health of the plant. Alternatively, the recombinantly expressed
apolipoprotein may fail to accumulate to effective levels, as
protective mechanisms may result in degradration of the
apolipoprotein. It thus is unclear whether and how the synthetic
capacity of plants may be harnessed to achieve the commercial
production of apolipoproteins in plants.
[0014] Thus in view of the shortcomings associated with the methods
for the recombinant production of apolipoproteins by the prior art,
there is a need in the art to improve methods for the production of
apolipoproteins.
SUMMARY OF THE INVENTION
[0015] The present invention relates to methods for the production
of apolipoprotein in plants. In particular the present invention
relates to methods for the production of apolipoprotein in plant
seeds.
[0016] Accordingly, the present invention provides a method for the
expression of an apolipoprotein in plants comprising: [0017] (a)
providing a chimeric nucleic acid construct comprising in the 5' to
3' direction of transcription as operably linked components: [0018]
(i) a nucleic acid sequence capable of controlling expression in
plant cells; and [0019] (ii) a nucleic acid sequence encoding an
apolipoprotein polypeptide; [0020] (b) introducing the chimeric
nucleic acid construct into a plant cell; and [0021] (c) growing
the plant cell into a mature plant capable of expressing the
apolipoprotein.
[0022] In accordance with the present invention plant seeds have
been found to be particularly suitable for the production of
apolipoprotein. Accordingly, the present invention provides a
method for expressing apolipoprotein in plant seeds comprising:
[0023] (a) providing a chimeric nucleic acid construct comprising
in the 5' to 3' direction of transcription as operably linked
components: [0024] (i) a nucleic acid sequence capable of
controlling expression in plant seed cells; and [0025] (ii) a
nucleic acid sequence encoding an apolipoprotein polypeptide;
[0026] (b) introducing the chimeric nucleic acid construct into a
plant cell; and [0027] (c) growing the plant cell into a mature
plant capable of setting seed wherein the seed expresses the
apolipoprotein.
[0028] In a further preferred embodiment the nucleic acid sequence
capable of controlling expression in a plant seed cell is a
seed-preferred promoter, such as the phaseolin promoter. In a
preferred embodiment, at least 0.25% of the total seed protein is
apolipoprotein.
[0029] In preferred embodiments of the present invention the
chimeric nucleic acid sequence further comprises a nucleic acid
sequence encoding a stabilizing polypeptide linked in reading frame
to the nucleic acid sequence encoding the apolipoprotein.
Preferably the stabilizing polypeptide is a polypeptide that in the
absence of the apolipoprotein can readily be expressed and stably
accumulates in a plant cell. The stabilizing protein may be plant
specific or non plant specific. Plant-specific stabilizing
polypeptides that can be used in accordance with the present
invention include oil body proteins and thioredoxins. Non-plant
specific stabilizing polypeptides that may be used in accordance
herewith include green fluorescent protein (GFP) and single chain
antibodies or fragments thereof. The plant-specific or non-plant
specific stabilizing polypeptide may be linked to the
apolipoprotein via a linker which can be cleaved to release the
apolipoprotein in its free native form.
[0030] The chimeric nucleic acid sequence further preferably
comprise a targeting signal in such a manner that the
apolipoprotein polypeptide accumulates in the endoplasmic reticulum
(ER) or in association with an ER-derived storage vesicle, for
example an oil body, within the plant cell. Accordingly, the
chimeric nucleic acid construct additionally may comprise a nucleic
acid sequence encoding a polypeptide which is capable of targeting
the apolipoprotein polypeptide to the ER or an ER derived storage
vesicle. Nucleic acid sequences that may be used to target the
apolipoprotein to the ER include for example nucleic acid sequences
encoding KDEL, HDEL, SDEL sequences. Nucleic acid sequences that
may be used to target the apolipoprotein to an oil body include
nucleic acid sequences encoding oil body proteins, such as
oleosins. In addition, in accordance with the present invention,
the apolipoprotein may be targeted to the oil body by expressing
the apolipoprotein in such a manner that the apolipoprotein does
not include a targeting signal, provided however, that the nucleic
acid sequence encoding the apolipoprotein comprises an
apolipoprotein pro-peptide.
[0031] In another preferred embodiment the chimeric nucleic acid
comprises a targeting signal in such a manner that the
apolipoprotein accumulates in the apoplast. Accordingly, in such an
embodiment the chimeric nucleic acid construct additional
preferably contains a nucleic acid sequence encoding a polypeptide
which is capable of targeting the apolipoprotein polypeptide to the
apoplast.
[0032] In yet a further preferred embodiment, the nucleic acid
sequence encoding the apolipoprotein is expressed in such a manner
that the apolipoprotein accumulates in the cytoplasm. In such an
embodiment the nucleic acid sequence does not comprise a targeting
signal.
[0033] In a further preferred embodiment the chimeric nucleic acid
construct is introduced into the plant cell under nuclear genomic
integration conditions. Under such conditions the chimeric nucleic
acid sequence is stably integrated in the plant's genome.
[0034] In a yet further preferred embodiment the nucleic acid
sequence encoding apolipoprotein is optimized for plant codon
usage. Preferred nucleic acid sequences used in accordance with the
present invention encode human, bovine or porcine Apolipoprotein
A-I and pro-Apolipoprotein A-I.
[0035] In another aspect, the present invention provides a method
of obtaining plant seed comprising apolipoprotein. Accordingly,
pursuant to the present invention a method is provided for
obtaining plant seed comprising: [0036] (a) providing a chimeric
nucleic acid construct comprising in the 5' to 3' direction of
transcription as operably linked components: [0037] (i) a nucleic
acid sequence capable of controlling expression in plant tissue
cells; and [0038] (ii) a nucleic acid sequence encoding an
apolipoprotein polypeptide; [0039] (b) introducing the chimeric
nucleic acid construct into a plant cell; [0040] (c) growing the
plant cell into a mature plant capable of setting seed; and [0041]
(d) obtaining seed from said plant wherein the seed comprises the
apolipoprotein.
[0042] The seeds may be used to obtain a population of progeny
plants each comprising a plurality of seeds expressing
apolipoprotein. The present invention also provides plants capable
of setting seed expressing apolipoprotein. In a preferred
embodiment of the invention, the plants capable of setting seed
comprise a chimeric nucleic acid sequence comprising in the 5' to
3' direction of transcription: [0043] (a) a first nucleic acid
sequence capable of controlling expression in a plant cell
operatively linked to; [0044] (b) a second nucleic acid sequence
encoding an apolipoprotein polypeptide, wherein the cell contains
the apolipoprotein.
[0045] In a preferred embodiment the chimeric nucleic acid sequence
is integrated in the plant's nuclear genome.
[0046] In a further preferred embodiment of the present invention
the plant that is used is an Arabidopsis plant and in a
particularly preferred embodiment the plant is a safflower
plant.
[0047] In yet another aspect, the present invention provides plant
seeds expressing apolipoprotein. In a preferred embodiment of the
present invention, the plant seeds comprise a chimeric nucleic acid
sequence comprising in the 5' to 3' direction of transcription:
[0048] (a) a first nucleic acid sequence capable of controlling
expression in a plant cell operatively linked to; [0049] (b) a
second nucleic acid sequence encoding an apolipoprotein
polypeptide.
[0050] The seeds are a source whence the desired apolipoprotein
polypeptide, which is synthesized by the seed cells, may be
extracted and obtained in a more or less pure form. The
apolipoprotein may be used to treat vascular diseases.
[0051] Other features and advantages of the present invention will
become readily apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples while indicating preferred embodiments of the
invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become readily apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The invention will now be described in relation to the
drawings in which:
[0053] FIG. 1. (A) Nucleotide sequence (SEQ ID NO:1) and deduced
amino acid sequence (SEQ ID NO:2) of Homo sapiens Apolipoprotein
A-I (Apo AI) (Kindly provided by Dr. Norman Wong, Calgary Alberta)
(Accession number NM.sub.--000039). Bold residues represent
pre-sequence signal peptide, underlined residues indicate
pro-sequence. (B) Nucleotide sequence (SEQ ID NO:3) and deduced
amino acid sequence (SEQ ID NO:4) for natural variant Apo
AI.sub.Milano (R173C). Bold/Italicized residues represents the
mutated amino acid. (C) Nucleotide sequence (SEQ ID NO:5) and
deduced amino acid sequence (SEQ ID NO:6) for natural variant Apo
AI Paris (R151C). Bold/Italicized residues represents the mutated
amino acid.
[0054] FIG. 2. Schematic drawing of all the binary constructs
created for apolipoprotein Apo AI expression in Arabidopsis
transgenic plants. (A) Constructs that are targeted to the cytosol
in the plant cell. (B) Constructs that are targeted to oil bodies
in the plant cell. (C-E) Constructs that are targeted to the
secretory pathway. (D & E) Constructs may contain additional
KDEL sequences to be retained in the endoplasmic reticulum. (D)
Constructs containing the pro-sequence of Apo AI or mature Apo AI.
(E) Constructs containing a cleavable sequence for the release of
pro-Apo AI or mature Apo AI. Legend describes the type of promoter,
signal peptide and coding sequence contained in each construct.
[0055] FIG. 3(A). Schematic drawing for the cloning strategy for
the GFP coding region to be used in the Apo AI-GFP translational
fusion constructs.
[0056] FIG. 3(B). Schematic drawing for the cloning strategy for
the pro- and mature coding regions of Apo AI and seed-specific Apo
AI-GFP translational fusion constructs.
[0057] FIG. 3(C). Schematic drawing for the cloning strategy for
creation of the binary vectors used to transform Agrobacterium
EHA101 cells for seed-specific cytosolic and oil-body based
targeting of Apo AI-GFP. Apo10 and Apo11 containing the mature and
pro-Apo AI-GFP, respectively, targeted to the cytosol. Apo12 and
Apo13 containing the pro- and mature Apo AI-GFP translational
fusion, respectively, targeted to oil bodies.
[0058] FIG. 3(D). Schematic drawing for the cloning strategy for
creation of the binary vectors used to transform Agrobacterium
EHA101 cells for seed-specific secretory pathway targeting of Apo
AI-GFP. Apo15 and Apo16 containing the mature and pro-Apo AI-GFP
translational fusion, respectively, targeted to the secretory
pathway.
[0059] FIG. 4(A). Schematic drawing for the cloning strategy for
creation of the binary vectors used to transform Agrobacterium
EHA101 cells for constitutive cytosolic targeting of Apo AI-GFP.
Apo17 and Apo18a containing the mature and pro-Apo AI-GFP
translational fusion, respectively, targeted to the cytosol.
[0060] FIG. 4(B). Schematic drawing for the cloning strategy for
constitutive cytosolic targeting of Apo AI-GFP. Apo18b containing
the pro-Apo AI-GFP translational fusion targeted to the
cytosol.
[0061] FIG. 5(A). Schematic drawing for the cloning strategy for
the pro- and mature coding regions of Apo AI and constitutive
expression of Apo AI-GFP translational fusion protein.
[0062] FIG. 5(B). Schematic drawing for the cloning strategy for
the creation of binary vectors used to transform Agrobacterium
EHA101 cells for constitutive secretory pathway targeting of Apo
AI-GFP. Apo19 and Apo20 containing the mature and pro-Apo AI-GFP
translational fusion, respectively, targeted to the secretory
pathway.
[0063] FIG. 6. Schematic drawing for the cloning strategy for the
mature form of the coding region of Apo AI. Apo21 for seed-specific
targeting to the cytosol, Apo23 for seed-specific targeting to oil
bodies, Apo25 for seed-specific targeting to oil-bodies and
purification with cleavage sequence klip8 and Apo29 for
seed-specific targeting to the secretory pathway.
[0064] FIG. 7. Schematic drawing for the cloning strategy for the
pro-form of the coding region of Apo AI. Apo22 for seed-specific
targeting to the cytosol, Apo24 for seed-specific targeting to oil
bodies, Apo30 for seed-specific targeting to the secretory pathway
and Apo26 for seed-specific targeting to oil-bodies and
purification with cleavage sequence klip8.
[0065] FIG. 8(A). Schematic drawing for the cloning strategy for
the pro-form of the coding region of Apo AI with the internal XhoI
sites removed and contains a KDEL signal peptide. Apo27 for
seed-specific targeting to the oil bodies as an in-frame fusion
with oleosin. Apo31 and Apo35 both targeted to the secretory
pathway, with Apo31 and Apo35 fused in-frame with the oleosin
antibody D9, and Apo35 accumulating in the endoplasmic reticulum
due to a KDEL signal peptide.
[0066] FIG. 8(B). Schematic drawing for the cloning strategy for
the pro-form of the coding region of Apo AI.sub.Milano. Apo27M for
seed-specific targeting of Apo AI.sub.Milano to the oil bodies as
an in-frame fusion with oleosin.
[0067] FIG. 9. Schematic drawing for the cloning strategy for the
pro-form of the coding region of Apo AI with the internal XhoI
sites removed. Apo28, targeted to oil bodies and able to be cleaved
at the klip8 sequence, Apo32 which targets Apo AI to the secretory
pathway fused in-frame to the oleosin antibody D9.
[0068] FIG. 10. Schematic drawing for the cloning strategy for the
pro- and mature forms of the coding region of Apo AI with the
internal XhoI sites removed containing an additional Met residue at
start of coding region. Apo34 (pro-Apo AI) and Apo33 (mature Apo
AI) which are targeted to the secretory pathway and are fused
in-frame with the oleosin antibody D9.
[0069] FIG. 11. Schematic drawing for the cloning strategy for the
pro-form of the coding region of Apo AI with the internal XhoI
sites containing a KDEL signal peptide. Apo36, targeted to the
secretory pathway and is fused in-frame with the oleosin antibody
D9, and accumulates in the endoplasmic reticulum due to a KDEL
signal peptide.
[0070] FIG. 12. Schematic drawing for the cloning strategy for the
pro- and mature forms of the coding region of Apo AI with the
internal XhoI sites removed containing an additional Met residue at
start of coding region and a KDEL signal peptide. Apo38 and Apo37
which are targeted to the secretory pathway and are fused in-frame
with the oleosin antibody D9, and accumulate in the endoplasmic
reticulum due to a KDEL signal peptide.
[0071] FIG. 13. Schematic drawing for the cloning strategy for the
pro- and mature forms of the coding region of Apo AI and a protease
cleavage site. Apo43, Apo44, Apo39 and Apo40 targeted to the
secretory pathway, fused in-frame with the oleosin antibody D9.
Apo43 and Apo44 would accumulate in the endoplasmic reticulum due
to a KDEL signal peptide.
[0072] FIG. 14. Schematic drawing for the cloning strategy for the
pro- and mature forms of the coding region of Apo AI, containing an
additional Met residue at start of coding region and a protease
cleavage site. Apo42, Apo46, Apo41, and Apo45 targeted to the
secretory pathway, fused in-frame with the oleosin antibody D9.
Apo46 and Apo45 would accumulate in the endoplasmic reticulum due
to a KDEL signal peptide.
[0073] FIG. 15. Schematic drawing for the cloning strategy for the
mature form of the coding region of Apo AI, containing an
additional Met residue at start of coding region and a protease
cleavage site. Apo47 is fused in-frame with the maize oleosin for
targeting to oil bodies.
[0074] FIG. 16. Westerns of total leaf protein (A) (25 ug) and
total seed protein (B) (50 ug) with polyclonal Apo AI antibody.
Apo17 is ubi-mat-Apo AI-GFP construct. c24 leaf (25 ug) and seed
protein (50 ug) were used as a negative control and 0.5 ug of human
blood serum protein was used as a positive control.
[0075] FIG. 17. Westerns of total leaf protein (A) (25 ug) and
total seed protein (B) (50 ug) with polyclonal Apo AI antibody.
Apo18a is ubi-pro-Apo AI-GFP construct. c24 leaf (25 ug) and seed
protein (50 ug) were used as a negative control and 0.5 ug of human
blood serum protein was used as a positive control.
[0076] FIG. 18. Westerns of total leaf protein (A) (25 ug) and
total seed protein (B) (50 ug) with polyclonal Apo AI antibody.
Apo19 is ubi-PRS-mat-Apo AI-GFP construct. c24 leaf (25 ug) and
seed protein (50 ug) were used as a negative control and 0.5 ug of
human blood serum protein was used as a positive control.
[0077] FIG. 19. Westerns of total leaf protein (A) (25 ug) and
total seed protein (B) (50 ug) with polyclonal Apo AI antibody.
Apo20 is ubi-PRS-pro-Apo AI-GFP construct. c24 leaf (25 ug) and
seed protein (50 ug) were used as a negative control and 0.5 ug of
human blood serum protein was used as a positive control.
[0078] FIG. 20. Westerns of total seed protein (50 ug) with
polyclonal GFP antibody. (A) Apo10 is pha-mat-Apo AI-GFP construct.
(B) Apo11 is pha-pro-Apo AI-GFP construct. c24 seed protein (50 ug)
was used as a negative control and 200 ng of purified GFP protein
was used as a positive control.
[0079] FIG. 21. Westerns of total seed protein (50 ug) with
polyclonal GFP antibody. (A) Apo12 is pha-oleosin-mat-Apo AI-GFP
construct. (B) Apo13 is pha-oleosin-pro-Apo AI-GFP construct. c24
seed protein (50 ug) was used as a negative control and 200 ng of
purified GFP protein was used as a positive control.
[0080] FIG. 22. Westerns of total seed protein (50 ug) with
polyclonal GFP antibody. (A) Apo15 is pha-PRS-mat-Apo AI-GFP
construct. (B) Apo16 is pha-PRS-pro-Apo AI-GFP construct. c24 seed
protein (50 ug) was used as a negative control and 200 ng of
purified GFP protein was used as a positive control.
[0081] FIG. 23. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo21 is pha-matApo AI. (B) Apo22
is pha-pro-Apo AI. c24 seed protein (50 ug) was used as a negative
control and 0.5 ug of human blood serum protein was used as a
positive control.
[0082] FIG. 24. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo23 is pha-oleo-matApo AI. (B)
Apo24 is pha-oleo-pro-Apo AI. c24 seed protein (50 ug) was used as
a negative control and 0.5 ug of human blood serum protein was used
as a positive control.
[0083] FIG. 25. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo25 is pha-oleo-klip8-matApo
AI(+Met). (B) Apo26 is pha-oleo-klip8-pro-Apo AI(+Met). c24 seed
protein (50 ug) was used as a negative control and 0.5 ug of human
blood serum protein was used as a positive control.
[0084] FIG. 26. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo28 is pha-oleo-klip8-pro-Apo AI.
(B) Apo29 is pha-PRS-mat-Apo AI. c24 seed protein (50 ug) was used
as a negative control and 0.5 ug of human blood serum protein was
used as a positive control.
[0085] FIG. 27. Western of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo30 is pha-PRS-pro-Apo AI. c24
seed protein (50 ug) was used as a negative control and 0.5 ug of
human blood serum protein was used as a positive control.
[0086] FIG. 28. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo32 is pha-PRS-D9 scFv-pro-Apo
AI. (B) Apo33 is pha-PRS-D9 scFv-mat-Apo AI(+met). c24 seed protein
(50 ug) was used as a negative control and 0.5 ug of human blood
serum protein was used as a positive control.
[0087] FIG. 29. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo34 is pha-PRS-D9 scFv-pro-Apo
AI(+met). (B) Apo35 is pha-PRS-D9 scFv-mat-Apo AI-KDEL. c24 seed
protein (50 ug) was used as a negative control and 0.5 ug of human
blood serum protein was used as a positive control.
[0088] FIG. 30. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo36 is pha-PRS-D9 scFv-pro-Apo
AI-KDEL. (B) Apo37 is pha-PRS-D9 scFv-mat-Apo AI(+met)-KDEL. c24
seed protein (50 ug) was used as a negative control and 0.5 ug of
human blood serum protein was used as a positive control.
[0089] FIG. 31. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo38 is pha-PRS-D9 scFv-pro-Apo
AI(+met)-KDEL. c24 seed protein (50 ug) was used as a negative
control and 0.5 ug of human blood serum protein was used as a
positive control. (B) Apo 29 is pha-PRS-D9 scFv-KLIP8-Apo AI. Wild
type seed was used as a negative control and 3 ug of human Apo AI
from normal human plasma (US Biologicals) was used as a positive
control.
[0090] FIG. 32. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo40 is pha-PRS-D9
scFv-klip8-pro-Apo AI. (B) Apo41 is pha-PRS-D9 scFv-klip8-mat-Apo
AI(+met). c24 seed protein (50 ug) was used as a negative control
and 0.5 ug of human blood serum protein was used as a positive
control.
[0091] FIG. 33. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo42 is pha-PRS-D9
scFv-klip8-pro-Apo AI(+met). c24 seed protein (50 ug) was used as a
negative control and 0.5 ug of human blood serum protein was used
as a positive control.
[0092] FIG. 34. Westerns of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo44 is pha-PRS-D9
scFv-klip8-pro-Apo AI-KDEL. (B) Apo45 is pha-PRS-D9
scFv-klip8-mat-Apo AI(+met). c24 seed protein (50 ug) was used as a
negative control and 0.5 ug of human blood serum protein was used
as a positive control.
[0093] FIG. 35. Western of total seed protein (50 ug) with
polyclonal Apo AI antibody. (A) Apo46 is pha-PRS-D9
scFv-klip8-pro-Apo AI(+met)-KDEL. c24 seed protein (50 ug) was used
as a negative control and 0.5 ug of human blood serum protein was
used as a positive control.
[0094] FIG. 36. Examination of untargeted and oil body targeted Apo
AI-GFP association with specific cellular fractions from seeds.
Western blot analysis using the polyclonal Apo AI antibody and
approximately equal quantities of total protein (50 .mu.g) isolated
from the aqueous (AQ) fraction, phosphate (PW) and urea (UW) washes
of oil bodies (PO and UO respectively, with approximately 20 .mu.g
of total oil bodies used) and the microsomal (ER) fraction from
mature seeds. Ponceau-S staining of the immunoblot shows relative
protein amounts loaded on the gel (upper panel). Human blood serum
(0.5 .mu.g) was used as a positive control for Apo AI expression.
(A) Apo10 is Apo AI-GFP. Apo11 is pro-Apo AI-GFP. (B) Apo12 is
oleosin-Apo AI-GFP and Apo13 is oleosin-pro-Apo AI-GFP.
[0095] FIG. 37. Examination of secreted pro- and mature Apo AI-GFP
association with specific cellular fractions from seeds. Western
blot analysis using the anti-Apo AI antibody and approximately
equal quantities of total protein (50 _g) isolated from the aqueous
(AQ) fraction, phosphate (PW) and urea (UW) washes of oil bodies
(PO and UO respectively, with approximately 20 .mu.g of total oil
body protein used) and the microsomal (ER) fraction from mature
seeds. Ponceau-S staining of the immunoblot shows relative protein
amounts loaded on the gel (upper panel). Human blood serum (0.5
.mu.g) was used as a positive control for Apo AI expression. Apo15
is PRS-Apo AI-GFP. Apo16 is PRS-pro-Apo AI-GFP.
[0096] FIG. 38. Constitutive expression of Apo AI-GFP translational
fusion constructs in leaves. Western blot analysis using the
anti-GFP antibody and approximately equal quantities of total
protein (50 rig) isolated from leaves. Ponceau-S staining of the
immunoblot shows relative protein amounts loaded on the gel (upper
panel). Wild-type (c24) plants were used as a negative control for
GFP expression. Purified GFP protein (200 ng) was used as a
positive control for GFP. UG-14 and UR2 are included as positive
controls for GFP protein accumulation in leaves. The expected
masses are as follows: Apo17=55.4 kDa; Apo18=56.4 kDa; Apo19=58.3
kDa; Apo20=59.3 kDa; UG-14=26.8 kDa; UR2=50 kDa.
[0097] FIG. 39. Seed-specific expression of untargeted and secreted
Apo AI in seeds. Western blot analysis using the anti-Apo AI
antibody and approximately equal quantities of total protein (50
.mu.g) isolated from mature seeds. Ponceau-S staining of the
immunoblot shows relative protein amounts loaded on the gel (upper
panel). Wild-type (c24) plants were used as a negative control for
Apo AI expression. Human blood serum (0.5 .mu.g) was used as a
positive control for Apo AI expression. The expected masses are as
follows: Apo21=28.3 kDa; Apo22=29.32 kDa; Apo23=46.9 kDa;
Apo24=47.8 kDa; Apo25=51.5 kDa; Apo26=52.5 kDa; Apo27=51.3 kDa;
Apo28=52.3 kDa; Apo29=31.3 kDa; Apo30=32.2 kDa.
[0098] FIG. 40. Examination of subcellular fractions of Apo22-3
(untargeted pro-Apo AI) T3 generation seeds. Western blot analysis
using the anti-Apo AI antibody and approximately equal quantities
of total protein (50 .mu.g) isolated from the aqueous (AQ)
fraction, phosphate (PW) and urea (UW) washes of oil bodies (PO and
UO respectively, with approximately 20 .mu.g of total oil bodies
used) from mature seeds. Ponceau-S staining of the immunoblot shows
relative protein amounts loaded on the gel (upper panel). Human
blood serum (0.5 .mu.g) was used as a positive control for Apo AI
expression. Molecular weight sizes are indicated on the left.
[0099] FIG. 41. Confocal micrographs of Apo AI-GFP seed-specific
constructs expressed in late cotyledonary stage embyro cells
stained with Nile Red. (A-C) Apo10 is untargeted mature Apo AI
fused to GFP. (D-F) Apo11 is untargeted pro-Apo AI fused to GFP.
(G-I) Apo12 is mature Apo AI fused to GFP targeted to oil bodies
using oleosin. (Column 1) Green channel. (Column 2) Red channel.
(Column 3) Merged channels. Bar=5 .mu.m.
[0100] FIG. 42. Confocal micrographs of Apo AI-GFP seed-specific
constructs expressed in late cotyledonary stage embryo cells
stained with Nile Red. (A-C) Apo13 is pro-Apo AI fused to GFP
targeted to oil bodies using oleosin. (D-F) Apo15 is mature Apo AI
fused to GFP targeted to the secretory pathway. (G-I) Apo16 is
pro-Apo AI fused to GFP targeted to the secretory pathway. (Column
1) Green channel. (Column 2) Red channel. (Column 3) Merged
channels. Bar=5 .mu.m.
[0101] FIG. 43. Confocal micrographs of constitutively expressed
untargeted and secreted Apo AI-GFP fusion protein in late
cotyledonary stage embryo cells stained with Nile Red. (A-C) Apo17
is untargeted mature Apo AI fused to GFP. (D-F) Apo18 is untargeted
pro-Apo AI fused to GFP. (G-H) Apo19 is mature Apo AI fused to GFP
targeted to the secretory pathway. (J-L) Apo20 is pro-Apo AI fused
to GFP targeted to the secretory pathway. (Column 1) Green channel.
(Column 2) Red channel. (Column 3) Merged channels. Bar=5
.mu.m.
[0102] FIG. 44. Confocal micrographs of Apo AI-GFP constitutive
constructs expressed in leaf epidermal cells. (A-C) Apo17 is
untargeted mature Apo AI fused to GFP. (D-F) Apo18 is untargeted
pro-Apo AI fused to GFP. (G-H) Apo19 is mature Apo AI fused to GFP
targeted to the secretory pathway. (J-L) Apo20 is pro-Apo AI fused
to GFP targeted to the secretory pathway. (Column 1) Green channel.
(Column 2) Red channel. (Column 3) Merged channels. Bar=5
.mu.m.
[0103] FIG. 45. Purification of Apo25, Apo26 and Apo28 by reverse
phase chromatography. (A) HPLC trace of ApoAI standard (B) HPLC
trace of Apo25 (C) HPLC trace of Apo26 (D) HPLC trace of Apo28.
Individual wavelengths used included 214, 254, 280 and 326 nm.
DETAILED DESCRIPTION OF THE INVENTION
[0104] As hereinbefore mentioned, the present invention relates to
methods for the production of apolipoprotein in transgenic plants.
The present inventors have surprisingly found that production of
apolipoproteins in plants is not only feasible but also offers
substantial advantages over the conventional methodologies. The raw
materials for plant based production are more stable, particularly
as the protein is produced in plant seeds, and, moreover, are free
of bacterial endotoxins. Thus the present invention provides a safe
source material for the manufacture of apolipoproteins. It has also
been discovered that recombinant expression of apolipoprotein can
yield the native apolipoprotein in more or less pure form at levels
that permits commercial scale manufacture of apolipoproteins.
Accordingly, pursuant to the present invention a method for the
expression of a nucleic acid sequence encoding apolipoprotein in
plants is provided in which the method comprises: [0105] (a)
providing a chimeric nucleic acid construct comprising in the 5' to
3' direction of transcription as operably linked components: [0106]
(i) a nucleic acid sequence capable of controlling expression in
plant cells; and [0107] (ii) a nucleic acid sequence encoding an
apolipoprotein polypeptide [0108] (b) introducing the chimeric
nucleic acid construct into a plant cell; and [0109] (c) growing
the plant cell into a mature plant expressing apolipoprotein.
[0110] The present inventors have found that high levels of
apolipoprotein expression may be achieved by expressing the
recombinant protein in plant seeds. Accordingly, the present
invention provides a method for expressing apolipoprotein in plant
seeds comprising: [0111] (a) providing a chimeric nucleic acid
construct comprising in the 5' to 3' direction of transcription as
operably linked components: [0112] (i) a nucleic acid sequence
capable of controlling expression in plant seed cells; and [0113]
(ii) a nucleic acid sequence encoding an apolipoprotein
polypeptide; [0114] (b) introducing the chimeric nucleic acid
construct into a plant cell; and [0115] (c) growing the plant cell
into a mature plant capable of setting seed wherein the seed
expresses the apolipoprotein.
TERMS AND DEFINITIONS
[0116] Unless defined otherwise, all technical and scientific terms
used herein shall have the same meaning as is commonly understood
by one skilled in the art to which the present invention belongs.
Where permitted, all patents, applications, published applications,
and other publications, including nucleic acid and polypeptide
sequences from GenBank, SwissPro and other databases referred to in
the disclosure are incorporated by reference in their entirety.
[0117] The term "nucleic acid sequence" as used herein refers to a
sequence of nucleoside or nucleotide monomers consisting of
naturally occurring bases, sugars and intersugar (backbone)
linkages. The term also includes modified or substituted sequences
comprising non-naturally occurring monomers or portions thereof.
The nucleic acid sequences of the present invention may be
deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences
(RNA) and may include naturally occurring bases including adenine,
guanine, cytosine, thymidine and uracil. The sequences may also
contain modified bases. Examples of such modified bases include aza
and deaza adenine, guanine, cytosine, thymidine and uracil; and
xanthine and hypoxanthine.
[0118] The terms "nucleic acid sequence encoding apolipoprotein"
and "nucleic acid sequence encoding an apolipoprotein polypeptide",
which may be used interchangeably herein, refer to any and all
nucleic acid sequences encoding an apolipoprotein polypeptide,
including any mammalian apolipoprotein polypeptide and any nucleic
acid sequences that encode pro-apolipoprotein and
pre-pro-apolipoprotein. As used herein "pro-apolipoprotein" refers
to an apolipoprotein polypeptide which includes a polypeptide which
is cleaved post-translationally. In native human apolipoprotein the
pro-peptide is a 6 amino acid residue polypeptide chain. The term
"pre-pro-apolipoprotein" refers to a pro-apolipoprotein molecule
additionally comprising an N-terminal signal sequence which
facilitates intracellular transport of the polypeptide chain.
Nucleic acid sequences encoding an apolipoprotein polypeptide
further include any and all nucleic acid sequences which (i) encode
polypeptides that are substantially identical to the apolipoprotein
polypeptide sequences set forth herein; or (ii) hybridize to any
apolipoprotein nucleic acid sequences set forth herein under at
least moderately stringent hybridization conditions or which would
hybridize thereto under at least moderately stringent conditions
but for the use of synonymous codons.
[0119] By the term "substantially identical" it is meant that two
polypeptide sequences preferably are at least 70% identical, and
more preferably are at least 85% identical and most preferably at
least 95% identical, for example 96%, 97%, 98% or 99% identical. In
order to determine the percentage of identity between two
polypeptide sequences the amino acid sequences of such two
sequences are aligned, using for example the alignment method of
Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by
Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the
highest order match is obtained between the two sequences and the
number of identical amino acids is determined between the two
sequences. Methods to calculate the percentage identity between two
amino acid sequences are generally art recognized and include, for
example, those described by Carillo and Lipton (SIAM J. Applied
Math., 1988, 48:1073) and those described in Computational
Molecular Biology, Lesk, e.d. Oxford University Press, New York,
1988, Biocomputing: Informatics and Genomics Projects. Generally,
computer programs will be employed for such calculations. Computer
programs that may be used in this regard include, but are not
limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12:
387) BLASTP, BLASTN and FASTA (Altschul et al., J. Molec. Biol.,
1990:215:403). A particularly preferred method for determining the
percentage identity between two polypeptides involves the Clustal W
algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994,
Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62
scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl.
Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10
and a gap extension penalty of 0.1, so that the highest order match
obtained between two sequences wherein at least 50% of the total
length of one of the two sequences is involved in the
alignment.
[0120] By "At least moderately stringent hybridization conditions"
it is meant that conditions are selected which promote selective
hybridization between two complementary nucleic acid molecules in
solution. Hybridization may occur to all or a portion of a nucleic
acid sequence molecule. The hybridizing portion is typically at
least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those
skilled in the art will recognize that the stability of a nucleic
acid duplex, or hybrids, is determined by the T.sub.m, which in
sodium containing buffers is a function of the sodium ion
concentration and temperature (T.sub.m=81.5.degree. C.-16.6
(Log.sub.10 [Na.sup.+])+0.41(% (G+C)-600/l), or similar equation).
Accordingly, the parameters in the wash conditions that determine
hybrid stability are sodium ion concentration and temperature. In
order to identify molecules that are similar, but not identical, to
a known nucleic acid molecule a 1% mismatch may be assumed to
result in about a 1.degree. C. decrease in T.sub.m, for example if
nucleic acid molecules are sought that have a >95% identity, the
final wash temperature will be reduced by about 5.degree. C. Based
on these considerations those skilled in the art will be able to
readily select appropriate hybridization conditions. In preferred
embodiments, stringent hybridization conditions are selected. By
way of example the following conditions may be employed to achieve
stringent hybridization: hybridization at 5.times. sodium
chloride/sodium citrate (SSC)/5.times.Denhardt's solution/1.0% SDS
at T.sub.m (based on the above equation)-5.degree. C., followed by
a wash of 0.2.times.SSC/0.1% SDS at 60.degree. C. Moderately
stringent hybridization conditions include a washing step in
3.times.SSC at 42.degree. C. It is understood however that
equivalent stringencies may be achieved using alternative buffers,
salts and temperatures. Additional guidance regarding hybridization
conditions may be found in: Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et
al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor
Laboratory Press, 1989, Vol. 3.
[0121] As used herein the terms "apolipoprotein" and
"apolipoprotein polypeptide" refer to any and all polypeptide
sequences of an apolipoprotein including all mammalian
apolipoprotein polypeptides and a polypeptide comprising a sequence
of amino acid residues which is (i) substantially identical to the
amino acid sequences constituting any apolipoprotein polypeptides
set forth herein or (ii) encoded by a nucleic acid sequence capable
of hybridizing under at least moderately stringent conditions to
any nucleic acid sequence encoding apolipoprotein set forth herein
or capable of hybridizing under at least moderately stringent
conditions to any nucleic acid sequence encoding apolipoprotein set
forth herein but for the use of synonymous codons. The terms
apolipoprotein and apolipoprotein polypeptide include
pro-apolipoprotein polypeptides. The apolipoprotein polypeptide is
preferably of human, porcine or bovine origin. In a preferred
embodiments these apolipoproteins include, but are not limited to,
Apolipoprotein A-I (Apo AI), Apolipoprotein A-IV (Apo AIV),
Apolipoprotein A-V (Apo AV) and Apolipoprotein E (Apo E).
[0122] The term "chimeric" as used herein in the context of nucleic
acid sequences refers to at least two linked nucleic acid sequences
which are not naturally linked. Chimeric nucleic acid sequences
include linked nucleic acid sequences of different natural origins.
For example a nucleic acid sequence constituting a plant promoter
linked to a nucleic acid sequence encoding human apolipoprotein is
considered chimeric. Chimeric nucleic acid sequences also may
comprise nucleic acid sequences of the same natural origin,
provided they are not naturally linked. For example a nucleic acid
sequence constituting a promoter obtained from a particular
cell-type may be linked to a nucleic acid sequence encoding a
polypeptide obtained from that same cell-type, but not normally
linked to the nucleic acid sequence constituting the promoter.
Chimeric nucleic acid sequences also include nucleic acid sequences
comprising any naturally occurring nucleic acid sequence linked to
any non-naturally occurring nucleic acid sequence.
[0123] Preparation of Recombinant Expression Vectors Comprising
Chimeric Nucleic Acid Sequences Encoding Apolipoprotein and a
Nucleic Acid Sequence Capable of Controlling Expression in a Plant
Cell
[0124] The nucleic acid sequences encoding apolipoprotein that may
be used in accordance with the methods and compositions provided
herein may be any nucleic acid sequence encoding an apolipoprotein
polypeptide, including any proapolipoprotein and
preproapolipoprotein.
[0125] There are a number of different apolipoproteins that are
found in human blood plasma, and they can act as signals, that
cause lipoproteins to act on certain tissues or that activate
enzymes that act on those lipoproteins (Lehninger, A. et al.
Principles of Biochemistry, second edition, New York, Worth
Publishers, 1993). These proteins include but are not limited to
alleles and isoforms of apolipoprotein A-I (Apo AI) (see for
example Sharpe C R et al., Nucleic Acids Res. 12 (9), 3917-3932
(1984)), Apo AII ((see for example Sharpe C R et al., Nucleic Acids
Res. 12 (9), 3917-3932 (1984)), Apo AIV (see for example
Elshourbagy N A et al, J. Biol. Chem. 261 (5), 1998-2002 (1986)),
Apo AV (see for example Hubacek et al. Physiol. Res. 2004.
53:225-228), Apo B-100 (see for example Law S W et al., Proc. Natl.
Acad. Sci. U.S.A. 83 (21), 8142-8146 (1986)), Apo B-48 (see for
example Powell L M et al., Cell 50 (6), 831-840 (1987)), Apo C-II
(see for example Sharpe C R et al., Nucleic Acids Res. 12 (9),
3917-3932 (1984)), Apo C-III (see for example Sharpe C R et al.,
Nucleic Acids Res. 12 (9), 3917-3932 (1984)), ApoC-IV (see for
example Allan C M et al., Genomics 28 (2), 291-300 (1995)), Apo D
(Drayna D et al. J. Biol. Chem. 261 (35), 16535-16539 (1986)), Apo
E (see for example Brewslow J L et al., J. Biol. Chem. 257 (24),
14639-14641 (1982)), Apo F (see for example Day J R et al.,
Biochem. Biophys. Res. Commun. 203 (2), 1146-1151 (1994)), Apo H
(see for example Mehdi, H., et al., Gene 108 (2), 293-298 (1991)
and Apo L (see for example Duchateau, P. N., et al., J. Biol. Chem.
272 (41), 25576-25582 (1997)). Exemplary nucleic acid sequences
encoding apolipoprotein are well known to the art and are generally
readily available from a diverse variety of mammalian sources
including human (see above), porcine (see for example Trieu V N et
al., Gene 134 (2), 267-270 (1993)), bovine (see for example Yang,
Y. W., et al. J. Mol. Evol. 32 (6), 469-475 (1991)), ovine (see for
example Robertson, J. A., et al., J. Steroid Biochem. Mol. Biol. 67
(4), 285-292 (1998)) and the like. Human apolipoprotein encoding
sequences that may be used include those encoding polypeptide
chains set forth as SEQ ID NO:1, 7 and 8. Further non-human
apolipoprotein encoding sequences that may be used in accordance of
the present invention are set forth in SEQ ID NO: 9-55 and 241-251.
The respective corresponding nucleic acid sequences encoding the
apolipoprotein polypeptide chains can be readily identified via the
Accession identifier numbers provided in Table 1. Using these
nucleic acid sequences, additional novel apolipoprotein encoding
nucleic acid sequences may be readily identified using techniques
known to those of skill in the art. For example libraries, such as
expression libraries, cDNA and genomic libraries, may be screened,
and databases containing sequence information from sequencing
projects may be searched for similar sequences. Alternative methods
to isolate additional nucleic acid sequences encoding
apolipoprotein polypeptides may be used, and novel sequences may be
discovered and used in accordance with the present invention. In
preferred embodiments nucleic acid sequences encoding
apolipoprotein are human, porcine and bovine apolipoprotein.
[0126] Numerous apolipoprotein analogs are known to the prior art
(see for example Cheung M C et al., Biochim Biophys Acta. 1988 May
2; 960(1):73-82 and Strobl W et al., Pediatr Res. 1988 August;
24(2):222-8) and may be used in accordance with the present
invention. Analogs that may be used herein include human
apolipoprotein molecules wherein a variety of natural and synthetic
mutations and modifications have been discovered including, but not
limited to, point mutations, deletion mutations, frameshift
mutations and chemical modifications. In accordance with the
present invention in a preferred embodiment, the natural variant
known as Apo AI-M is used. Examples of mutations and modifications
that may be used in accordance with the present invention include,
but are not limited to, those set forth in Table 2.
[0127] In preferred embodiments, the nucleic acid sequence encoding
apolipoprotein that is used is pro-apolipoprotein.
[0128] Alterations to the nucleic acid sequence encoding
apolipoprotein to prepare apolipoprotein analogs may be made using
a variety of nucleic acid modification techniques known to those
skilled in the art, including for example site directed
mutagenesis, targeted mutagenesis, random mutagenesis, the addition
of organic solvents, gene shuffling or a combination of these and
other techniques known to those of skill in the art (Shraishi et
al., 1988, Arch. Biochem. Biophys, 358: 104-115; Galkin et al.,
1997, Protein Eng. 10: 687-690; Carugo et al., 1997, Proteins 28:
10-28; Hurley et al., 1996, Biochem, 35 : 5670-5678; Holmberg et
al., 1999, Protein Eng. 12 : 851-856).
[0129] In accordance herewith the nucleic acid sequence encoding
apolipoprotein is linked to a nucleic acid sequence capable of
controlling expression of the apolipoprotein polypeptide in a plant
cell. Accordingly, the present invention also provides a nucleic
acid sequence encoding apolipoprotein linked to a promoter capable
of controlling expression in a plant cell. Nucleic acid sequences
capable of controlling expression in plant cells that may be used
herein include any plant derived promoter capable of controlling
expression of polypeptides in plants. Generally, promoters obtained
from dicotyledonous plant species will be used when a
dicotyledonous plant is selected in accordance herewith, while a
monocotyledonous plant promoter will be used when a
monocotyledonous plant species is selected. In one embodiment, a
promoter is used which results in the expression of the
apolipoprotein polypeptide in the entire plant. Constitutive
promoters that may be used include, for example, the 35S
cauliflower mosaic virus (CaMV) promoter (Rothstein et al., 1987,
Gene 53: 153-161), the rice actin promoter (McElroy et al., 1990,
Plant Cell 2:163-171; U.S. Pat. No. 6,429,357), a ubiquitin
promoter, such as the corn ubiquitin promoter (U.S. Pat. Nos.
5,879,903; 5,273,894), and the parsley ubiquitin promoter
(Kawalleck, P. et al., 1993, Plant Mol. Biol. 21:673-684).
[0130] In particularly preferred embodiments of the present
invention, the apolipoprotein is produced in plant seeds.
Production in plants seeds offers flexibility in storage and
shipment of apolipoprotein as a raw material, since apolipoprotein
retains its activity upon extraction from stored seed. Furthermore,
the amount of biomass that needs to be subjected to extraction is
limited due to the relatively low water content present in plant
seeds. Accordingly, in a preferred embodiment of the present
invention the plant cell is a seed cell and the plant is grown into
a mature plant capable of setting seed wherein the seed expresses
the apolipoprotein. In a further preferred embodiment the nucleic
acid sequence capable of controlling expression in a plant cell is
a seed-preferred promoter, such as the phaseolin promoter. In such
an embodiment a promoter which results in preferential expression
of the apolipoprotein polypeptide in seed tissue is used.
"Seed-preferred promoters" in this regard are promoters which
control expression of a recombinant protein (i.e. apolipoprotein)
so that preferably at least 80% of the total amount of recombinant
protein present in the mature plant is present in the seed. More
preferably at least 90% of the total amount of recombinant protein
present in the mature plant is present in the seed. Most preferably
at least 95% of the total amount of recombinant protein present in
the mature plant is present in the seed. Seed-preferred promoters
that may be used in this regard include, for example, the bean
phaseolin promoter (Sengupta-Gopalan et al., 1985, Proc. Natl.
Acad. Sci. USA 82: 3320-3324); the Arabidopsis 18 kDa oleosin
promoter (U.S. Pat. No. 5,792,922) or the flax oleosin promoter (WO
01/16340); the flax legumin like seed storage protein (linin)
promoter (WO 01/16340); the flax 2S storage protein promoter (WO
01/16340); an endosperm preferred promoter such as the Amy32b
promoter (Rogers and Milliman, J. Biol. Chem., 1984, 259:
12234-12240, the Amy6-4 promoter (Kursheed and Rogers, J. Biol.
Chem., 1988, 263: 18953-18960 or the Aleurain promoter (Whittier et
al., 1987, Nucleic Acids Res., 15: 2515-2535) or the bean arcelin.
promoter (Jaeger G D, et al., 2002, Nat. Biotechnol. December;
20:1265-8). New promoters useful in various plants are constantly
discovered. Numerous examples of plant promoters may be found in
Ohamuro et al. (Biochem. of Pints., 1989, 15: 1-82).
[0131] In preferred embodiments of the present invention the
chimeric nucleic acid sequence further comprises a nucleic acid
sequence encoding a stabilizing polypeptide linked in reading frame
to the nucleic acid sequence encoding the apolipoprotein. The
stabilizing polypeptide is used to facilitate protein folding
and/or enhance the stable accumulation of the apolipoprotein in
plant cells. In addition the stabilizing polypeptide may be used to
target the apolipoprotein to a desired location within the plant
cell and/or facilitate purification of the apolipoprotein.
Preferably the stabilizing polypeptide is a polypeptide that in the
absence of the apolipoprotein can readily be expressed and stably
accumulates in transgenic plant cells. The stabilizing polypeptide
may be a plant specific or non-plant specific polypeptide.
Plant-specific stabilizing polypeptides that can be used in
accordance with the present invention include oil body proteins
(See below) and thioredoxins, for example, the thioredoxin shown in
SEQ ID NO:56. Non-plant specific stabilizing polypeptides that may
be used in accordance herewith include green fluorescent protein
(GFP) (Davis and Vierstra, 1996, Weeds World 3(2):43-48) (SEQ ID
NO:57) and single chain antibodies or fragments thereof. Preferably
non-plant specific stabilizing polypeptides are codon optimized for
optimal expression in plants.
[0132] Single chain antibodies or antibodies that are preferably
used herein include single chain antibodies or fragments thereof
that facilitate purification of the apolipoprotein. In accordance
herewith, for example, a single chain antibody or fragment thereof
which is capable of specific association with an oil body or oil
body protein may be used, thereby permitting copurification of the
apolipoprotein with the oil body fraction which can readily be
obtained from plant seeds. Preferably the single chain antibody is
capable of associating with an oil body protein obtainable from the
seed in which the apolipoprotein is expressed, i.e. in an
embodiment of the invention in which Arabidopsis plant cells are
used, a single chain antibody or fragment thereof is selected which
is capable of associating with an Arabidopsis oil body protein. In
a further preferred embodiment, the single chain antibody is a
single chain FV antibody capable of specifically associating with
the 18 kDa oleosin from Arabidopsis thaliana (D9scFv). In the most
preferred embodiment, the single chain antibody is shown in SEQ ID
NO:240. The term "single chain antibody fragment" (scFv) or
"antibody fragment" as used herein means a polypeptide containing a
variable light (V.sub.L) domain linked to a variable heavy
(V.sub.H) domain by a peptide linker (L), represented by
V.sub.L-L-V.sub.H. The order of the V.sub.L and V.sub.H domains can
be reversed to obtain polypeptides represented as
V.sub.H-L-V.sub.L. "Domain" is a segment of protein that assumes a
discrete function, such as antigen binding or antigen recognition.
The single chain antibody fragments for use in the present
invention can be derived from the light and/or heavy chain variable
domains of any antibody. Preferably, the light and heavy chain
variable domains are specific for the same antigen. Most preferably
the antigen is an oil body protein. The individual antibody
fragments which are joined to form a multivalent single chain
antibody may be directed against the same antigen or can be
directed against different antigens. Methodologies to create single
chain antibodies are well known to the art. For example single
chain antibodies can be created by screening single chain (scFV)
phage display libraries.
[0133] Methodologies to create single chain antibodies from phage
display libraries are well known to the art. McCarrerty et al.
(Nature 348: 552-554) demonstrated the use of a phage-display
system in which fragments of antibodies were expressed as a fusion
protein with a fd phage vector to allow for the expression of
single chain antibodies on the surface of the phage. The production
of a single chain antibody phage display library can be achieved
using for example, the Recombinant Phage Antibody System developed
by Amersham Biosciences and Cambridge Antibody Technology. A more
detailed protocol is available from Amersham Biosciences which is
sold as in 3 parts including a mouse scFV molecule, and expression
module and a detection module. Briefly, the protocol for the
production of single chain antibodies is as follows. Messenger RNA
can be obtained from either a mouse hybridoma or mouse spleen cells
from a mouse that has been immunized with the antigen of interest.
The mouse hybridoma represents the most abundant source for the
antibody gene to be cloned as it expressed the heavy and light
chain genes for a single antibody but antibodies can also be cloned
using spleen cells from an immunized mouse. The mRNA is converted
to cDNA using a reverse transcriptase and random hexamer primers.
The use of random hexamers will result in cDNA molecules that are
sufficient in length to clone the variable regions of the heavy and
light chain molecules. After the cDNA molecules are created,
primary PCR reactions are performed to amplify the heavy and light
variable regions separately. Primers are designed to amplify the
heavy or light chain variable region by hybridizing to opposite
ends of the chain. Once the variable regions are amplified, the PCR
reactions are subjected to agarose gel electrophoresis and gel
purified to remove the primers and any extraneous PCR products.
Once the heavy and light chain variable regions have been purified
they are assembled into a single gene using a linker. The linker
region is designed to ensure that the correct reading frame is
maintained between the heavy and light chain. For example, the
variable heavy (V.sub.H) and variable light (V.sub.L) chains may be
linked using a (Gly.sub.4Ser).sub.3 linker to obtain a single chain
antibody fragment (scFv) of approximately 750 base pairs in length.
Once the heavy and light chains are assembled with the linker a
secondary PCR reaction is performed to amplify the assembled scFV
DNA fragments. Primers should be designed to introduce restriction
sites to allow for cloning into phagemid expression vectors. For
example Sfi I and Not I sites can be added to the 5' and 3' end of
these scFv gene for cloning into the pCANTAB 5 E vector (Amersham
Biosciences). Once PCR is complete, the DNA fragments should be
purified to remove unincorporated primers and dNTPs. This can be
achieved using spun-column purification. Once the DNA fragments
have been purified and quantified the fragments are digested with
the appropriate restriction enzymes to allow for cloning into the
appropriate expression vector. The DNA fragments are subsequently
ligated into an expression vector, for example pCANTAB 5E (Amersham
Biosciences) and introduced into competent E. coli cells. The cells
should be grown on appropriate selection media to ensure that only
cells containing the expression vector will grow (i.e. using a
specific carbon source and antibiotic selection). Once the E. coli
is grown, the phagemid-containing colonies are infected with a M13
helper phage (i.e. KO7--Amersham Biosciences) to yield recombinant
phage which display the scFv fragments. The M13 phage will initiate
phage replication and complete phage particles will be produced and
released from the cells, expressing scFv species on their surface.
The phage displaying the correct scFv antibodies are identified by
panning using the specific antigen. To eliminate the non-specific
phage, the culture of recombinant phage can be transferred to an
antigen-coated support (i.e. a flask or a tube), and washed. Only
those phage displaying the correct scFv will be bound to the
support. A susceptible strain of E. coli is subsequently infected
with the phage bound to the antigen-coated support. The phage can
be enriched by rescuing with the helper phage and panning against
the antigen multiple times or can be plated directly onto a solid
medium without further enrichment. The E. coli cells that have been
infected with the phage selected against the appropriate antigen
are plated and individual colonies are picked. Phage, from the
individual colonies, are then assayed using for example the ELISA
assay (enzyme-linked immunosorbent assay). Phage antibodies which
are positive using the ELISA assay can then be used to infect E.
coli HB2151 cells for the production of soluble recombinant
antibodies. Once the appropriate clones are selected the sequence
of the scFv antibody gene can be identified and used for the
present invention.
[0134] The stabilizing protein may be linked to the apolipoprotein
via a linker which can be cleaved to release the apolipoprotein in
its free native form. Linkers that may be included in this regard
include peptide sequences recognized by Factor Xa, IgA protease, or
entorokinase. In a particularly preferred embodiment the linker
encodes a chymosin pro-sequence which may be cleaved with mature
chymosin as set forth in PCT Patent Application WO 98/49326.
[0135] In preferred embodiments the chimeric nucleic acid sequence
further comprises a "targeting signal". Targeting signal as used
herein means any amino acid sequence capable of directing the
apolipoprotein polypeptide, when expressed, to a desired location
within the plant cell. Suitable targeting signals that may be used
herein include those capable of targeting the apolipoprotein
polypeptide to the endoplasmic reticulum or a storage vesicle
derived from the endoplasmic reticulum, such as an oil body, and
the apoplast.
[0136] In order to achieve accumulation of the apolipoprotein in
the ER or an ER-derived storage vesicle, the polypeptide encoding
the polypeptide encoding the apolipoprotein is linked to a
targeting signal which causes the apolipoprotein to be retained in
the ER or an ER-derived storage vesicle. In a preferred embodiment
of the present invention, the targeting signal that is capable of
retaining the apolipoprotein in the ER contains a C-terminal
ER-retention motif. Examples of such C-terminal ER-retention motifs
include KDEL, HDEL, DDEL, ADEL and SDEL sequences. Other examples
include HDEF (Lehmann et al., 2001, Plant Physiol. 127(2):
436-439), or two arginine residues close to the N-terminus located
at positions 2 and 3, 3 and 4, or 4 and 5 (Abstract from Plant
Biology 2001 Program, ASPB, July 2001, Providence, R.I., USA).
Nucleic acid sequences encoding a C-terminal retention motif are
preferably linked to the nucleic acid sequence encoding the
apolipoprotein in such a manner that the polypeptide capable of
retaining the apolipoprotein in the ER is linked to the C-terminal
end of the apolipoprotein polypeptide.
[0137] In embodiments of the present invention in which the
apolipoprotein is retained in the ER the chimeric nucleic acid
sequence additionally may contain a nucleic sequence which targets
the nucleic acid sequence to the endomembrane system ("signal
peptide"). In embodiments of the present invention in which the
apolipoprotein polypeptide is retained in the ER using a sequence,
such as KDEL, HDEL or SDEL polypeptide, it is particularly
desirable to include a nucleic acid sequence encoding a signal
peptide. Exemplary signal peptides that may be used herein include
the tobacco pathogenesis related protein (PR-S) signal sequence
(SEQ. ID. NO:58) (Sijmons et al., 1990, Bio/technology, 8:217-221),
lectin signal sequence (Boehn et al., 2000, Transgenic Res,
9(6):477-86), signal sequence from the hydroxyproline-rich
glycoprotein from Phaseolus vulgaris (Yan et al., 1997, Plant
Phyiol. 115(3):915-24 and Corbin et al., 1987, Mol Cell Biol
7(12):4337-44), potato patatin signal sequence (Iturriaga, G et
al., 1989, Plant Cell 1:381-390 and Bevan et al., 1986, Nuc. Acids
Res. 41:4625-4638.) and the barley alpha amylase signal sequence
(Rasmussen and Johansson, 1992, Plant Mol. Biol. 18(2):423-7).
Example No. 3 herein shows accumulation of the apolipoprotein in
the ER.
[0138] In a further preferred embodiment, the apoliprotein
polypeptide is linked to a polypeptide that is capable of retaining
the apolipoprotein polypeptide in an ER-derived storage vesicle. In
a preferred embodiment, the ER derived storage vesicle is an oil
body and the apolipoprotein is linked to an oil body protein. Oil
body proteins that may be used in this regard include any protein
that naturally associates with an oil body (see SEQ ID NOs:59-137
in Table 3). The respective corresponding nucleic acid sequences
encoding the oil body protein polypeptide chains can be readily
identified via the Accession identifier numbers provided in Table
3. Using these nucleic acid sequences, additional novel oil body
proteins encoding nucleic acid sequences may be readily identified
using techniques known to those of skill in the art. For example
libraries, such as expression libraries, cDNA and genomic
libraries, may be screened, and databases containing sequence
information from sequencing projects may be searched for similar
sequences. Alternative methods to isolate additional nucleic acid
sequences encoding oil body protein polypeptides may be used, and
novel sequences may be discovered and used in accordance with the
present invention. Oil body proteins that are particularly
preferred are oleosins, for example a corn oleosin (Bowman-Vance et
al., 1987, J. Biol. Chem. 262: 11275-11279; Qu et al., 1990, J.
Biol. Chem. 265: 2238-2243 or Brassica (Lee et al., 1991, Plant
Physiol. 96: 1395-1397), caleosins, see for example Genbank
accession number AF067857) and steroleosins (Lin et al., 2002 Plant
Physiol. 128(4):1200-11). In a further preferred embodiment, the
oil body protein is a plant oleosin and shares sequence similarity
with other plant oleosins such as the oleosin isolated from
Arabidopsis thaliana (SEQ ID NO:138) or Brassica napus (SEQ ID
NO:139). In another embodiment, the oil body protein is a caleosin
or calcium binding protein from plant, fungal or other sources and
shares sequence homology with plant caleosins such as the caleosin
isolated from Arabidopsis thaliana (SEQ ID NO:140 and SEQ ID
NO:141) In another embodiment the oil body protein is a steroleosin
(SEQ ID NO:142), a sterol binding dehydrogenase (Lin L-J et al,
(2002) Plant Physiol 128: 1200-1211). This embodiment of the
present invention is exemplified in Example No. 3.
[0139] In addition, in accordance with the present invention, the
apolipoprotein may also be targeted to an oil body by expressing
the apolipoprotein in such a manner that the apolipoprotein does
not include a targeting signal, provided however, that the nucleic
acid sequence encoding the apolipoprotein comprises an
apolipoprotein pro-peptide. This embodiment of the present
invention is exemplified in Example No. 3.
[0140] Polypeptides capable of retaining the apolipoprotein in the
ER or an ER derived storage vesicle are typically not cleaved and
the apolipoprotein may accumulate in the form of a fusion protein,
which is, for example, typically the case when a KDEL retention
signal is used to retain the polypeptide in the ER or when an oil
body protein is used to retain the polypeptide in an oil body.
[0141] In another embodiment of the present invention the
apolipoprotein polypeptide is expressed in such a manner the
polypeptide accumulates in the apoplast. In order to achieve such
accumulation the chimeric nucleic acid sequence preferable
comprises a targeting sequence capable of directing the
apoliprotein polypeptide to the ER ("signal peptide"). Exemplary
signal peptides that may be used herein include the hereinbefore
mentioned tobacco pathogenesis related protein (PR-S) signal
sequence (SEQ. ID. NO:58) (Sijmons et al., 1990, Bio/technology,
8:217-221), lectin signal sequence (Boehn et al., 2000, Transgenic
Res, 9(6):477-86), signal sequence from the hydroxyproline-rich
glycoprotein from Phaseolus vulgaris (Yan et al., 1997, Plant
Phyiol. 115(3):915-24 and Corbin et al., 1987, Mol Cell Biol
7(12):4337-44), potato patatin signal sequence (Iturriaga, G et
al., 1989, Plant Cell 1:381-390 and Bevan et al., 1986, Nuc. Acids
Res. 41:4625-4638.) and the barley alpha amylase signal sequence
(Rasmussen and Johansson, 1992, Plant Mol. Biol. 18(2):423-7). Such
targeting signals may in vivo be cleaved off from the
apolipoprotein polypeptide, which for example is typically the case
when an apoplast targeting signal, such as the tobacco pathogenesis
related protein-S (PR-S) signal sequence (Sijmons et al., 1990,
Bio/technology, 8:217-221) is used. Other signal peptides can be
predicted using the SignalP World Wide Web server
(http://www.cbs.dtu.dk/services/SignalP/) which predicts the
presence and location of signal peptide cleavage sites in amino
acid sequences from different organisms. In general there is little
conservation of the primary amino acid sequence, although general
physiochemical properties are conserved to some extent. The generic
structure of signal peptides has 3 regions, the short
amino-terminal "n-region" contains positively charged residues, the
central hydrophobic "h-region" ranges in size from 7 to 15 amino
acids and the carboxy-terminal "c-region" contains polar amino
acids and a cleavage site that is recognized by membrane bound
signal peptidase enzymes (Nakai K., 2000, Advances in Protein Chem
54:277-344). A targeting signal that also may be used in accordance
herewith includes the native apolipoprotein signal sequence (18
amino acids in length in case of the human sequence). In preferred
embodiments hereof an N-terminally located apoplast targeting
sequence, such as the hereinbefore mentioned tobacco PR-S sequence
is used combined with a C-terminally located ER retention sequence
such as the KDEL sequence.
[0142] In yet a further preferred embodiment, the nucleic acid
sequence encoding the apolipoprotein is expressed in such a manner
that the apolipoprotein accumulates in the cytoplasm. In such an
embodiment the nucleic acid sequence does not comprise a targeting
signal. Preferably in such an embodiment the apolipoprotein
comprises a further stabilizing polypeptide, such as green
fluorescent protein (GFP).
[0143] The chimeric nucleic acid sequence may also comprise N-
and/or C-terminal polypeptide extensions. Such extensions may be
used to stabilize and/or assist in folding of the apolipoprotein
polypeptide chain or they may facilitate targeting to a compartment
in the cell, for example the oil body. Polypeptide extensions that
may be used in this regard include for example a nucleic acid
sequence encoding a single chain antibody, or a nucleic acid
sequence encoding green fluorescent protein (Davis and Vierstra,
1996, Weeds World 3(2):43-48), or combinations of such
polypeptides. Single chain antibody extensions that are
particularly desirable include those that permit association of the
apoliprotein with an oil body in order to facilitate purification
of the apolipoprotein in association with the oil body fraction.
Such extensions are preferably included in embodiments of the
present invention in which the apolipoprotein is expressed in the
plant seed and targeted within the seed cell to the ER or to the
apoplast.
[0144] In a further embodiment, a cleavage site may be located
upstream of the N-terminus or downstream of the C-terminus of the
Apolipoprotein A-I peptide allowing for the Apolipoprotein A-I
polypeptide to be cleaved from the fusion partner, thereby
obtaining isolated Apolipoprotein A-I. Examples of such cleavage
sites can be found in WO 98/49326 (Method for the cleavage of
fusion proteins) and related applications and LaVallie et al.
(1994) Enzymatic and chemical cleavage of fusion proteins In
Current Protocols in Molecular Biology pp 16.4.5-16.4.17, John
Wiley and Sons, Inc., New York N.Y. In a preferred embodiment, the
cleavage site is KLIP 8 (SEQ ID NO:143) which is cleavable by
aspartic proteases including chymosin. In a further preferred
embodiment, an extra methionine residue is added to the N-terminus
of the Apo AI polypeptide or pro-Apo AI polypeptide.
[0145] The invention further provides methods for the separation of
heterologous proteins from host cell components by partitioning of
the oil body fraction and subsequent release of the heterologous
protein via specific cleavage of the heterologous protein--oil body
protein fusion. Optionally a cleavage site may be located upstream
of the N-terminus and downstream of the C-terminus of the
heterologous polypeptide allowing the fusion polypeptide to be
cleaved and separated by phase separation into its component
peptides.
[0146] The nucleic acid sequence encoding apolipoprotein may be
altered, to improve expression levels for example, by optimizing
the nucleic acid sequence in accordance with the preferred codon
usage for a particular plant cell type which is selected for the
expression of the apolipoprotein polypeptide, or by altering motifs
known to destabilize mRNAs (see for example: PCT Patent Application
97/02352). Comparison of the codon usage of the nucleic acid
sequence encoding the apolipoprotein polypeptide with the codon
usage of the plant cell type will enable the identification of
codons that may be changed. Construction of synthetic genes by
altering the codon usage is described in for example PCT Patent
Application 93/07278.
[0147] In a preferred embodiment, the nucleic acid sequence
encoding apolipoprotein that is used is represented by SEQ. ID. NO
1, SEQ. ID. NO. 3 or SEQ. ID. NO. 5.
[0148] Certain genetic elements capable of enhancing expression of
the apolipoprotein polypeptide may be used herein. These elements
include the untranslated leader sequences from certain viruses,
such as the AMV leader sequence (Jobling and Gehrke, 1987, Nature,
325: 622-625) and the intron associated with the maize ubiquitin
promoter (U.S. Pat. No. 5,504,200). Generally the chimeric nucleic
acid sequence will be prepared so that genetic elements capable of
enhancing expression will be located 5' to the nucleic acid
sequence encoding the apolipoprotein polypeptide.
[0149] In accordance with the present invention the chimeric
nucleic acid sequences comprising a promoter capable of controlling
expression in plant linked to a nucleic acid sequence encoding an
apolipoprotein polypeptide can be integrated into a recombinant
expression vector which ensures good expression in the cell.
Accordingly, the present invention includes a recombinant
expression vector comprising in the 5' to 3' direction of
transcription as operably linked components: [0150] (i) a nucleic
acid sequence capable of controlling expression in plant cells; and
[0151] (ii) a nucleic acid sequence encoding an apolipoprotein
polypeptide; wherein the expression vector is suitable for
expression in a plant cell. The term "suitable for expression in a
plant cell" means that the recombinant expression vector comprises
the chimeric nucleic acid sequence of the present invention linked
to genetic elements required to achieve expression in a plant cell.
Genetic elements that may be included in the expression vector in
this regard include a transcriptional termination region, one or
more nucleic acid sequences encoding marker genes, one or more
origins of replication and the like. In preferred embodiments, the
expression vector further comprises genetic elements required for
the integration of the vector or a portion thereof in the plant
cell's nuclear genome, for example the T-DNA left and right border
sequences which facilitate the integration into the plant's nuclear
genome in embodiments of the invention in which plant cells are
transformed using Agrobacterium. In a further preferred embodiment
said plant cell is a plant seed cell.
[0152] As hereinbefore mentioned, the recombinant expression vector
generally comprises a transcriptional terminator which besides
serving as a signal for transcription termination further may serve
as a protective element capable of extending the mRNA half life
(Guarneros et al., 1982, Proc. Natl. Acad. Sci. USA, 79: 238-242).
The transcriptional terminator is generally from about 200
nucleotides to about 1000 nucleotides and the expression vector is
prepared so that the transcriptional terminator is located 3' of
the nucleic acid sequence encoding apolipoprotein. Termination
sequences that may be used herein include, for example, the
nopaline termination region (Bevan et al., 1983, Nucl. Acids. Res.,
11: 369-385), the phaseolin terminator (van der Geest et al., 1994,
Plant J. 6: 413-423), the arcelin terminator (Jaeger G D, et al.,
2002, Nat. Biotechnol. 20:1265-8), the terminator for the octopine
synthase genes of Agrobacterium tumefaciens or other similarly
functioning elements. Transcriptional terminators may be obtained
as described by An (An, 1987, Methods in Enzym. 153: 292).
[0153] Pursuant to the present invention the expression vector may
further contain a marker gene. Marker genes that may be used in
accordance with the present invention include all genes that allow
the distinction of transformed cells from non-transformed cells,
including all selectable and screenable marker genes. A marker gene
may be a resistance marker such as an antibiotic resistance marker
against, for example, kanamycin (U.S. Pat. No. 6,174,724),
ampicillin, G418, bleomycin, hygromycin which allows selection of a
trait by chemical means or a tolerance marker against a chemical
agent, such as the normally phytotoxic sugar mannose (Negrotto et
al., 2000, Plant Cell Rep. 19: 798-803). Other convenient markers
that may be used herein include markers capable of conveying
resistance against herbicides such as glyphosate (U.S. Pat. Nos.
4,940,935; 5,188,642), phosphinothricin (U.S. Pat. No. 5,879,903)
or sulphonyl ureas (U.S. Pat. No. 5,633,437). Resistance markers,
when linked in close proximity to nucleic acid sequence encoding
the apolipoprotein polypeptide, may be used to maintain selection
pressure on a population of plant cells or plants that have not
lost the nucleic acid sequence encoding the apolipoprotein
polypeptide. Screenable markers that may be employed to identify
transformants through visual inspection include
.beta.-glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and 5,599,670)
and green fluorescent protein (GFP) (Niedz et al., 1995, Plant Cell
Rep., 14: 403).
[0154] Recombinant vectors suitable for the introduction of nucleic
acid sequences into plants include Agrobacterium and Rhizobium
based vectors, such as the Ti and Ri plasmids, including for
example pBIN19 (Bevan, Nucl. Acid. Res., 1984, 22: 8711-8721),
pGKB5 (Bouchez et al., 1993, C R Acad. Sci. Paris, Life Sciences,
316:1188-1193), the pCGN series of binary vectors (McBride and
Summerfelt, 1990, Plant Mol. Biol., 14:269-276) and other binary
vectors (e.g. U.S. Pat. No. 4,940,838).
[0155] The recombinant expression vectors of the present invention
may be prepared in accordance with methodologies well known to
those skilled in the art of molecular biology. Such preparation
will typically involve the bacterial species Escherichia coli as an
intermediairy cloning host. The preparation of the E. coli vectors
as well as the plant transformation vectors may be accomplished
using commonly known technique's such as restriction digestion,
ligation, gelectrophoresis, DNA sequencing, the Polymerase Chain
Reaction (PCR) and other methodologies. A wide variety of cloning
vectors is available to perform the necessary steps required to
prepare a recombinant expression vector. Among the vectors with a
replication system functional in E. coli, are vectors such as
pBR322, the pUC series of vectors, the M13mp series of vectors,
pBluescript etc. Typically, these cloning vectors contain a marker
allowing selection of transformed cells. Nucleic acid sequences may
be introduced in these vectors, and the vectors may be introduced
in E. coli grown in an appropriate medium. Recombinant expression
vectors may readily be recovered from cells upon harvesting and
lysing of the cells. Further, general guidance with respect to the
preparation of recombinant vectors may be found in, for example:
Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold
Spring Harbor Laboratory Press, 1989, Vol. 3.
[0156] Preparation of Plants Comprising Seed Capable of Expressing
Apolipoprotein
[0157] In accordance with the present invention the chimeric
nucleic acid sequence is introduced into a plant cell and the cells
are grown into mature plants, wherein the plant expresses the
apolipoprotein polypeptide.
[0158] In accordance herewith any plant species or plant cell may
be selected. Particular cells used herein include cells obtainable
from Arabidopsis thaliana, Brazil nut (Betholettia excelsa); castor
bean (Riccinus communis); coconut (Cocus nucifera); coriander
(Coriandrum sativum); cotton (Gossypium spp.); groundnut (Arachis
Hypogaea); jojoba (Simmondsia chinensis); linseed/flax (Linum
usitatissimum); maize (Zea mays); mustard (Brassica spp. and
Sinapis alba); oil palm (Elaeis guineeis); olive (Olea eurpaea);
rapeseed (Brassica spp.); rice (Oryza sativa); safflower (Carthamus
tinctorius); soybean (Glycine max); squash (Cucurbita maxima);
barley (Hordeum vulgare); wheat (Traeticum aestivum); duckweed
(Lemnaceae sp.) and sunflower (Helianthus annuus).
[0159] In accordance herewith in a preferred embodiment plant
species or plant cells from oil seed plants are used. Oil seed
plants that may be used herein include peanut (Arachis hypogaea);
mustard (Brassica spp. and Sinapis alba); rapeseed (Brassica spp.);
chickpea (Cicer arietinum); soybean (Glycine max); cotton
(Gossypium hirsutum); sunflower (Helianthus annuus); (Lentil Lens
culinaris); linseed/flax (Linum usitatissimum); white clover
(Trifolium repens); olive (Olea eurpaea); oil palm (Elaeis
guineeis); safflower (Carthamus tinctorius) and narbon bean (Vicia
narbonesis).
[0160] In accordance herewith in a particularly preferred
embodiment Arabidopsis, flax or safflower is used.
[0161] Methodologies to introduce plant recombinant expression
vectors into a plant cell, also referred to herein as
"transformation", are well known to the art and typically vary
depending on the plant cell that is selected. General techniques to
introduce recombinant expression vectors in cells include,
electroporation; chemically mediated techniques, for example
CaCl.sub.2 mediated nucleic acid uptake; particle bombardment
(biolistics); the use of naturally infective nucleic acid
sequences, for example virally derived nucleic acid sequences, or
Agrobacterium or Rhizobium derived sequences, polyethylene glycol
(PEG) mediated nucleic acid uptake, microinjection and the use of
silicone carbide whiskers.
[0162] In preferred embodiments, a transformation methodology is
selected which will allow the integration of the chimeric nucleic
acid sequence in the plant cell's genome, and preferably the plant
cell's nuclear genome. In accordance herewith this is considered
particularly desirable as the use of such a methodology will result
in the transfer of the chimeric nucleic acid sequence to progeny
plants upon sexual reproduction. Transformation methods that may be
used in this regard include biolistics and Agrobacterium mediated
methods.
[0163] Transformation methodologies for dicotyledenous plant
species are well known. Generally, Agrobacterium mediated
transformation is used because of its high efficiency, as well as
the general susceptibility by many, if not all, dicotyledenous
plant species. Agrobacterium transformation generally involves the
transfer of a binary vector, such as one of the hereinbefore
mentioned binary vectors, comprising the chimeric nucleic acid
sequence of the present invention from E. coli to a suitable
Agrobacterium strain (e.g. EHA101 and LBA4404) by, for example,
tri-parental mating with an E. coli strain carrying the recombinant
binary vector and an E. coli strain carrying a helper plasmid
capable of mobilizing the binary vector to the target Agrobacterium
strain, or by DNA transformation of the Agrobacterium strain
(Hofgen et al., Nucl. Acids. Res., 1988, 16:9877). Other techniques
that may be used to transform dicotyledenous plant cells include
biolistics (Sanford, 1988, Trends in Biotechn. 6:299-302);
electroporation (Fromm et al., 1985, Proc. Natl. Acad. Sci. USA.,
82:5824-5828); PEG mediated DNA uptake (Potrykus et al., 1985, Mol.
Gen. Genetics, 199:169-177); microinjection (Reich et al.,
Bio/Techn., 1986, 4:1001-1004); and silicone carbide whiskers
(Kaeppler et al., 1990, Plant Cell Rep., 9:415-418) or in planta
transformation using, for example, a flower dipping methodology
(Clough and Bent, 1998, Plant J., 16:735-743).
[0164] Monocotyledonous plant species may be transformed using a
variety of methodologies including particle bombardment (Christou
et al., 1991, Biotechn. 9:957-962; Weeks et al., Plant Physiol.,
1993, 102:1077-1084; Gordon-Kamm et al., Plant Cell, 1990,
2:5603-618); PEG mediated DNA uptake (European Patents 0292 435;
0392 225) or Agrobacterium mediated transformation (Goto-Fumiyuki
et al., 1999, Nature-Biotech. 17:282-286).
[0165] The exact plant transformation methodology may vary somewhat
depending on the plant species and the plant cell type (e.g.
seedling derived cell types such as hypocotyls and cotyledons or
embryonic tissue) that is selected as the cell target for
transformation. As hereinbefore mentioned in a particularly
preferred embodiment safflower is used. A methodology to obtain
safflower transformants is available in Baker and Dyer (Plant Cell
Rep., 1996, 16:106-110). Additional plant species specific
transformation protocols may be found in: Biotechnology in
Agriculture and Forestry 46: Transgenic Crops I (Y. P. S. Bajaj
ed.), Springer-Verlag, New York (1999), and Biotechnology in
Agriculture and Forestry 47: Transgenic Crops II (Y. P. S. Bajaj
ed.), Springer-Verlag, New York (2001).
[0166] Following transformation, the plant cells are grown and upon
the emergence of differentiating tissue, such as shoots and roots,
mature plants are regenerated. Typically a plurality of plants is
regenerated. Methodologies to regenerate plants are generally plant
species and cell type dependent and will be known to those skilled
in the art. Further guidance with respect to plant tissue culture
may be found in, for example: Plant Cell and Tissue Culture, 1994,
Vasil and Thorpe Eds., Kluwer Academic Publishers; and in: Plant
Cell Culture Protocols (Methods in Molecular Biology 111), 1999,
Hall Eds, Humana Press.
[0167] In one aspect, the present invention provides a method of
obtaining plant seed comprising apolipoprotein. Accordingly, the
present invention provides a method for obtaining plant seed
comprising apolipoprotein comprising: [0168] (a) providing a
chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components: [0169]
(i) a nucleic acid sequence capable of controlling expression in
plant seed cells; and [0170] (ii) a nucleic acid sequence encoding
an apolipoprotein polypeptide; [0171] (b) introducing the chimeric
nucleic acid construct into a plant cell; [0172] (c) growing the
plant cell into a mature plant; and [0173] (d) obtaining seed from
said plant wherein the seed comprises apolipoprotein.
[0174] The seeds may be used to obtain a population of progeny
plants each comprising a plurality of seeds expressing
apolipoprotein. In a preferred embodiment at least approximately
0.25% of the total seed protein is apolipoprotein. More preferably
least approximately 0.5% of the total seed protein is
apolipoprotein. Most preferably at least approximately 1.0% of the
total seed protein is apolipoprotein.
[0175] In preferred embodiments, a plurality of transformed plants
is obtained, grown, and screened for the presence of the desired
chimeric nucleic acid sequence, the presence of which in putative
transformants may be tested by, for example, growth on a selective
medium, where herbicide resistance markers are used, by direct
application of the herbicide to the plant, or by Southern blotting.
If the presence of the chimeric nucleic acid sequence is detected,
transformed plants may be selected to generate progeny and
ultimately mature plants comprising a plurality of seeds comprising
the desired chimeric nucleic acid sequence. Such seeds may be used
to isolate apolipoprotein or they may be planted to generate two or
more subsequent generations. It will generally be desirable to
plant a plurality of transgenic seeds to obtain a population of
transgenic plants, each comprising seeds comprising a chimeric
nucleic acid sequence encoding apolipoprotein. Furthermore, it will
generally be desirable to ensure homozygosity in the plants to
ensure continued inheritance of the recombinant polypeptide.
Methods for selecting homozygous plants are well known to those
skilled in the art. Methods for obtaining homozygous plants that
may be used include the preparation and transformation of haploid
cells or tissues followed by the regeneration of haploid plantlets
and subsequent conversion to diploid plants for example by the
treatment with colchine or other microtubule disrupting agents.
Plants may be grown in accordance with otherwise conventional
agricultural practices.
[0176] In another aspect, the present invention also provides
plants capable of setting seed expressing apolipoprotein. In a
preferred embodiment of the invention, the plants capable of
setting seed comprise a chimeric nucleic acid sequence comprising
in the 5' to 3' direction of transcription: [0177] (a) a first
nucleic acid sequence capable of controlling expression in a plant
seed cell operatively linked to; [0178] (b) a second nucleic acid
sequence encoding an apolipoprotein polypeptide, wherein the seed
contains apolipoprotein.
[0179] In a preferred embodiment the chimeric nucleic acid sequence
is stably integrated in the plant's nuclear genome.
[0180] In yet another aspect, the present invention provides plant
seeds expressing apolipoprotein. In a preferred embodiment of the
present invention, the plant seeds comprise a chimeric nucleic acid
sequence comprising in the 5' to 3' direction of transcription:
[0181] (a) a first nucleic acid sequence capable of controlling
expression in a plant seed cell operatively linked to; [0182] (b) a
second nucleic acid sequence encoding an apolipoprotein
polypeptide.
[0183] The apolipoprotein polypeptide may be present in a variety
of different types of seed cells including, for example, the
hypocotyls and the embryonic axis, including in the embryonic roots
and embryonic leafs, and where monocotyledonous plant species,
including cereals and corn, are used in the endosperm tissue.
[0184] Once the plants have been obtained the apolipoprotein
protein may be extracted and obtained from the plant in a more or
less pure form.
[0185] Accordingly, the present invention provides a method for
preparing substantially pure apolipoprotein comprising: [0186] (a)
providing a chimeric nucleic acid construct comprising in the 5' to
3' direction of transcription as operably linked components: [0187]
(i) a nucleic acid sequence capable of controlling expression in
plant seed cells; and [0188] (ii) a nucleic acid sequence encoding
an apolipoprotein polypeptide; [0189] (b) introducing the chimeric
nucleic acid construct into a plant cell; [0190] (c) growing the
plant cell into a mature plant; and obtaining seed from said plant
wherein the seed comprises apolipoprotein; and [0191] (e)
separating apolipoprotein from the plant seed constituents to
obtain substantially pure apolipoprotein.
[0192] In order to separate the apolipoprotein from the seed
constituents, plant seeds may be ground using any comminuting
process resulting in a substantial disruption of the seed cell
membrane and cell walls. Both dry and wet milling conditions (U.S.
Pat. No. 3,971,856; Lawhon et al., 1977, J. Am. Oil Chem. Soc.,
63:533-534) may be used. Suitable milling equipment in this regard
include colloid mills, disc mills, IKA mills, industrial scale
homogenizers and the like. The selection of the milling equipment
will depend on the seed type and throughput requirements. Solid
seed contaminant such as seed hulls, fibrous materials, undissolved
carbohydrates, proteins and other water insoluble contaminants may
be removed from the seed fraction using for example size-exclusion
based methodologies, such as filtering or gravitational based
processes such as centrifugation. In preferred embodiments, the use
of organic solvents commonly used in oil extraction, such as
hexane, is avoided because such solvents may damage the
apolipoprotein polypeptide. As hereinbefore mentioned in preferred
embodiments of the present invention the apoliprotein is prepared
in a manner that permits association of the apolipoprotein
polypeptide with seed oil bodies. Accordingly, seed oil bodies
comprising the apolipoprotein may be prepared following comminuting
of the seed using for example the methodologies detailed in U.S.
Pat. No. 6,146,645. Thus the present invention also includes
substantially pure oil bodies comprising apolipoprotein obtained
from plant seed. The oil bodies may be used as a refined plant seed
fraction to further purify apolipoprotein. Substantially pure
apolipoprotein may be recovered from seed using a variety of
additional purification methodologies such as centrifugation based
techniques; size exclusion based methodologies, including for
example membrane ultrafiltration and crossflow ultrafiltration; and
chromatographic techniques, including for example ion-exchange
chromatography, size exclusion chromatography, affinity
chromatography, high performance liquid chromatography (HPLC), fast
protein liquid chromatography (FPLC), hydrophobic interaction
chromatography and the like. Generally, a combination of such
techniques will be used to obtain substantially pure
apolipoprotein. A preferred methodology to obtain substantially
pure apoliprotein in its native form from transgenic plant seeds is
further detailed in Example 6 herein. Thus the present invention
also includes substantially pure apolipoprotein obtained from a
plant.
[0193] Pharmaceutical apolipoprotein formulations may be prepared
from the purified apolipoprotein and such formulations may be used
to treat vascular diseases. Generally the purified apolipoprotein
will be admixed with a pharmaceutically acceptable carrier or
diluent in amounts sufficient to exert a therapeutically useful
effect in the absence of undesirable side effects on the patient
treated. To formulate an apolipoprotein composition, the weight
fraction of apolipoprotein is dissolved, suspended, dispersed or
otherwise mixed in a selected carrier or diluent at an effective
concentration such that the treated condition is ameliorated. The
pharmaceutical apolipoprotein formulations are preferably
formulated for single dosage administration. Therapeutically
effective doses for the parenteral delivery of human apolipoprotein
are well known to the art. Where apolipoprotein analogs are used or
other modes of delivery are used therapeutically effective doses
may be readily empirically determined by those of skill in the art
using known testing protocols or by extrapolation of in-vivo or
in-vitro test data. It is understood however that concentrations
and dosages may vary in accordance with the severity of the
condition alleviated. It is further understood that for any
particular subject, specific dosage regimens may be adjusted over
time according to individual judgement of the person administering
or supervising administration of the formulations.
[0194] Pharmaceutical solutions or suspensions may include for
example a sterile diluent such as, for example, water, lactose,
sucrose, dicalcium phosphate, or carboxymethyl cellulose. Carriers
that may be used include water, saline solution, aqueous dextrose,
glycerol, glycols, ethanol and the like, to thereby form a solution
or suspension. If desired the pharmaceutical compositions may also
contain non-toxic auxiliary substances such a wetting agents;
emulsifying agents; solubilizing agents; antimicrobial agents, such
as benzyl alcohol and methyl parabens; antioxidants, such as
ascorbic acid and sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); pH buffering agents such as
actetate, citrate or phosphate buffers; and combinations
thereof.
[0195] The final formulation of the apolipoprotein preparation will
generally depend on the mode of apolipoprotein delivery. The
apolipoprotein prepared in accordance with the present invention
may be delivered in any desired manner; however parenteral, oral
and nasal forms of delivery are considered the most likely used
modes of delivery. Parenteral preparations can be enclosed in
ampoules, disposable syringes or single or multiple dose vials made
of glass, plastic or other such suitable materials.
EXAMPLES
[0196] The following examples are offered by way of illustration
and not by way of limitation.
Example 1
Construction of Apolipoprotein A-I Clones
[0197] Apo10
[0198] Apo10 (SEQ ID NO:144) is a clone designed to express in a
seed-specific manner which is constructed as per FIGS. 3(A), 3(B)
and 3(C). As seen in FIG. 2, the Apo10 clone consists of a
seed-specific promoter and terminator (phaseolin) driving the
expression of a fusion protein (SEQ ID NO:145) between mature Apo
AI and GFP. To construct this clone forward primer 1186 (SEQ ID
NO:146 (5'-GGATCCCCtTGGCTAGTAAAGG-3') removed a NcoI site from the
start of GFP (template derived from the vector pVS-GFP). Reverse
primer 1187 (SEQ ID. NO:147)
(5'-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3') added PstI, XbaI
and HindIII sites after the stop codon. The PCR fragment was
ligated into the EcoRI cloning vector Topo (Invitrogen) creating
plasmid G1 (FIG. 3(A)). Forward primer 1190 (SEQ ID NO:148)
(5'-CCATGGggCGGCATTTCTGGCAGCAAGATG-3') amplifies the mature
sequence of Apo AI and adds a NcoI site to the start of gene.
Reverse primer 1189 (SEQ ID NO:149)
(5'-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes the stop codon of the
gene and adds a BamHI site to assist in creating a in-frame
translation fusion with GFP (Plasmid G1). The template for these
primers was a pKS+ based vector (Strategene) containing the entire
coding sequence for human Apo AI gene. The PCR fragment was ligated
into the EcoRI site of the Topo cloning vector (Invitrogen)
creating Plasmid M2. Plasmid M2 contained the mature sequence of
Apo AI was cut with restriction enzymes NcoI and BamHI. Plasmid G'1
contains the GFP coding sequence and was cut with BamHI and XbaI
(see FIG. 3(B)). The fragments of M2 and G'1 were ligated together
into the NcoI and XbaI sites of the plasmid SBS2090 (see FIG. 3(B))
to create the plasmid 2M4. Plasmid 2M4 was cut with NcoI and
HindIII to remove the Apo AI-GFP fusion cassette and the fragments
were used subsequently to clone into the NcoI/HindIII sites of
binary vector pSBS4006 (see FIG. 3(C)). Note that this plasmid
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) and a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium.
[0199] Apo11
[0200] Apo11 (SEQ ID NO:150) is a clone designed to express in a
seed-specific manner which is constructed as per FIGS. 3(A), 3(B)
and 3(C). As seen in FIG. 2, the Apo11 clone consists of a
seed-specific promoter and terminator (phaseolin) driving the
expression of a fusion protein (SEQ ID NO:151) between pro-Apo AI
and GFP. To construct this clone, forward primer 1186 (SEQ ID
NO:146) (5'-GGATCCCCtTGGCTAGTAAAGG-3') removed a NcoI site from the
start of GFP (template derived from the vector pVS-GFP). Reverse
primer 1187 (SEQ ID NO:147)
(5'-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3') added PstI, XbaI
and HindIII sites after the stop codon. The PCR fragment was
ligated into the EcoRI cloning vector Topo (Invitrogen) creating
plasmid G1 (FIG. 3(A)). Forward primer 1191 (SEQ ID NO:152)
(5'-CCATGGATGAACCCCCCCAGAGCCCCTG-3') amplifies the pro-sequence of
Apo AI and adds a NcoI site to the start of gene. Reverse primer
1189 (SEQ ID NO:149) (5'-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes
the stop codon of the gene and adds a BamHI site to assist in
creating a translation fusion with GFP (Plasmid G1). The template
for these primers was a pKS+ based vector (Strategene) containing
the entire coding sequence for human Apo AI gene. The PCR fragments
were each separately ligated into the EcoRI site of the Topo
cloning vector (Invitrogen) creating Plasmid P2. Plasmid P2
contained the proApo AI sequence was cut with restriction enzymes
NcoI and BamHI. Plasmid G'1 contains the GFP coding sequence and
was cut with BamHI and XbaI (see FIG. 3(B)). The fragments of P2
and G'1 were ligated together into the NcoI and XbaI sites of the
plasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2P5. Plasmid
2P5 was cut with NcoI and HindIII to remove the pro-Apo AI-GFP
fusion cassette and the fragments were used subsequently to clone
into the NocI/HindIII sites binary vector pSBS4006 (see FIG. 3(C).
Note that this plasmid contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) and a pat gene conferring
host plant phosphinothricine resistance (Wohlleben et al., 1988,
Gene 70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium.
[0201] Apo12
[0202] Apo12 (SEQ ID NO:153) is a clone designed to express in a
seed-specific manner which is constructed as per FIGS. 3(A), 3(B)
and 3(C). As seen in FIG. 2, the Apo12 clone consists of a
seed-specific promoter and terminator (phaseolin) driving the
expression of a fusion protein (SEQ ID NO:154) between an oleosin
(van Rooijen, G. J., et al. Plant Mol. Biol. 18 (6), 1177-1179
(1992)), mature Apo AI and GFP. To construct this clone forward
primer 1186 (SEQ ID NO:146) (5'-GGATCCCCtTGGCTAGTAAAGG-3') removed
a NcoI site from the start of GFP (template derived from the vector
pVS-GFP). Reverse primer 1187 (SEQ ID NO:147)
(5'-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3') added PstI, XbaI
and HindIII sites after the stop codon. The PCR fragment was
ligated into the EcoRI cloning vector Topo (Invitrogen) creating
plasmid G1 (FIG. 3(A)). Forward primer 1190 (SEQ ID NO: 148)
(5'-CCATGGggCGGCATTTCTGGCAGCAAGATG-3') amplifies the mature
sequence of Apo AI and adds a NcoI site to the start of gene.
Reverse primer 1189 (SEQ ID NO:149)
(5'-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes the stop codon of the
gene and adds a BamHI site to assist in creating a in-frame
translation fusion with GFP (Plasmid G1). The template for these
primers was a pKS+ based vector (Strategene) containing the entire
coding sequence for human Apo AI gene. The PCR fragment was ligated
into the EcoRI site of the Topo cloning vector (Invitrogen)
creating Plasmid M2. Plasmid M2 contained the mature sequence of
Apo AI was cut with restriction enzymes NcoI and BamHI. Plasmid G'1
contains the GFP coding sequence and was cut with BamHI and XbaI
(see FIG. 3(B)). The fragments of M2 and G'1 were ligated together
into the NcoI and XbaI sites of the plasmid SBS2090 (see FIG. 3(B))
to create the plasmid 2M4. Plasmid 2M4 was cut with NcoI and
HindIII to remove the Apo AI-GFP fusion cassette and the fragments
were used subsequently to clone into the NcoI/HindIII sites of
binary vector pSBS4008 (see FIG. 3(C)). Note that this plasmid
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the Arabidopsis oleosin
gene (van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6),
1177-1179) for fusion with the Apo AI/GFP fusion. The plasmid also
contains a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37)) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium.
[0203] Apo13
[0204] Apo13 (SEQ ID NO:155) is a clone designed to express in a
seed-specific manner which is constructed as per FIGS. 3(A), 3(B)
and 3(C). As seen in FIG. 2, the Apo13 clone consists of a
seed-specific promoter and terminator (phaseolin) driving the
expression of a fusion protein (SEQ ID NO:156) between oleosin,
pro-Apo AI and GFP. To construct this clone, forward primer 1186
(SEQ ID NO:146) (5'-GGATCCCCtTGGCTAGTAAAGG-3') removed a NcoI site
from the start of GFP (template derived from the vector pVS-GFP).
Reverse primer 1187 (SEQ ID NO:147)
(5'-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3') added PstI, XbaI
and HindIII sites after the stop codon. The PCR fragment was
ligated into the EcoRI cloning vector Topo (Invitrogen) creating
plasmid G1 (FIG. 3(A)). Forward primer 1191 (SEQ ID NO:152)
(5'-CCATGGATGAACCCCCCCAGAGCCCCTG-3') amplifies the pro-sequence of
Apo AI and adds a NcoI site to the start of gene. Reverse primer
1189 (SEQ ID NO:149) (5'-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes
the stop codon of the gene and adds a BamHI site to assist in
creating a translation fusion with GFP (Plasmid G1). The template
for these primers was a pKS+ based vector (Strategene) containing
the entire coding sequence for human Apo AI gene. The PCR fragments
were each separately ligated into the EcoRI site of the Topo
cloning vector (Invitrogen) creating Plasmid P2. Plasmid P2
contained the pro-Apo AI sequence was cut with restriction enzymes
NcoI and BamHI. Plasmid G'1 contains the GFP coding sequence and
was cut with BamHI and XbaI (see FIG. 3(B)). The fragments of P2
and G'1 were ligated together into the NcoI and XbaI sites of the
plasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2P5. Plasmid
2P5 was cut with NcoI and HindIII to remove the pro-Apo AI-GFP
fusion cassette and the fragments were used subsequently to clone
into the NocI/HindIII sites of binary vector pSBS4008 (see FIG.
3(C)). Note that this plasmid contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the Arabidopsis oleosin gene (van Rooijen G. J. et al. 1992,
Plant Mol. Biol. 18 (6), 1177-1179) for fusion with the Apo AI/GFP
fusion. The plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium.
[0205] Apo15
[0206] Apo15 (SEQ ID NO:157) is a clone designed to express in a
seed-specific manner which is constructed as per FIGS. 3(A), 3(B)
and 3(D). As seen in FIG. 2, the Apo15 clone consists of a
seed-specific promoter and terminator (phaseolin) driving the
expression of a fusion protein (SEQ ID NO:158) between mature Apo
AI and GFP. The fusion protein was targeted for expression through
the secretory pathway using the tobacco pathogen related sequence
(PRS) signal peptide (Sijmons et al., 1990, Bio/technology,
8:217-221). To construct this clone forward primer 1186 (SEQ ID
NO:146) (5'-GGATCCCCtTGGCTAGTAAAGG-3') removed a NcoI site from the
start of GFP (template derived from the vector pVS-GFP). Reverse
primer 1187 (SEQ ID NO: 147)
(5'-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3') added PstI, XbaI
and HindIII sites after the stop codon. The PCR fragment was
ligated into the EcoRI cloning vector Topo (Invitrogen) creating
plasmid G1 (FIG. 3(A)). Forward primer 1190 (SEQ ID NO:148)
(5'-CCATGGggCGGCATTTCTGGCAGCAAGATG-3') amplifies the mature
sequence of Apo AI and adds a NcoI site to the start of gene.
Reverse primer 1189 (SEQ ID NO:149)
(5'-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes the stop codon of the
gene and adds a BamHI site to assist in creating a in-frame
translation fusion with GFP (Plasmid G1). The template for these
primers was a pKS+ based vector (Strategene) containing the entire
coding sequence for human Apo AI gene. The PCR fragment was ligated
into the EcoRI site of the Topo cloning vector (Invitrogen)
creating Plasmid M2. Plasmid M2 contained the mature sequence of
Apo AI was cut with restriction enzymes NcoI and BamHI. Plasmid G'1
contains the GFP coding sequence and was cut with BamHI and XbaI
(see FIG. 3(B)). The fragments of M2 and G'1 were ligated together
into the NcoI and XbaI sites of the plasmid SBS2090 (see FIG. 3(B))
to create the plasmid 2M4. Plasmid 2M4 was cut with NcoI and
HindIII to remove the Apo AI-GFP fusion cassette and the fragments
were used subsequently to clone into the NcoI/HindIII sites of
binary vector pSBS4011 (see FIG. 3(D)). Note that this plasmid
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) fused to the PRS signal peptide. The plasmid also
contains a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant. Mol. Bio., 21:673-684)) for transformation
into Agrobacterium.
[0207] Apo16
[0208] Apo16 (SEQ ID NO:159) is a clone designed to express in a
seed-specific manner which is constructed as per FIGS. 3(A), 3(B)
and 3(D). As seen in FIG. 2, the Apo16 clone consists of a
seed-specific promoter and terminator (phaseolin) driving the
expression of a fusion protein (SEQ ID NO:160) between pro-Apo AI
and GFP. The fusion protein was targeted for expression through the
secretory pathway using the tobacco pathogen related sequence (PRS)
signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221).
To construct this clone, forward primer 1186 (SEQ ID NO:146)
(5'-GGATCCCCtTGGCTAGTAAAGG-3') removed a NcoI site from the start
of GFP (template derived from the vector pVS-GFP). Reverse primer
1187 (SEQ ID NO: 147)
(5'-AAGCTTTCTAGACTGCAGTCATGACTTATTTGTATAGTTC-3') added PstI, XbaI
and HindIII sites after the stop codon. The PCR fragment was
ligated into the EcoRI cloning vector Topo (Invitrogen) creating
plasmid G1 (FIG. 3(A)). Forward primer 1191 (SEQ ID NO:152)
(5'-CCATGGATGAACCCCCCCAGAGCCCCTG-3') amplifies the pro-sequence of
Apo AI and adds a NcoI site to the start of gene. Reverse primer
1189 (SEQ ID NO:149) (5'-GGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes
the stop codon of the gene and adds a BamHI site to assist in
creating a translation fusion with GFP (Plasmid G1). The template
for these primers was a pKS+ based vector (Strategene) containing
the entire coding sequence for human Apo AI gene. The PCR fragments
were each separately ligated into the EcoRI site of the Topo
cloning vector (Invitrogen) creating Plasmid P2. Plasmid P2
contained the pro-Apo AI sequence was cut with restriction enzymes
NcoI and BamHI. Plasmid G'1 contains the GFP coding sequence and
was cut with BamHI and XbaI (see FIG. 3(B)). The fragments of P2
and G'1 were ligated together into the NcoI and XbaI sites of the
plasmid SBS2090 (see FIG. 3(B)) to create the plasmid 2P5. Plasmid
2P5 was cut with NcoI and HindIII to remove the pro-Apo AI-GFP
fusion cassette and the fragments were used subsequently to clone
into the NcoI/HindIII sites of binary vector pSBS4011 (see FIG.
3(D)). Note that this plasmid contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) fused to the PRS signal
peptide.
[0209] The plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium.
[0210] Apo17
[0211] Apo17 (SEQ ID NO:161) is clone designed to express in a
constitutive manner which is constructed as per FIG. 4(A). As seen
in FIG. 2, the Apo17 clone consists of a constitutive promoter and
terminator (ubiquitin) driving the expression of a fusion protein
(SEQ ID NO:162) between Apo AI and GFP. To construct this clone,
plasmid 2M4 (see FIG. 3(B)) was cut with NcoI and PstI to remove
the Apo AI-GFP fusion cassette and the fragment was ligated into
the NcoI and PstI sites of the plasmid pKUO3' to create plasmid
KU2M4. The pKUO3' plasmid contains a Brassica napus oleosin gene
which is removed when the vector is cut with NcoI and PstI
resulting in the Apo AI/GFP fusion construct under the control of a
parsley ubiquitin promoter and ubiquitin terminator (Kawalleck, P.
et al., 1993, Plant Mol. Biol. 21:673-684). The KU2M4 plasmid was
cut with KpnI and ligated into the KpnI site of the binary vector
SBS3000 (FIG. 4. Note that this plasmid contains a pat gene
conferring host plant phosphinothricine resistance (Wohlleben et
al., 1988, Gene 70:25-37) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. The Apo17 clone contains a mature Apo AI-GFP
translational fusion and is targeted to the cytosol.
[0212] Apo18a
[0213] Apo18a (SEQ ID NO:163) is clone designed to express in a
constitutive manner which is constructed as per FIG. 4. As seen in
FIG. 2, the Apo18a clone consists of a constitutive promoter and
terminator (ubiquitin) driving the expression of a fusion protein
(SEQ ID NO:164) between pro-Apo AI and GFP. To construct this
clone, plasmid 2P5 (see FIG. 3(B)) was cut with NcoI and PstI to
remove the pro-Apo AI-GFP fusion cassette and the fragment was
ligated into the NcoI and PstI sites of the plasmid pKUO3' to
create plasmid KU2P5. The pKUO3' plasmid contains a Brassica napus
oleosin gene which is removed when the vector is cut with NcoI and
PstI resulting in the pro-Apo AI/GFP fusion construct under the
control of a parsley ubiquitin promoter and ubiqutin terminator
(Kawalleck, P. et al., 1993, Plant Mol. Biol. 21:673-684). The
KU2P5 plasmid was cut with KpnI and ligated into the KpnI site of
the binary vector SBS3000 (FIG. 4. Note that this plasmid contains
a pat gene conferring host plant phosphinothricine resistance
(Wohlleben et al., 1988, Gene 70:25-37)) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. The Apo18a clone contains a pro-Apo AI-GFP
translational fusion and is targeted to the cytosol.
[0214] Apo18b
[0215] Apo18b (SEQ ID NO:163) is clone designed to express in a
constitutive manner which is constructed as per FIG. 4(B). As seen
in FIG. 2, the Apo18b clone consists of a constitutive promoter and
terminator (ubiquitin) driving the expression of a fusion protein
(SEQ ID NO:164) between pro-Apo AI and GFP. Note that clones Apo18a
and Apo18b both consist have a constitutive promoter (ubiquitin)
driving the expression a fusion protein (SEQ ID NO:164) between
pro-Apo AI and GFP. The difference between the 2 clones is that
Apo18a is inserted into the KpnI site of binary vector pSBS3000
(which contains the pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. In contrast,
Apo18b is inserted into the KpnI site of binary vector pSBS5001
(which contains the pmi gene (Miles et al., 1984, Gene 21:41-48),
encoding for phosphomannose isomerase which allows for positive
selection on mannose containing selection media. The pmi gene is
under the control of the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. To construct
this clone, the Apo18a binary vector was cut with KpnI remove the
pro-Apo AI-GFP fusion cassette and the fragment was ligated into
the KpnI site of the plasmid SBS5001 for expression in
Agrobacterium. The Apo18b clone contains a pro-Apo AI-GFP
translational fusion and is targeted to the cytosol.
[0216] Apo19
[0217] Apo19 (SEQ ID NO:165) is clone designed to express in a
constitutive manner which is constructed as per FIGS. 5(A) and
5(B). As seen in FIG. 2, the Apo19 clone consists of a constitutive
promoter and terminator (ubiquitin) driving the expression of a
fusion protein (SEQ ID NO:166) between Apo AI and GFP. The fusion
protein was targeted for expression through the secretory pathway
using the tobacco pathogen related sequence (PRS) signal peptide
(Sijmons et al., 1990, Bio/technology, 8:217-221). To construct
this clone, Apo15 is used as a template. Forward primer 1177 (SEQ
ID NO:167) (5'-GCAGCATTCATGAACTTCCTTAAGTCTTTCC-3') amplifies the
start of the plant presequence (PRS) which contains a BspHI site at
the start codon. Reverse primer 1178 (SEQ ID NO:168)
(5'-GGTGGTGGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes the stop codon
of the gene and adds a BamHI site to assist in creating an in-frame
translation fusion with GFP. Extra bases were left on the ends of
both primers to facilitate restriction enzyme digestion. The G1
plasmid (see FIG. 3(A)) was digested with BamHI and PstI for
ligation into pubiP+iS3' which contains the ubiquitin promoter and
terminator from Petroselinum crispum (Kawalleck et al., 1993,
Plant. Mol. Bio., 21:673-684). The PRS-Apo AI PCR fragment was
digested with BspHI and BamHI and ligated with the G1 fragment into
the plasmid pubiP+iS3' to create plasmid 19-6. Plasmid 19-6 was
digested with EcoRI to remove the expression cassette and the
cassette was then ligated into the plasmid pKUO3'K (FIG. 4(A))
between the EcoRI sites (removing the existing cassette), creating
plasmid KU19-6. KU19-6 was digested with KpnI and the fragment was
ligated into the KpnI sites of the plasmid SBS3000 (FIG. 5(B)).
Note that this plasmid contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. The Apo19 clone
contains a mature Apo AI-GFP translational fusion, respectively,
targeted to the secretory pathway.
[0218] Apo20
[0219] Apo20 (SEQ ID NO:169) is clone designed to express in a
constitutive manner which is constructed as per FIGS. 5(A) and
5(B). As seen in FIG. 2, the Apo20 clone consists of a constitutive
promoter and terminator (ubiquitin) driving the expression of a
fusion protein (SEQ ID NO:170) between pro-Apo AI and GFP. The
fusion protein was targeted for expression through the secretory
system using the tobacco pathogen related sequence (PRS) signal
peptide (Sijmons et al., 1990, Bio/technology, 8:217-221). To
construct this clone, Apo16 is used as a template. Forward primer
1177 (SEQ ID NO:167) (5'-GCAGCATTCATGAACTTCCTTAAGTCTTTCC-3')
amplifies the start of the plant presequence (PRS) which contains a
BspHI site at the start codon. Reverse primer 1178 (SEQ ID NO:168)
(5'-GGTGGTGGATCCcCTGGGTGTTGAGCTTCTTAGTG-3') removes the stop codon
of the gene and adds a BamHI site to assist in creating an in-frame
translation fusion with GFP. Extra bases were left on the ends of
both primers to facilitate restriction enzyme digestion. The G1
plasmid (see FIG. 3(A)) was digested with BamHI and PstI for
ligation into pubiP+iS3' which contains the ubiquitin promoter and
terminator from Petroselinum crispum (Kawalleck et al., 1993,
Plant. Mol. Bio., 21:673-684). The PRS-pro-Apo AI PCR fragment was
digested with BspHI and BamHI and ligated with the G1 fragment into
the plasmid pubiP+iS3' to create plasmid 20-11. Plasmid 20-11 was
digested with EcoRI to remove the expression cassette and the
cassette was then ligated into the plasmid pKUO3'K (FIG. 4(A))
between the EcoRI sites (removing the existing cassette), creating
plasmid KU20-11. KU20-11 was digested with KpnI and the fragment
was ligated into the KpnI sites of the plasmid SBS3000 (FIG. 5(B).
Note that this plasmid contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37)) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. The Apo20 clone
contains a pro-Apo AI-GFP translational fusion, respectively,
targeted to the secretory pathway.
[0220] Apo21
[0221] Apo21 (SEQ ID NO:171) is a seed-preferred clone which is
constructed as per FIG. 6. As seen in FIG. 2, the Apo21 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of Apo AI (SEQ ID NO:172). To construct this
clone forward primer 1203 (SEQ ID NO:173)
(5'-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3') adds a NcoI site to the
start of mature Apo AI. Reverse primer 1206 (SEQ ID NO: 174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The PCR
fragment was digested with NcoI and HindIII and ligated into the
plasmid SBS2090 creating plasmid 5-3 (FIG. 6). Plasmid 5-3 was
digested with NcoI and HindIII and the Apo AI fragment was ligated
into the NcoI/HindIII sites of SBS4006 (FIG. 3(C)). SBS4006
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) and a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37)) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo21 is a clone
for seed-specific targeting of Apo AI to the cytosol.
[0222] Apo22
[0223] Apo22 (SEQ ID NO:175) is a seed-preferred clone which is
constructed as per FIG. 7. As seen in FIG. 2, the Apo22 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of pro-Apo AI (SEQ ID NO:176). To construct
this clone forward primer 1201 (SEQ ID NO: 177)
(5'-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3') adds an NcoI site to
the start of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The PCR
fragment was digested with NcoI and HindIII and ligated into the
plasmid SBS2090 (FIG. 3(B)) creating plasmid 4-2 (FIG. 7). Plasmid
4-2 was digested with NcoI and HindIII and the pro-Apo AI fragment
was ligated into the NcoI/HindIII sites of SBS4006 (FIG. 3(C)).
SBS4006 contains the .beta.-phaseolin promoter/terminator from
Phaseolus vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc.
USA 80:1897-1901) and a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684)) for transformation into Agrobacterium. Apo22 is a
clone for seed-specific targeting of pro-Apo AI to the cytosol.
[0224] Apo23
[0225] Apo23 (SEQ ID NO:178) is a seed-preferred clone which is
constructed as per FIG. 6. As seen in FIG. 2, the Apo23 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of oleosin/Apo AI (SEQ ID NO:179). To
construct this clone forward primer 1203 (SEQ ID NO: 173)
(5'-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3') adds an NcoI site to the
start of mature Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The PCR
fragment was digested with NcoI and HindIII and ligated into the
plasmid SBS2090 creating plasmid 5-3 (FIG. 6). Plasmid 5-3 was
digested with NcoI and HindIII and the Apo AI fragment was ligated
into the NcoI/HindIII sites of SBS4008 (FIG. 3(C)). SBS4008
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the Arabidopsis oleosin
gene (van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6),
1177-1179) and a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37)) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant. Mol. Bio., 21:673-684)) for transformation
into Agrobacterium. Apo23 is a clone for seed-specific targeting of
oleosin/Apo AI to the oil bodies.
[0226] Apo24
[0227] Apo24 (SEQ ID NO:180) is a seed-preferred clone which is
constructed as per FIG. 7. As seen in FIG. 2, the Apo24 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of oleosin/pro-Apo AI (SEQ ID NO:181). To
construct this clone forward primer 1201 (SEQ ID NO: 177)
(5'-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3') adds an NcoI site to
the start of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The PCR
fragment was digested with NcoI and HindIII and ligated into the
plasmid SBS2090 creating plasmid 4-2 (FIG. 7). Plasmid 4-2 was
digested with NcoI and HindIII and the pro-Apo AI fragment was
ligated into the NcoI/HindIII sites of SBS4008 (FIG. 3(C)). SBS4008
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the Arabidopsis oleosin
gene (van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6),
1177-1179) and a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo24 is a clone for seed-specific targeting of
oleosin/pro-Apo AI to the oil bodies.
[0228] Apo25
[0229] Apo25 (SEQ ID NO:182) is a seed-preferred clone which is
constructed as per FIG. 6. As seen in FIG. 2, the Apo25 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of oleosin/klip8/met/Apo AI (SEQ ID NO:183).
This construct has a klip8 cleavage sequence (SEQ ID NO:143) to
facilitate cleavage of the fusion protein with chymosin. To
construct this clone forward primer 1203 (SEQ ID NO:173)
(5'-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3') adds an NcoI site to the
start of mature Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The PCR
fragment was digested with NcoI and HindIII and ligated into the
plasmid SBS2090 (FIG. 3(B)) creating plasmid 5-3 (FIG. 6). Plasmid
5-3 was digested with NcoI and HindIII and the Apo AI fragment was
ligated into the NcoI/HindIII sites of SBS4010 (FIG. 7). SBS4010
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the Arabidopsis oleosin
gene (van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6),
1177-1179) and a klip8 cleavage site. The plasmid also contains a
pat gene conferring host plant phosphinothricine resistance
(Wohlleben et al., 1988, Gene 70:25-37)) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium.
[0230] Apo25 is a clone for seed-specific targeting of
oleosin/klip8/met/Apo AI to the oil bodies and purification using
the klip8 cleavage sequence.
[0231] Apo26
[0232] Apo26 (SEQ ID NO:184) is a seed-preferred clone which is
constructed as per FIG. 7. As seen in FIG. 2, the Apo26 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of oleosin/klip8/met/pro-Apo AI (SEQ ID
NO:185). This construct has a klip8 cleavage sequence (SEQ ID
NO:143) to facilitate cleavage of the fusion protein with chymosin.
To construct this clone forward primer 1201 (SEQ ID NO:177)
(5'-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3') adds an NcoI site to
the start of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The PCR
fragment was digested with NcoI and HindIII and ligated into the
plasmid SBS2090 (FIG. 3(B)) creating plasmid 4-2 (FIG. 7). Plasmid
4-2 was digested with NcoI and HindIII and the pro-met-Apo AI
fragment was ligated into the BspHI/HindIII sites of SBS4010 (FIG.
7). SBS4010 contains the .beta.-phaseolin promoter/terminator from
Phaseolus vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc.
USA 80:1897-1901) controlling the expression of the Arabidopsis
oleosin gene (van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18
(6), 1177-1179) and a klip8 cleavage site. The plasmid also
contains a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo26 is a clone for seed-specific targeting of
oleosin/klip8/met/pro-Apo AI to the oil bodies and purification
using the klip8 cleavage sequence.
[0233] Apo27
[0234] Apo27 (SEQ ID NO:186) is a seed-preferred clone which is
constructed as per FIG. 8(A). As seen in FIG. 2, the Apo27 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of oleosin/klip8/Apo AI (SEQ ID NO:187).
Apo27 was targeted for expression to the oil bodies using the
Arabidopsis oleosin sequence (van Rooijen G. J. et al. 1992, Plant
Mol. Biol. 18 (6), 1177-1179) and has a klip8 cleavage sequence
(SEQ ID NO:143) to facilitate cleavage of the fusion protein with
chymosin. To construct this clone forward primer 1200 (SEQ ID
NO:188) (5'-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3') adds an
XhoI site and extra nucleotides to facilitate in-frame cloning into
the klip8 (SEQ ID NO:143) cleavage sequence to the start of pro-Apo
AI. Reverse primer 1206 (SEQ ID NO: 174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. The template for these primers was the
Apo33 plasmid which contains the pro-form of Apo AI without
internal XhoI sites and additional Met residue. The PCR fragments
were cut with XhoI and ligated into the XhoI/EcoRV sites of pKS+
creating the plasmids 6-3. Plasmid 6-3 was cut with XhoI and
HindIII and ligated into the XhoI/HindIII sites of binary bector
SBS4010 (FIG. 7.) Note SBS4010 contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the Arabidopsis oleosin gene (van Rooijen G. J. et al. 1992,
Plant Mol. Biol. 18 (6), 1177-1179) and a klip8 cleavage site. The
plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37)) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684)) for transformation into Agrobacterium. Apo27 is a
clone for seed-specific targeting of Apo AI to oil bodies and
purification with cleavage sequence klip8.
[0235] Apo27M
[0236] Apo27M (SEQ ID NO:189) is a seed-preferred clone which is
constructed as per FIG. 8(B) As seen in FIG. 2, the Apo27M clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of oleosin/klip8/met/Apo AI-M (SEQ ID
NO:190). This construct has a klip8 cleavage sequence (SEQ ID
NO:143) to facilitate cleavage of the fusion protein with chymosin.
To construct this clone, forward primer 1202
(5'-GCAGCACTCGAGcaagttcGATGAACCCCCCCAGAGCCC-3') (SEQ ID NO:191)
adds an XhoI site and extra nucleotides to facilitate in-frame
cloning into the klip8 cleavage sequence to the start of mat-Apo
AI. Forward primer 1225 (5'-CGCCAGtGCTTGGCCGCGCGCCTTG-3') (SEQ ID
NO:192) is a blunt ended primer which makes a base pair mutation
from C to T to change an Arg residue into a Cys residue. Reverse
primer 1206 (5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA
GCG-3') (SEQ ID NO:174) adds a HindIII site after the stop codon
and adds a silent mutation to remove the second XhoI site. Both
primers contained extra bases on the 5' ends to facilitate
restriction enzyme digestion. The template for these primers was
the plasmid P-10 which already had its XhoI sites mutated. The
double-stranded template was removed by DpnI digestion. The PCR
fragment was digested with XhoI and HindIII and ligated into the
plasmid pKS+ XhoI/HindIII sites creating plasmid ApoM. ApoM was
digested with XhoI and HindIII and the fragment was ligated into
the XhoI/HindIII sites of the plasmid SBS4010 (FIG. 7). SBS4010
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the Arabidopsis oleosin
gene (van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6),
1177-1179) and a klip8 cleavage site. The plasmid also contains a
pat gene conferring host plant phosphinothricine resistance
(Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo27M is a clone for seed-specific targeting of
oleosin/klip8/met/Apo AI-M to the oil bodies and purification using
the klip8 cleavage sequence.
[0237] Apo 28
[0238] Apo28 (SEQ ID NO:193) is a seed-preferred clone which is
constructed as per FIG. 9. As seen in FIG. 2, the Apo28 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of oleosin/klip8/pro-Apo AI (SEQ ID NO:194).
To construct this clone forward primer 1200 (SEQ ID NO: 188)
(5'-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3') adds an XhoI site
and extra nucleotides to facilitate in-frame cloning into the klip8
cleavage sequence to the start of pro-Apo AI. Forward primer 1205
(SEQ ID NO:195) (5'-CCAAGCCCGCGCTaGAGGACCTCCG-3') is a blunt ended
primer which adds a silent mutation to remove the first XhoI site.
Reverse primer 1206 (SEQ ID NO: 174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The
template for these primers was a pKS+ based vector (Stratagene)
which contained the entire coding sequence for human Apo AI gene.
The double-stranded template was removed by DpnI digestion. The PCR
fragment was digested with XhoI and HindIII and ligated into the
plasmid pKS+ XhoI/HindIII sites creating plasmid P-10. P-10 was
digested with XhoI and HindIII and the fragment was ligated into
the XhoI/HindIII sites of the plasmid SBS4010 (FIG. 7). Note
SBS4010 contains the .beta.-phaseolin promoter/terminator from
Phaseolus vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc.
USA 80:1897-1901) controlling the expression of the Arabidopsis
oleosin gene (van Rooijen G. J. et al. 1992, Plant Mol. Biol. 18
(6), 1177-1179) and a klip8 cleavage site. The plasmid also
contains a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37)) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant. Mol. Bio., 21:673-684)) for transformation
into Agrobacterium. Apo28 is a pro-Apo AI clone targeted to oil
bodies and able to be cleaved at the klip8 sequence.
[0239] Apo29
[0240] Apo29 (SEQ ID NO:196) is a seed-preferred clone which is
constructed as per FIG. 6. As seen in FIG. 2, the Apo29 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS-Apo AI (SEQ ID NO:197). Apo29 was
targeted for expression to the through the secretory pathway using
the tobacco pathogen related sequence (PRS) signal peptide (Sijmons
et al., 1990, Bio/technology, 8:217-221). To construct this clone
forward primer 1203 (SEQ ID NO:173)
(5'-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3') adds an NcoI site to the
start of mature Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The PCR
fragment was digested with NcoI and HindIII and ligated into the
plasmid SBS2090 creating plasmid 5-3 (FIG. 6). Plasmid 5-3 was
digested with NcoI and HindIII and the Apo AI fragment was ligated
into the NcoI/HindIII sites of SBS4011 (FIG. 3D). SBS4011 contains
the .beta.-phaseolin promoter/terminator from Phaseolus vulgaris
(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)
controlling the expression of the Arabidopsis oleosin gene (van
Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) and
the PRS signal peptide (Sijmons et al., 1990, Bio/technology,
8:217-221). The plasmid also contains a pat gene conferring host
plant phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37)) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo29 is a clone
for seed-specific targeting of Apo AI to the secretory pathway.
[0241] Apo30
[0242] Apo30 (SEQ ID NO:198) is a seed-preferred clone which is
constructed as per FIG. 7. As seen in FIG. 2, the Apo30 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS-pro-Apo AI (SEQ ID NO:199). Pro-Apo
AI was targeted for expression to the secretory pathway using the
tobacco pathogen related sequence (PRS) signal peptide (Sijmons et
al., 1990, Bio/technology, 8:217-221). To construct this clone
forward primer 1201 (SEQ ID NO:177)
(5'-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3') adds an NcoI site to
the start of pro-Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate RE digestion. The PCR fragment was
digested with NcoI and HindIII and ligated into the plasmid SBS2090
creating plasmid 4-2 (FIG. 7). Plasmid 4-2 was digested with NcoI
and HindIII and the Apo AI fragment was ligated into the
NcoI/HindIII sites of pSBS4011 (FIG. 3D). SBS4011 contains the
.beta.-phaseolin promoter/terminator from Phaseolus vulgaris
(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)
controlling the expression of the Arabidopsis oleosin gene (van
Rooijen G. J. et al. 1992, Plant Mol. Biol. 18 (6), 1177-1179) and
the PRS signal peptide (Sijmons et al., 1990, Bio/technology,
8:217-221). The plasmid also contains a pat gene conferring host
plant phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37)) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684)) for transformation into Agrobacterium. Apo30 is a
clone for seed-specific targeting of pro-Apo AI to the secretory
pathway.
[0243] Apo31
[0244] Apo31 (SEQ ID NO:200) is a seed-preferred clone which is
constructed as per FIG. 8(A). As seen in FIG. 2, the Apo28 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/Apo AI (SEQ ID NO:201) fusion
protein. D9 ScFV/Apo AI was targeted for expression to the
secretory pathway using the tobacco pathogen related sequence (PRS)
signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221).
To construct this clone forward primer 1200 (SEQ ID NO:188)
(5'-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3') adds an XhoI site
to the start of pro-Apo AI. Reverse primer 1206 (SEQ ID NO: 174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. The template for these primers was the
Apo33 plasmid which contains the pro-form of Apo AI without
internal XhoI sites and additional Met residue. The PCR fragments
were cut with XhoI and ligated into the XhoI/EcoRV sites of pKS+
creating the plasmid 6-3. Plasmid 6-3 was cut with XhoI and HindIII
and ligated into the XhoI/HindIII sites of binary bector SBS4055
(FIG. 9). Note SBS4055 contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the PRS signal sequence fused inframe to the D9 scFV/Apo AI
insert. The plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo31 is a clone
for seed-specific targeting of Apo AI to the secretory pathway and
purification with the oleosin D9 scFV antibody.
[0245] Apo32
[0246] Apo32 (SEQ ID NO:202) is a seed-preferred clone which is
constructed as per FIG. 9. As seen in FIG. 2, the Apo32 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of D9 scFV/pro-Apo AI (SEQ ID NO:203). To
construct this clone forward primer 1200 (SEQ ID NO: 188)
(5'-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3') adds an XhoI site
to the start of pro-Apo AI. Forward primer 1205 (SEQ ID NO:195)
(5'-CCAAGCCCGCGCTaGAGGACCTCCG-3') is a blunt ended primer which
adds a silent mutation to remove the first XhoI site. Reverse
primer 1206 (SEQ ID NO: 174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The
template for these primers was a pKS+ based vector (Stratagene)
which contained the entire coding sequence for human Apo AI gene.
The double-stranded template was removed by DpnI digestion. The PCR
fragment was digested with XhoI and HindIII and ligated into the
plasmid pKS+ XhoI/HindIII sites creating plasmid P-10. P-10 was
digested with XhoI and HindIII and the fragment was ligated into
the XhoI/HindIII sites of the plasmid SBS4055 (FIG. 9). Note
SBS4055 contains the .beta.-phaseolin promoter/terminator from
Phaseolus vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc.
USA 80:1897-1901) controlling the expression of the PRS signal
sequence fused in frame to the D9 scFV antibody. The plasmid also
contains a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37)) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo32 is a clone targeting pro-Apo AI to the
sectretory pathway fused in-frame to the oleosin antibody D9 to aid
in purification.
[0247] Apo33
[0248] Apo33 (SEQ ID NO:204) is a seed-preferred clone which is
constructed as per FIG. 10. As seen in FIG. 2, the Apo33 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/met/Apo AI/ (SEQ ID NO:205)
fusion protein. D9 ScFV/met/Apo AI was targeted for expression to
the secretory pathway using the tobacco pathogen related sequence
(PRS) signal peptide (Sijmons et al., 1990, Bio/technology,
8:217-221). To construct this clone forward primer 1203 (SEQ ID NO:
173) (5'-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3') adds an NcoI site
to the start of mature Apo AI. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The
template for these primers was the P-10 plasmid (FIG. 9) which
contains the pro-form of Apo AI without internal XhoI sites. The
PCR fragment was digested with NcoI and HindIII and ligated into
the NcoI/HindIII sites of pSBS2090 creating plasmid 20-2. The
plasmid was cut with NcoI and HindIII and the fragment ligated into
the pSBS4010 vector BspHI/HindIII sites creating plasmid 4010+20-2.
The plasmid was cut with XhoI and HindIII and the fragment was
ligated into the XhoI/HindIII sites of the binary vector SBS4055
(FIG. 10). Note SBS4055 contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the PRS signal sequence fused inframe to the D9 scFV antibody.
The plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo33 is a
seed-specific clone which targets Apo AI to the secretory pathway
and fused in-frame with the oleosin antibody D9 to aid in
purification.
[0249] Apo34
[0250] Apo34 (SEQ ID NO:206) is a seed-preferred clone which is
constructed as per FIG. 10. As seen in FIG. 2, the Apo34 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/met/pro-Apo AI/ (SEQ ID
NO:207) fusion protein. D9 ScFV/met/pro-Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221). To construct this clone forward primer
1201 (SEQ ID NO: 177) (5'-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3')
adds an NcoI site to the start of pro-Apo AI. Reverse primer 1206
(SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. Both primers contained extra bases on
the 5' ends to facilitate restriction enzyme digestion. The
template for these primers was the P-10 plasmid (FIG. 9) which
contains the pro-form of Apo AI without internal XhoI sites. The
PCR fragment was digested with NcoI and HindIII and ligated into
the NcoI/HindIII sites of SBS2090 (FIG. 3(B)) creating plasmid
19-2. The plasmid was cut with NcoI and HindIII and the fragment
ligated into the SBS4010 vector BspHI/HindIII sites creating
plasmid 4010+19-2. The plasmid was cut with XhoI and HindIII and
the fragment was ligated into the XhoI/HindIII sites of the binary
vector SBS4055 (FIG. 10). Note SBS4055 contains the
.beta.-phaseolin promoter/terminator from Phaseolus vulgaris
(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)
controlling the expression of the PRS signal sequence fused inframe
to the D9 scFV antibody. The plasmid also contains a pat gene
conferring host plant phosphinothricine resistance (Wohlleben et
al., 1988, Gene 70:25-37)) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo34 is a seed-specific clone which targets
met/pro-Apo AI to the secretory pathway and fused in-frame with the
oleosin antibody D9 to aid in purification.
[0251] Apo35
[0252] Apo35 (SEQ ID NO:208) is a seed-preferred clone which is
constructed as per FIG. 8. As seen in FIG. 2, the Apo35 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/Apo AI/KDEL (SEQ ID NO:209)
fusion protein. D9 ScFV/Apo AI was targeted for expression to the
secretory pathway using the tobacco pathogen related sequence (PRS)
signal peptide (Sijmons et al., 1990, Bio/technology, 8:217-221)
and KDEL retention signal (Munro and Pelham, 1987, Cell 48:899-907)
is used to retain the polypeptide in the ER. To construct this
clone forward primer 1200 (SEQ ID NO:188)
(5'-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3') adds an XhoI site
to the start of pro-Apo AI. Reverse primer 1208 (SEQ ID NO:210)
(5'-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3)' adds a KDEL
sequence before the stop codon and a HindIII site after the stop
codon. The template for these primers was the Apo33 plasmid which
contains the pro-form of Apo AI without internal XhoI sites and
additional Met residue. The PCR fragments were cut with XhoI and
ligated into the XhoI/EcoRV sites of pKS+ creating the plasmid 8-5.
Plasmid 8-5 was cut with XhoI and HindIII and ligated into the
XhoI/HindIII sites of binary bector SBS4055 (FIG. 9). Note SBS4055
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the PRS signal sequence
fused inframe a D9 scFV insert. The plasmid also contains a pat
gene conferring host plant phosphinothricine resistance (Wohlleben
et al., 1988, Gene 70:25-37) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo35 is a clone for seed-specific targeting of Apo
AI to the secretory pathway with retention in the ER and
purification with the oleosin D9 scFV antibody.
[0253] Apo36
[0254] Apo36 (SEQ ID NO:211) is a seed-preferred clone which is
constructed as per FIG. 11. As seen in FIG. 2, the Apo36 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/pro-Apo AI/KDEL (SEQ ID
NO:212) fusion protein. D9 ScFV/pro-Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221) and KDEL retention signal (Munro and
Pelham, 1987, Cell 48:899-907) is used to retain the polypeptide in
the ER. To construct this clone forward primer 1200 (SEQ ID NO:188)
(5'-GCAGCACTCGAGcaagttcCGGCATTTCTGGCAGCAAGA-3') adds an XhoI site
and extra nucleotides to facilitate in-frame cloning into the klip8
cleavage sequence to the start of pro-Apo AI. Reverse primer 1208
(SEQ ID NO:210) (5'-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3')
adds a KDEL sequence before the stop codon and a HindIII site after
the stop codon. The template for these primers was the P-10 (FIG.
9) plasmid which contains the pro-form of Apo AI without internal
XhoI sites. The PCR fragment was cut with XhoI and HindIII and
ligated into the XhoI/HindIII sites of pKS+creating the plasmid
7-12. Plasmid 7-12 was cut with XhoI and HindIII and ligated into
the XhoI/HindIII sites of the binary vector SBS4055 (FIG. 9). Note
SBS4055 contains the .beta.-phaseolin promoter/terminator from
Phaseolus vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc.
USA 80:1897-1901) controlling the expression of the PRS signal
sequence fused inframe a D9 scFV insert. The plasmid also contains
a pat gene conferring host plant phosphinothricine resistance
(Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo36 is a clone for the seed-specific expression of
pro-Apo AI targeted to the secretory pathway and is fused in-frame
with the oleosin antibody D9, and accumulates in the endoplasmic
reticulum due to a KDEL signal peptide.
[0255] Apo37
[0256] Apo37 (SEQ ID NO:213) is a seed-preferred clone which is
constructed as per FIG. 12. As seen in FIG. 2, the Apo37 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/met/Apo AI/KDEL (SEQ ID
NO:214) fusion protein. D9 ScFV/met/Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221) and KDEL retention signal (Munro and
Pelham, 1987, Cell 48:899-907) is used to retain the polypeptide in
the ER. To construct this clone forward primer 1203 (SEQ ID NO:173)
(5'-GCAGCACCATGGATGAACCCCCCCAGAGCCCCTG-3') adds an NcoI site to the
start of mature Apo AI. Reverse primer 1208 (SEQ ID NO:210)
(5'-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3') adds a KDEL
sequence before the stop codon and a HindIII site after the stop
codon. The template for these primers was the P-10 plasmid (FIG. 9)
which contains the pro-form of Apo AI without internal XhoI sites.
The PCR fragment was ligated into the EcoRV sites of pKS+ plasmid
creating plasmid 10-2. The plasmid was cut with NcoI and HindIII
and the fragment was ligated into the SBS4010 vector BspHI/HindIII
sites creating plasmid 4010+10-2. The plasmid was cut with XhoI and
HindIII and the fragment was ligated into the XhoI/HindIII sites of
binary vector SBS4055 (FIG. 9). Note SBS4055 contains the
.beta.-phaseolin promoter/terminator from Phaseolus vulgaris
(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)
controlling the expression of the PRS signal sequence fused inframe
a D9 scFV insert. The plasmid also contains a pat gene conferring
host plant phosphinothricine resistance (Wohlleben et al., 1988,
Gene 70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo37 is a clone
which targets met/Apo AI to the secretory pathway and is fused
in-frame with the oleosin antibody D9, and accumulate in the
endoplasmic reticulum due to a KDEL signal peptide.
[0257] Apo38
[0258] Apo38 (SEQ ID NO:215) is a seed-preferred clone which is
constructed as per FIG. 12. As seen in FIG. 2, the Apo38 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/met/pro-Apo AI/KDEL (SEQ ID
NO:216) fusion protein. D9 ScFV/met/pro-Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221) and KDEL retention signal (Munro and
Pelham, 1987, Cell 48:899-907) is used to retain the polypeptide in
the ER. To construct this clone forward primer 1201 (SEQ ID NO:177)
(5'-GCAGCACCATGGggCGGCATTTCTGGCAGCAAGATG-3') adds an NcoI site to
the start of mature Apo AI. Reverse primer 1208 (SEQ ID NO:210)
(5'-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3') adds a KDEL
sequence before the stop codon and a HindIII site after the stop
codon. The template for these primers was the P-10 plasmid (FIG. 9)
which contains the pro-form of Apo AI without internal XhoI sites.
The PCR fragment was ligated into the EcoRV sites of pKS+ plasmid
creating plasmid 9-2. The plasmid was cut with NcoI and HindIII and
the fragment was ligated into the SBS4010 vector BspHI/HindIII
sites creating plasmid 4010+9-2. The plasmid was cut with XhoI and
HindIII and the fragment was ligated into the XhoI/HindIII sites of
binary vector SBS4055 (FIG. 9). Note SBS4055 contains the
.beta.-phaseolin promoter/terminator from Phaseolus vulgaris
(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)
controlling the expression of the PRS signal sequence fused inframe
a D9 scFV insert. The plasmid also contains a pat gene conferring
host plant phosphinothricine resistance (Wohlleben et al., 1988,
Gene 70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo38 is a clone
which targets met/pro-Apo AI to the secretory pathway and is fused
in-frame with the oleosin antibody D9, and accumulate in the
endoplasmic reticulum due to a KDEL signal peptide.
[0259] Apo39
[0260] Apo39 (SEQ ID NO:217) is a seed-preferred clone which is
constructed as per FIG. 13. As seen in FIG. 2, the Apo39 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/Apo AI (SEQ ID NO:218)
fusion protein. D9 ScFV/klip8/Apo AI was targeted for expression to
the secretory pathway using the tobacco pathogen related sequence
(PRS) signal peptide (Sijmons et al., 1990, Bio/technology,
8:217-221) and KDEL retention signal (Munro and Pelham, 1987, Cell
48:899-907) is used to retain the polypeptide in the ER. This clone
has a klip8 cleavage sequence (SEQ ID NO:143) to facilitate
cleavage of the fusion protein with chymosin. To construct this
clone forward primer 1207 (SEQ ID NO:219)
5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3') sequence and adds a SalI
site to the start codon. Reverse primer 1206 (SEQ ID NO:174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent (amplifies
the start of the klip8) mutation to remove the second XhoI site.
The template for the PCR reaction was the plasmid Apo27 (FIG.
8(A)). The PCR product was cut with SalI and HindIII and ligated
into pKS+ creating the plasmid 13-1. The plasmid was cut with SalI
and HindIII and ligated into the XhoI/HindIII sites of plasmid
SBS4055 (FIG. 9). Note SBS4055 contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the PRS signal sequence fused inframe a D9 scFV insert. The
plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37)) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684)) for transformation into Agrobacterium. Apo39 is a
clone for the seed-preferred expression of D9 scFV/klip8/Apo AI
targeted to the secretory pathway. Purification of Apo AI is
achieved using the D9 scFV antibody which has affinity for the
oleosin on the oil body. The protein can be cleaved using chymosin
which will cleave the klip8 site.
[0261] Apo40
[0262] Apo40 (SEQ ID NO:220) is a seed-preferred clone which is
constructed as per FIG. 13. As seen in FIG. 2, the Apo40 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/pro-Apo AI (SEQ ID
NO:221) fusion protein. D9 ScFV/klip8/pro-Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221). This clone has a klip8 cleavage
sequence (SEQ ID NO:143) to facilitate cleavage of the fusion
protein with chymosin. To construct this clone forward primer 1207
(SEQ ID NO:219) 5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3') sequence
and adds a SalI site to the start codon. Reverse primer 1206 (SEQ
ID NO:174) (5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA
GCG-3') adds a HindIII site after the stop codon and adds a silent
mutation to remove the second XhoI site. The template for the PCR
reaction was the plasmid Apo27 (FIG. 8(A)). The PCR product was cut
with SalI and HindIII and ligated into pKS+ creating the plasmid
14-5. The plasmid was cut with SalI and HindIII and ligated into
the XhoI/HindIII sites of plasmid SBS4055 (FIG. 9). Note SBS4055
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the PRS signal sequence
fused inframe a D9 scFV insert. The plasmid also contains a pat
gene conferring host plant phosphinothricine resistance (Wohlleben
et al., 1988, Gene 70:25-37) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo40 is a clone for the seed-preferred expression
of D9 scFV/klip8/pro-Apo AI targeted to the secretory pathway.
Purification of pro-Apo AI is achieved using the D9 scFV antibody
which has affinity for the oleosin on the oil body. The protein can
be cleaved using chymosin which will cleave the klip8 site.
[0263] Apo 41
[0264] Apo41 (SEQ ID NO:222) is a seed-preferred clone which is
constructed as per FIG. 14. As seen in FIG. 2, the Apo41 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/met/Apo AI (SEQ ID
NO:223) fusion protein. D9 ScFV/klip8/met/Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221). This clone has a klip8 cleavage
sequence (SEQ ID NO:143) to facilitate cleavage of the fusion
protein with chymosin. To construct this clone forward primer 1207
(SEQ ID NO:219) (5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3')
amplifies the start of the klip8 sequence and adds a SalI site to
the start codon. Reverse primer 1206 (SEQ ID NO: 174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. The template for the PCR reaction was
the plasmid Apo25 (FIG. 6). The PCR product was cut with SalI and
HindIII and ligated into pKS+ creating the plasmid 11-1. The
plasmid was cut with SalI and HindIII and ligated into the
XhoI/HindIII site of binary vector SBS4055 (FIG. 9). Note SBS4055
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the PRS signal sequence
fused inframe a D9 scFV insert. The plasmid also contains a pat
gene conferring host plant phosphinothricine resistance (Wohlleben
et al., 1988, Gene 70:25-37) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684)) for transformation into
Agrobacterium. Apo41 is a clone for the seed-preferred expression
of D9 scFV/klip8/met/Apo AI targeted to the secretory pathway.
Purification of met/Apo AI is achieved using the D9 scFV antibody
which has affinity for the oleosin on the oil body. The protein can
be cleaved using chymosin which will cleave the klip8 site.
[0265] Apo42
[0266] Apo42 (SEQ ID NO:224) is a seed-preferred clone which is
constructed as per FIG. 14. As seen in FIG. 2, the Apo42 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/met/pro-Apo AI (SEQ ID
NO:225) fusion protein. D9 ScFV/klip8/met/pro-Apo AI was targeted
for expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221). This clone has a klip8 cleavage
sequence (SEQ ID NO:143) to facilitate cleavage of the fusion
protein with chymosin. To construct this clone forward primer 1207
(SEQ ID NO:210) (5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3')
amplifies the start of the klip8 sequence and adds a SalI site to
the start codon. Reverse primer 1206 (SEQ ID NO: 174)
(5'-GTGGTGAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTCCTCcAGA GCG-3') adds
a HindIII site after the stop codon and adds a silent mutation to
remove the second XhoI site. The template for the PCR reaction was
the plasmid Apo26 (FIG. 7). The PCR product was cut with SalI and
HindIII and ligated into pKS+ creating the plasmid 12-1. The
plasmid was cut with SalI and HindIII and ligated into the
XhoI/HindIII site of binary vector SBS4055 (FIG. 9). Note SBS4055
contains the .beta.-phaseolin promoter/terminator from Phaseolus
vulgaris (Slightom et al., 1983, Proc. Natl. Acad. Sc. USA
80:1897-1901) controlling the expression of the PRS signal sequence
fused inframe a D9 scFV insert. The plasmid also contains a pat
gene conferring host plant phosphinothricine resistance (Wohlleben
et al., 1988, Gene 70:25-37)) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant. Mol. Bio., 21:673-684) for transformation into
Agrobacterium. Apo42 is a clone for the seed-preferred expression
of D9 scFV/klip8/met/pro-Apo AI targeted to the secretory pathway.
Purification of met/Apo AI is achieved using the D9 scFV antibody
which has affinity for the oleosin on the oil body. The protein can
be cleaved using chymosin which will cleave the klip8 site.
[0267] Apo43
[0268] Apo43 (SEQ ID NO:226) is a seed-preferred clone which is
constructed as per FIG. 13. As seen in FIG. 2, the Apo43 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/Apo AI/KDEL (SEQ ID
NO:227) fusion protein. D9 ScFV/klip8/Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221) and KDEL retention signal (Munro and
Pelham, 1987, Cell 48:899-907) is used to retain the polypeptide in
the ER. This clone has a klip8 cleavage sequence (SEQ ID NO:143) to
facilitate cleavage of the fusion protein with chymosin. To
construct this clone forward primer 1207 (SEQ ID NO: 219)
5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3') sequence and adds a SalI
site to the start codon. Reverse primer 1208 (SEQ ID NO:210)
(5'-AAGCTTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3') adds a KDEL
sequence before the stop codon and a HindIII site after the stop
codon. The template for the PCR reaction was the plasmid Apo27
(FIG. 8(A)). The PCR product was cut with SalI and HindIII and
ligated into pKS+ creating the plasmid 17-1. The plasmid was cut
with SalI and HindIII and ligated into the XhoI/HindIII sites of
plasmid SBS4055 (FIG. 9). Note SBS4055 contains the
.beta.-phaseolin promoter/terminator from Phaseolus vulgaris
(Slightom et al., 1983, Proc. Natl. Acad. Sc. USA 80:1897-1901)
controlling the expression of the PRS signal sequence fused inframe
a D9 scFV insert. The plasmid also contains a pat gene conferring
host plant phosphinothricine resistance (Wohlleben et al., 1988,
Gene 70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo43 is a clone
for the seed-preferred expression of D9 scFV/klip8/Apo AI/KDEL
targeted to the secretory pathway. Apo43 will accumulate in the ER
due to the KDEL signal peptide. Purification of Apo AI is achieved
using the D9 scFV antibody which has affinity for the oleosin on
the oil body. The protein can be cleaved using chymosin which will
cleave the klip8 site.
[0269] Apo44
[0270] Apo44 (SEQ ID NO:228) is a seed-preferred clone which is
constructed as per FIG. 13. As seen in FIG. 2, the Apo44 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/pro-Apo AI/KDEL (SEQ ID
NO:229) fusion protein. D9 ScFV/klip8/pro-Apo AI/KDEL was targeted
for expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221) and KDEL retention signal (Munro and
Pelham, 1987, Cell 48:899-907) is used to retain the polypeptide in
the ER. This clone has a klip8 cleavage sequence (SEQ ID NO:143) to
facilitate cleavage of the fusion protein with chymosin. To
construct this clone forward primer 1207 (SEQ ID NO:219)
5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3') sequence and adds a SalI
site to the start codon. Reverse primer 1208 (SEQ ID NO:210)
(5'-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3') adds a KDEL
sequence before the stop codon and a HindIII site after the stop
codon. The template for the PCR reaction was the plasmid Apo28
(FIG. 9). The PCR product was cut with SalI and HindIII and ligated
into pKS+ creating the plasmid 18-2. The plasmid was cut with SalI
and HindIII and ligated into the XhoI/HindIII sites of plasmid
SBS4055 (FIG. 9). Note SBS4055 contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the PRS signal sequence fused inframe a D9 scFV insert. The
plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo44 is a clone
for the seed-preferred expression of D9 scFV/klip8/pro-Apo AI/KDEL
targeted to the secretory pathway. Apo44 will accumulate in the ER
due to the KDEL signal peptide. Purification of pro-Apo AI is
achieved using the D9 scFV antibody which has affinity for the
oleosin on the oil body. The protein can be cleaved using chymosin
which will cleave the klip8 site.
[0271] Apo45
[0272] Apo45 (SEQ ID NO:230) is a seed-preferred clone which is
constructed as per FIG. 14. As seen in FIG. 2, the Apo45 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/met/Apo AI/KDEL (SEQ ID
NO:231) fusion protein. D9 ScFV/klip8/met/Apo AI was targeted for
expression to the secretory pathway using the tobacco pathogen
related sequence (PRS) signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221) and KDEL retention signal (Munro and
Pelham, 1987, Cell 48:899-907) is used to retain the polypeptide in
the ER. This clone has a klip8 cleavage sequence (SEQ ID NO:143) to
facilitate cleavage of the fusion protein with chymosin. To
construct this clone forward primer 1207 (SEQ ID NO:219)
(5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3') amplifies the start of
the klip8 sequence and adds a SalI site to the start codon. Reverse
primer 1208 (SEQ ID NO: 210)
(5'-AAGCTTTCAtagctcatctttCTGGGTGTTGAGCTTCTTAG-3') adds a KDEL
sequence before the stop codon and a HindIII site after the stop
codon. The template for the PCR reaction was the plasmid Apo25
(FIG. 6). The PCR product was cut with SalI and HindIII and ligated
into pKS+ creating the plasmid 15-1. This plasmid was cut with SalI
and HindIII and ligated into the XhoI/HindIII site of binary vector
SBS4055 (FIG. 9). Note SBS4055 contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the PRS signal sequence fused inframe a D9 scFV insert. The
plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo45 is a clone
for the seed-preferred expression of D9 scFV/klip8/met/Apo AI/KDEL
targeted to the secretory pathway. Apo45 will accumulate in the ER
due to the KDEL signal peptide. Purification of Apo AI is achieved
using the D9 scFV antibody which has affinity for the oleosin on
the oil body. The protein can be cleaved using chymosin which will
cleave the klip8 site.
[0273] Apo46
[0274] Apo46 (SEQ ID NO:232) is a seed-preferred clone which is
constructed as per FIG. 14. As seen in FIG. 2, the Apo46 clone
consists of a seed-preferred promoter and terminator (phaseolin)
driving the expression of PRS/D9 scFV/klip8/met/pro-Apo AI/KDEL
(SEQ ID NO:233) fusion protein. D9 ScFV/klip8/met/pro-Apo AI was
targeted for expression to the secretory pathway using the tobacco
pathogen related sequence (PRS) signal peptide (Sijmons et al.,
1990, Bio/technology, 8:217-221) and KDEL retention signal (Munro
and Pelham, 1987, Cell 48:899-907) is used to retain the
polypeptide in the ER. This clone has a klip8 cleavage sequence
(SEQ ID NO:143) to facilitate cleavage of the fusion protein with
chymosin. To construct this clone forward primer 1207 (SEQ ID
NO:219) (5'-GCAGCAGTCGACtATGGCTGAGATCACCCGCATTC-3') amplifies the
start of the klip8 sequence and adds a SalI site to the start
codon. Reverse primer 1208 (SEQ ID NO: 210)
(5'-AAGCTTTCAtagctcatctUCTGGGTGTTGAGCTTCTTAG-3') adds a KDEL
sequence before the stop codon and a HindIII site after the stop
codon. The template for the PCR reaction was the plasmid Apo26
(FIG. 7). The PCR product was cut with SalI and HindIII and ligated
into pKS+ creating the plasmid 16-4. The plasmid was cut with SalI
and HindIII and ligated into the XhoI/HindIII site of binary vector
SBS4055 (FIG. 9). Note SBS4055 contains the .beta.-phaseolin
promoter/terminator from Phaseolus vulgaris (Slightom et al., 1983,
Proc. Natl. Acad. Sc. USA 80:1897-1901) controlling the expression
of the PRS signal sequence fused inframe a D9 scFV insert. The
plasmid also contains a pat gene conferring host plant
phosphinothricine resistance (Wohlleben et al., 1988, Gene
70:25-37) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio.,
21:673-684) for transformation into Agrobacterium. Apo46 is a clone
for the seed-preferred expression of D9 scFV/klip8/met/pro-Apo AI
targeted to the secretory pathway and retained in the ER.
Purification of Apo AI is achieved using the D9 scFV antibody which
has affinity for the oleosin on the oil body. The protein can be
cleaved using chymosin which will cleave the klip8 site.
[0275] Apo47
[0276] Apo47 (SEQ ID NO:234) is a seed-preferred clone which is
constructed as per FIG. 15. As seen in FIG. 2, the Apo47 clone
consists of a seed-preferred promoter and terminator (linin)
driving the expression of maize oleosin/klip8/met/Apo AI (SEQ ID
NO:235). This construct has a klip8 cleavage sequence (SEQ ID
NO:143) to facilitate cleavage of the fusion protein with chymosin.
To construct this clone, forward primer 1226
(5'-GCAGCACCATGGCTGATCACCACCG-3') (SEQ ID NO: 236) is used to
amplify the coding sequence of maize oleosin from pSBS2377 and adds
an NcoI site to the start of gene in combination with reverse
primer 1227 (5'-GTGGTGAAGCTTAGACCCCTGCGCC-3') (SEQ ID NO:237) which
removes the stop codon of the gene and adds a HindIII site to
assist in creating an in-frame translation fusion with
klip8/met/Apo AI. The coding sequence of klip8/met/Apo AI from
Apo25 is amplified using forward primer 1228
(5'-GCAGCAAAGCTTATGGCTGAGATCAC-3') (SEQ ID NO:238) which amplifies
the sequence and adds a HindIII site to the start of gene. Reverse
primer 1229 (5'-GTGTGGGATCCTCACTGGGTGTTG-3') (SEQ ID NO:239) adds a
BamHI site after the stop codon. The PCR fragments containing the
maize oleosin cDNA and klip8/met/Apo AI were ligated into the EcoRI
sites of the Topo cloning vector (Invitrogen). Plasmid MzOleo was
cut with restriction enzymes NcoI and HindIII. Plasmid
klip8/met/Apo AI was cut with HindIII and BamHI. The fragments of
MzOleo and klip8/met/Apo AI were ligated together into the NcoI and
BamHI sites of the plasmid SBS5709 to create the plasmid Apo47.
pSBS5709 contains the linin promoter/terminator from WO 01/16340
flanking a multiple cloning site. pSBS5709 also contains the pmi
gene (Miles et al., 1984, Gene 21:41-48), encoding for
phosphomannose isomerase which allows for positive selection on
mannose containing selection media. The pmi gene is under the
control of the ubiquitin promoter/terminator from Petroselinum
crispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684)) for
transformation into Agrobacterium. Apo47 is a clone for
seed-specific targeting of maize oleosin/klip8/met/Apo AI to the
oil bodies and purification using the klip8 cleavage sequence.
Example 2
Agrobacterium and Arabidopsis Transformation
[0277] Arabidopsis thaliana cv. Columbia (C24) is used for all the
experiments. Seeds are planted on the surface of a soil mixture
(two-thirds Redi-earth and one-third perlite with a pH=6.7) or an
Arabidopsis soil mixture supplied by Lehle Seeds (perlite,
vermiculite, peat, terra-green, with a pH=5.5) in 4 inch pots. The
seedlings are allowed to grow to a rosette stage of 6-8 leaves to a
diameter of approximately 2.5 cm. These seedlings are transplanted
into 4 inch pots containing the above soil mixture, covered with
window screen material which has five 1 cm diameter holes cut into
the mesh; one in each of the corners, and one in the center. The
pots are placed inside a dome at 4.degree. C. for four days for a
cold treatment and subsequently moved to 24.degree. C. growth room
with constant light at about 150 .mu.E and 50% relative humidity.
The plants are irrigated at 2-3 day interval and fertilized weekly
with 1% of Peters 20-20-20. Each pot contains five plants. When
plants reach about 2 cm in height, the primary bolts are cut to
encourage the growth of secondary and tertiary bolts. 4 to 5 days
after cutting the primary bolts, the plants are ready to be
infected with Agrobacterium. The plasmid was transformed into
electrocompetent Agrobacterium EHA101. The pots with Arabidopsis
plants are inverted and infected with 500 ml of a re-suspension an
overnight Agrobacterium culture containing the plant transformation
vector of interest for 20 seconds. It is critical that the
Agrobacterium culture contains 5% sucrose and 0.05% of the
surfactant Silwet L-77 (Lehle Seeds). The pots are subsequently
covered with a trans-parent plastic dome for 24 hours to maintain
higher humidity. The plants are allowed to grow to maturity and
seeds (untransformed and transformed) are harvested. For selection
of transgenic lines, the putative transformed seeds are sterilized
in a quick wash of 70% ethanol, then 20% commercial bleach for 15
min and then rinsed at least four times with ddH.sub.2O. About 1000
sterilized seeds are mixed with 0.6% top agar and evenly spread on
a half strength MS plate (Murashige and Skoog, 1962, Physiologia
Plantarum 15: 473-497) containing 0.5% sucrose and 80 .mu.M of the
herbicide phosphinothricin (PPT) DL. The plates are then placed in
a growth room with light regime 8 hr dark and 16 hr light at
24.degree. C. After 7 to 10 days, putative transgenic seedlings are
green and growing whereas untransformed seedlings are bleached.
After the establishment of roots the putative transgenic seedlings
are individually transferred to pots (the individually plants are
irrigated in 3 day interval and fertilized with 1% Peters 20-20-20
in 5 day interval) and allowed to grow to maturity. The pots are
covered with a transparent plastic dome for three days to protect
the sensitive seedlings. After 7 days the seedlings are covered
with a seed collector from Lehle Seeds to prevent seed loss due to
scattering. Seeds from these transgenic plants are harvested
individually and ready for analysis.
Total Leaf Extract Preparation
[0278] An Arabidopsis leaf was frozen with liquid nitrogen and
ground in a 1.5 ml microfuge tube using a drill. 200-250 .mu.l of
0.5M Tris-HCl, pH 7.5 was added and the sample put on ice. 20% SDS
was added to a final concentration of 2%. The sample was boiled for
5 minutes and the extract was spun in a microfuge at maximum speed
for 5 minutes. The liquid was removed to another microfuge tube and
stored at -20.degree. C. Soluble proteins were quantified using the
BCA Protein assay (Pierce) and analyzed on a 15% SDS-PAGE followed
by Western blotting. An anti-Apo AI or anti-GFP rabbit antiserum
was used as the primary antibody; and anti-rabbit-IgG [H+L]-AP
conjugate (Bio-Rad) was used as the secondary antibody.
Total Seed Extract Preparation
[0279] Approximately 40 Arabidopsis seeds (T2 seed) were ground in
50 uL buffer (50 mM Tris pH 7.5) in microfuge tube using Stir-Pak
laboratory mixer. 20% SDS was subsequently added to the sample to a
final concentration of 2% and the sample was boiled for 5 minutes
and centrifuged at maximum speed for 5 minutes. For loading onto an
SDS-PAGE gel, SDS-PAGE 2X loading buffer (100 mM Tris pH 6.8, 20%
glycerol, 4% SDS, 2 mg/mL bromophenol blue, 200 mM DTT) and 1M DTT
were added to sample, boiled for 5 minutes and centrifuged at
maximum speed for 2 minutes.
Example 3
Western Blot Analysis for Apolipoprotein Expression
Constitutive Expression
[0280] Apo17
[0281] As seen in Example 1, Apo17 (SEQ ID NO:27) is a fusion
protein between mature Apo AI and GFP. An ubiquitin promoter and
terminator are used for constitutive expression of the construct.
Western blot analysis (FIG. 16(A)) using a polyclonal Apo AI
antibody detected very low amounts (less than 0.1% of total leaf
protein) the Apo-AI/GFP fusion protein in a total leaf extract at a
molecular weight of approximately 55 kDa in 9 of the 12 clones.
However substantial expression (at least 1% of total seed protein)
of the Apo17 fusion protein was detected in a total seed extract
(FIG. 16(B)) at approximately 55 kDa in 11 out of 12 clones
tested.
[0282] Apo18a
[0283] As seen in Example 1, Apo18 (SEQ ID NO:29) is a fusion
protein between pro-Apo AI and GFP. An ubiquitin promoter and
terminator are used for expression of the construct. Western blot
analysis (FIG. 17(A)) using a polyclonal Apo AI antibody detected
very low amounts (less than 0.1% of total leaf protein) of the
pro-Apo AI/GFP fusion protein in a total leaf extract at a
molecular weight of approximately 56 kDa in 3 of the 12 clones
tested. However substantial expression (at least 1% of total seed
protein) of the Apo18 fusion protein was detected in a total seed
extract (FIG. 17(B)) at approximately 56 kDa in all clones
tested.
[0284] Apo19
[0285] As seen in Example 1, Apo19 (SEQ ID NO:31) is a fusion
protein between Apo AI and GFP. An ubiquitin promoter and
terminator are used for expression of the construct and the PRS
signal peptide is used for the targeted expression of the fusion
protein to the ER. Western blot analysis (FIG. 18(A)) using a
polyclonal Apo AI antibody detected very low levels (less than 0.1%
of total leaf protein) of the Apo AI/GFP fusion protein in a total
leaf extract at a molecular weight of approximately 58 kDa in 10 of
the 12 clones tested. However substantial expression (at least 1%
of total seed protein) of the Apo19 fusion protein was detected in
a total seed extract (FIG. 18(B)) at approximately 58 kDa in 17 of
the 18 clones tested.
[0286] Apo20
[0287] As seen in Example 1, Apo20 (SEQ ID NO:35) is a fusion
protein between pro-Apo AI and GFP. An ubiquitin promoter and
terminator are used for expression of the construct and the PRS
signal peptide is used for the targeted expression of the fusion
protein to the ER. Western blot analysis (FIG. 19(A)) using a
polyclonal Apo AI antibody detected very low levels (less than 0.1%
of total leaf protein) of the pro-Apo AI/GFP fusion protein in a
total leaf extract at a molecular weight of approximately 59 kDa in
3 of the 12 clones tested. However substantial expression (at least
1% of total seed protein) of the Apo20 fusion protein was detected
in a total seed extract (FIG. 19(B)) at approximately 59 kDa in 16
of the 18 clones tested.
Seed-Specific Expression
[0288] Apo10
[0289] As seen in Example 1, Apo10 (SEQ ID NO:10) is a fusion
protein between mature Apo AI and GFP. A phaseolin promoter and
terminator are used for seed-specific expression of the construct.
Western blot analysis (FIG. 20(A)) using a polyclonal GFP antibody
detected the Apo AI/GFP fusion protein in a total seed extract at a
molecular weight of approximately 55 kDa in 7 of the 10 clones
tested.
[0290] Apo11
[0291] As seen in Example 1, Apo11 (SEQ ID NO:16) is a fusion
protein between pro-Apo AI and GFP. A phaseolin promoter and
terminator are used for seed-specific expression of the construct.
Western blot analysis (FIG. 20(B)) using a polyclonal GFP antibody
detected the pro-Apo AI/GFP fusion protein in a total seed extract
at a molecular weight of approximately 56 kDa in 10 of the 14
clones tested.
[0292] Apo12
[0293] As seen in Example 1, Apo12 (SEQ ID NO:19) is a fusion
protein between oleosin, mature Apo AI and GFP. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct. Western blot analysis (FIG. 21(A)) using a
polyclonal GFP antibody detected the Apo AI/GFP fusion protein in a
total seed extract at a molecular weight of approximately 74 kDa in
all of the 17 clones tested.
[0294] Apo13
[0295] As seen in Example 1, Apo13 (SEQ ID NO:21) is a fusion
protein between oleosin, pro-Apo AI and GFP. A phaseolin promoter
and terminator are used for seed-specific expression of the
construct. Western blot analysis (FIG. 21(B)) using a polyclonal
GFP antibody detected the oleosin/pro-Apo AI/GFP fusion protein in
a total seed extract at a molecular weight of approximately 75 kDa
in all 18 of the clones tested.
[0296] Apo15
[0297] As seen in Example 1, Apo15 (SEQ ID NO:23) is a fusion
protein between mature Apo AI and GFP. A phaseolin promoter and
terminator are used for seed-specific expression of the construct
and the PRS signal peptide is used for the targeted expression of
the fusion protein to the secretory system. Western blot analysis
(FIG. 22(A)) using a polyclonal GFP antibody detected the Apo
AI/GFP fusion protein in a total seed extract at a molecular weight
of approximately 58 kDa in 9 of the 10 clones tested.
[0298] Apo16
[0299] As seen in Example 1, Apo16 (SEQ ID NO:25) is a fusion
protein between pro-Apo AI and GFP. A phaseolin promoter and
terminator are used for seed-specific expression of the construct
and the PRS signal peptide is used for the targeted expression of
the fusion protein to the secretory pathway. Western blot analysis
(FIG. 22(B)) using a polyclonal GFP antibody detected the pro-Apo
AI/GFP fusion protein in a total seed extract at a molecular weight
of approximately 59 kDa in 10 of the 13 clones tested.
[0300] Apo21
[0301] As seen in Example 1, Apo21 (SEQ ID NO:37) is Apo AI. A
phaseolin promoter and terminator are used for seed-specific
expression of the construct. Western blot analysis (FIG. 23(A))
using a polyclonal Apo AI antibody was used to detect the Apo AI
protein in a total seed extract. The expected molecular weight was
approximately 28 kDa. In the 12 clones tested, a number of
different proteins were detected with the Apo AI antibody which
ranged from approximately 25 kDa to upwards of 55 kDa. It should be
noted that the expression of Apo AI was detrimental to the health
of the plants (i.e. stunted siliques and the absence of seeds).
[0302] Apo22
[0303] As seen in Example 1, Apo22 (SEQ ID NO:41) is pro-Apo AI. A
phaseolin promoter and terminator are used for seed-specific
expression of the construct. Western blot analysis (FIG. 23(B))
using a polyclonal Apo AI antibody was used to detect the Apo AI
protein in a total seed extract. The expected molecular weight was
approximately 29 kDa. In the 6 clones tested, a number of different
proteins were detected with the Apo AI antibody which ranged from
approximately 25 kDa to upwards of 55 kDa. Clone 22-3 has a protein
of the appropriate molecular weight. It should be noted that the
expression of pro-Apo AI was somewhat detrimental to the health of
the plants with an intermediate phenotype when compared to the
health of plants expressing constructs Apo21, Apo29 and Apo30
versus Apo23.
[0304] Apo23
[0305] As seen in Example 1, Apo23 (SEQ ID NO:44) is a fusion
protein between oleosin and Apo AI. A phaseolin promoter and
terminator are used for seed-specific expression of the construct.
Western blot analysis (FIG. 24(A)) using a polyclonal Apo AI
antibody detected the oleosin/Apo AI fusion protein in a total seed
extract at a molecular weight of approximately 47 kDa in 4 of the 5
clones tested.
[0306] Apo24
[0307] As seen in Example 1, Apo24 (SEQ ID NO:46) is a fusion
protein between oleosin and pro-Apo AI. A phaseolin promoter and
terminator are used for seed-specific expression of the construct.
Western blot analysis (FIG. 24(B)) using a polyclonal Apo AI
antibody detected the oleosin/Apo AI fusion protein in a total seed
extract at a molecular weight of approximately 48 kDa in all 7
clones tested.
[0308] Apo25
[0309] As seen in Example 1, Apo25 (SEQ ID NO:48) is a fusion
protein between oleosin and Apo AI(+Met) with a klip8 cleavage
sequence separating the two components. A phaseolin promoter and
terminator are used for seed-specific expression of the construct.
Western blot analysis (FIG. 25(A)) using a polyclonal Apo AI
antibody detected the oleosin-klip8-Apo AI(+Met) fusion protein in
a total seed extract at a molecular weight of approximately 51 kDa
in all 4 of the clones tested.
[0310] Apo26
[0311] As seen in Example 1, Apo26 (SEQ ID NO:51) is a fusion
protein between oleosin and pro-Apo AI(+Met) with a klip8 cleavage
sequence separating the two components. A phaseolin promoter and
terminator are used for seed-specific expression of the construct.
Western blot analysis (FIG. 25(B)) using a polyclonal Apo AI
antibody detected the oleosin-klip8-proApo AI(+Met) fusion protein
in a total seed extract at a molecular weight of approximately 52
kDa in all 11 of the clones tested.
[0312] Apo28
[0313] As seen in Example 1, Apo28 (SEQ ID NO:60) is a fusion
protein between oleosin and pro-Apo AI with a klip8 cleavage
sequence separating the two components. A phaseolin promoter and
terminator are used for seed-specific expression of the construct.
Western blot analysis (FIG. 26(A)) using a polyclonal Apo AI
antibody detected the oleosin-klip8-pro-Apo AI fusion protein in a
total seed extract at a molecular weight of approximately 52 kDa in
all 7 of the clones tested.
[0314] Apo29
[0315] As seen in Example 1, Apo29 (SEQ ID NO:63) is Apo AI. A
phaseolin promoter and terminator are used for seed-specific
expression of the construct and the PRS signal peptide is used for
the targeted expression of the protein to the secretory pathway.
Western blot analysis (FIG. 26(B)) using a polyclonal Apo AI
antibody was used to detect the Apo AI protein in a total seed
extract. The expected molecular weight was approximately 31 kDa. In
the 10 clones tested, only 1 clone had a protein detected but the
molecular weight was in the range of 37 kDa. It should be noted
that the expression of Apo AI was detrimental to the health of the
plants (i.e. stunted siliques and the absence of seeds).
[0316] Apo30
[0317] As seen in Example 1, Apo30 (SEQ ID NO:65) is pro-Apo AI. A
phaseolin promoter and terminator are used for seed-specific
expression of the construct and the PRS signal peptide is used for
the targeted expression of the fusion protein to the secretory
pathway. Western blot analysis (FIG. 27) using a polyclonal Apo AI
antibody was used to detect the Apo AI protein in a total seed
extract. The expected molecular weight was approximately 32 kDa. In
the 13 clones tested, a number of different proteins were detected
with the Apo AI antibody which ranged from approximately 25 kDa to
upwards of 55 kDa. It should be noted that the expression of
pro-Apo AI was detrimental to the health of the plants (i.e.
stunted siliques and the absence of seeds).
[0318] Apo32
[0319] As seen in Example 1, Apo32 (SEQ ID NO:69) is a fusion
protein between the D9 scFV antibody and pro-Apo AI. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct and the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway. Western
blot analysis (FIG. 28(A)) using a polyclonal Apo AI antibody
detected the D9 scFV-pro-Apo AI fusion protein in a total seed
extract at a molecular weight of approximately 59 kDa in all 5 of
the clones tested.
[0320] Apo33
[0321] As seen in Example 1, Apo33 (SEQ ID NO:71) is a fusion
protein between the D9 scFV antibody and Apo AI(+met). A phaseolin
promoter and terminator are used for seed-specific expression of
the construct and the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway. Western
blot analysis (FIG. 28(B)) using a polyclonal Apo AI antibody
detected the D9 scFV-Apo AI(+met) fusion protein in a total seed
extract at a molecular weight of approximately 59 kDa in 1 of the 8
clones tested.
[0322] Apo34
[0323] As seen in Example 1, Apo34 (SEQ ID NO:73) is a fusion
protein between the D9 scFV antibody and pro-Apo AI(+met). A
phaseolin promoter and terminator are used for seed-specific
expression of the construct and the PRS signal peptide is used for
the targeted expression of the fusion protein to the secretory
pathway. Western blot analysis (FIG. 29) using a polyclonal Apo AI
antibody detected the D9 scFV-pro-Apo AI(+met) fusion protein in a
total seed extract at a molecular weight of approximately 59 kDa in
6 of the 7 clones tested.
[0324] Apo36
[0325] As seen in Example 1, Apo36 (SEQ ID NO:78) is a fusion
protein between the D9 scFV antibody and pro-Apo AI. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct, the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway and KDEL
retention signal is used to retain the polypeptide in the ER.
Western blot analysis (FIG. 30(A)) using a polyclonal Apo AI
antibody detected the D9 scFV-pro-Apo AI-KDEL fusion protein in a
total seed extract at a molecular weight of approximately 60 kDa in
all 13 of the clones tested.
[0326] Apo37
[0327] As seen in Example 1, Apo37 (SEQ ID NO:80) is a fusion
protein between the D9 scFV antibody and Apo AI(+met). A phaseolin
promoter and terminator are used for seed-specific expression of
the construct, the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway and KDEL
retention signal is used to retain the polypeptide in the ER.
Western blot analysis (FIG. 30(B)) using a polyclonal Apo AI
antibody detected the D9 scFV-Apo AI(+met)-KDEL fusion protein in a
total seed extract at a molecular weight of approximately 59 kDa in
all 15 of the clones tested.
[0328] Apo38
[0329] As seen in Example 1, Apo38 (SEQ ID NO:82) is a fusion
protein between the D9 scFV antibody and pro-Apo AI(+met). A
phaseolin promoter and terminator are used for seed-specific
expression of the construct, the PRS signal peptide is used for the
targeted expression of the fusion protein to the secretory pathway
and KDEL retention signal is used to retain the polypeptide in the
ER. Western blot analysis (FIG. 31A) using a polyclonal Apo AI
antibody detected the D9 scFV-pro-Apo AI(+met)-KDEL fusion protein
in a total seed extract at a molecular weight of approximately 60
kDa in 9 of the 11 clones tested.
[0330] Apo39
[0331] As seen in Example 1, Apo39 (SEQ ID NO:83) is a fusion
protein between the D9 scFV antibody, KLIP8 and Apo AI. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct, the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway and KLIP8
is used as a cleavable linker. Western blot analysis (FIG. 31B)
using a polyclonal Apo AI antibody detected the D9 scFV-KLIP8-Apo
AI fusion protein in a total seed extract at a molecular weight of
approximately 55 kDa in all 12 of the clones tested.
[0332] Apo40
[0333] As seen in Example 1, Apo40 (SEQ ID NO:87) is a fusion
protein between the D9 scFV antibody and pro-Apo AI with a klip8
cleavage sequence separating the two components. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct and the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway. Western
blot analysis (FIG. 32(A)) using a polyclonal Apo AI antibody
detected the D9 scFV-klip8-pro-Apo AI fusion protein in a total
seed extract at a molecular weight of approximately 63 kDa in 12 of
the 13 clones tested.
[0334] Apo41
[0335] As seen in Example 1, Apo41 (SEQ ID NO:89) is a fusion
protein between the D9 scFV antibody and Apo AI(+met) with a klip8
cleavage sequence separating the two components. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct and the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway. Western
blot analysis (FIG. 32(B)) using a polyclonal Apo AI antibody
detected the D9 scFV-klip8-Apo AI(+met) fusion protein in a total
seed extract at a molecular weight of approximately 63 kDa in 8 of
the 9 clones tested.
[0336] Apo42
[0337] As seen in Example 1, Apo42 (SEQ ID NO:91) is a fusion
protein between the D9 scFV antibody and pro-Apo AI(+met) with a
klip8 cleavage sequence separating the two components. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct and the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway. Western
blot analysis (FIG. 33(A)) using a polyclonal Apo AI antibody
detected the D9 scFV-klip8-pro-Apo AI(+met) fusion protein in a
total seed extract at a molecular weight of approximately 64 kDa in
all of the 13 clones tested.
[0338] Apo44
[0339] As seen in Example 1, Apo44 (SEQ ID NO:95) is a fusion
protein between the D9 scFV antibody and pro-Apo AI. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct, the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway and KDEL
retention signal is used to retain the polypeptide in the ER.
Western blot analysis (FIG. 34(A)) using a polyclonal Apo AI
antibody detected the D9 scFV-pro-Apo AI-KDEL fusion protein in a
total seed extract at a molecular weight of approximately 64 kDa in
4 of the 15 clones tested.
[0340] Apo45
[0341] As seen in Example 1, Apo45 (SEQ ID NO:97) is a fusion
protein between the D9 scFV antibody and Apo AI(+met). A phaseolin
promoter and terminator are used for seed-specific expression of
the construct, the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway and KDEL
retention signal is used to retain the polypeptide in the ER.
Western blot analysis (FIG. 34(B)) using a polyclonal Apo AI
antibody detected the D9 scFV-Apo AI(+met)-KDEL fusion protein in a
total seed extract at a molecular weight of approximately 63 kDa in
all 12 of the clones tested.
[0342] Apo46
[0343] As seen in Example 1, Apo46 (SEQ ID NO:99) is a fusion
protein between the D9 scFV antibody and pro-Apo AI(+met) with a
klip8 cleavage sequence separating the two components. A phaseolin
promoter and terminator are used for seed-specific expression of
the construct, the PRS signal peptide is used for the targeted
expression of the fusion protein to the secretory pathway and KDEL
retention signal is used to retain the polypeptide in the ER.
Western blot analysis (FIG. 35(A)) using a polyclonal Apo AI
antibody detected the D9 scFV-klip8-pro-Apo AI(+met)-KDEL fusion
protein in a total seed extract at a molecular weight of
approximately 64 kDa in 10 of the 11 clones tested.
Example 4
Western Blot Analysis for Apolipoprotein Localization
[0344] The isolation of oil bodies was performed as previously
described (van Rooijen & Moloney, 1995) with the following
modifications. Briefly, 250 mg of dry mature seeds were surface
sterilized with 70% ethanol, rinsed twice with sterile water and
once with a phosphate buffer (100 mM phosphate buffer pH 8 with
0.5M NaCl). After washing, the seeds were resuspended in phosphate
buffer for analytical analysis and then ground using a sterilized
mortar and pestle. After grinding, the sample was transferred to a
centrifuge bottle and centrifuged for 15 min at 10,000 g at RT.
After centrifugation, the fat pad containing the oil bodies was
removed from the aqueous phase (AQ) and transferred to a 1.5 mL
microfuge tube. The oil bodies were resuspended in a low stringency
phosphate buffer (100 mM phosphate buffer pH 8 with 0.5M NaCl). The
sample was centrifuged for 15 minutes at 10,000 g at 4.degree. C.
and the undertatant removed. The undernatant (PW) and phosphate
washed oil bodies (PO) were tested for the presence of the
apolipoprotein. The oil bodies were subsequently resuspended in a
high stringency urea buffer (8M Urea in 100 mM Na-Carbonate buffer
pH 8). The sample was centrifuged for 15 min at 10,000 g at
4.degree. C. and the undernatant removed. The undernatant (UW) and
urea washed oil bodies (UO) were tested for the presence of the
apolipoprotein. Note that oil bodies are treated with low and high
stringency washes in order to remove proteins which associate with
the oil bodies. Oleosin resists high stringency washes and remains
with the oil body fraction. The microsomal fraction (ER) was
obtained by grinding approximately 250 mg of dry seeds in 4 mL of
microsome grinding buffer (0.5M Sucrose, 0.2M Hepes-NaOH buffer, pH
7.4), into a slurry with a mortar and pestle. The slurry was
centrifuged at 10,000 g for 30 min at 4.degree. C. The supernatant
was transferred into new tubes, and recentrifuged at 10,000 g for
another 30 min at 4.degree. C. to completely remove oil bodies. The
supernatant was then centrifuged for 2 hrs at 100,000 g at
4.degree. C. using an ultracentrifuge with a swinging bucket rotor.
The pellet was washed with microsome grinding buffer, quickly
centrifuged for 5 min for 10,000 g and resuspended in 10-15 uL of
microsome grinding buffer and stored at -20.degree. C.
Untargeted Constructs
[0345] As seen in FIG. 36A, pro-(Apo11) and mature (Apo10) Apo
AI-GFP fusions, without additional targeting signals, were examined
in the different fractions. Apo10 shows that while the mature Apo
AI-GFP fusion protein is detected in all cellular fractions, there
appears to be more protein accumulation with the oil bodies washed
with phosphate buffer and the urea wash fraction. This suggests
that while Apo10 does possess some affinity to oil bodies, a high
stringency wash is sufficient to remove it from the surface of the
oil bodies. Apo11 shows that the presence of the native
pro-sequence of Apo AI targets the Apo AI-GFP fusion protein to the
oil body fraction to a greater extent than when the pro-peptide is
missing, and that the protein remains bound to the oil-bodies even
when high stringency washes are used. It appears that the pro-Apo
AI peptide acts as an `anchoring` sequence to maintain pro-Apo AI
on the surface on the oil bodies. Multiple lower molecular weight
bands can be detected in these fractions, which may be degradation
products.
Oil Body Targeted Constructs
[0346] This pattern of tight association of Apo AI-GFP fusion
protein with oil bodies can also be seen in the oil body targeted
constructs (FIG. 36B). Pro-(Apo13) and mature (Apo12) Apo AI-GFP
fusions are fused in-frame to the C-terminus of oleosin, which
serves as a targeting signal to oil bodies. As seen previously for
the untargeted pro-Apo AI-GFP fusion, the protein is predominantly
associated with the phosphate and urea-washed oil body fractions.
Multiple lower molecular weight bands are also detected in these
fractions, indicating that there is something unique to the oil
body associated fractions which leads to an accumulation of
possible degradation products.
Secretory System Targeted Constructs
[0347] The PRS signal peptide (Sijmons et al., 1990,
Bio/technology, 8:217-221) which targets proteins to the secretory
pathway was added as an in-frame translational fusion to the
pro-(Apo16) and mature (Apo15) forms of Apo AI-GFP (FIG. 37).
Normally, this results in the secretion of the fusion protein into
the extracellular space (apoplast) of plant cells. However, when
the cellular fractions of the two constructs were examined, the Apo
AI-GFP fusion protein was detected in all of the fractions, with
more protein being detected in the oil body fractions. A band the
size of the recombinant fusion protein can also be observed in the
oil body fractions when the Ponceau-S stained immunoblot is
examined (FIG. 37, upper panel). The presence of the native
pro-sequence of Apo AI did not appear to change the secreted fusion
protein's association with a specific cellular fraction. The plant
presequence recognition signal (PRS) peptide appears to interfere
with the `anchoring` characteristic of the pro-Apo AI peptide.
Constitutive Expression of Apo AI-GFP in the Leaves
[0348] Leaf tissue that constitutively expressed the untargeted and
secreted pro- and mature forms of Apo AI-GFP was examined to
determine if the Apo AI-GFP fusion protein could accumulate in
non-oil producing tissues. Leaf tissues were homogenized (as
described in example 2) from the transgenic plants lines
constitutively expressing the untargeted pro-(Apo18) and mature
(Apo17) and the secreted pro-(Apo20) and mature (Apo19) forms of
Apo AI-GFP and used for immunoblotting. Leaf material was also
sampled from wild-type (c24) plants for a negative control, and
from UR2 (ubiquitin-driven oleosin-GFP) plants and UG-14
(ubiquitin-driven GFP) plants for positive controls for leaf
expression. Similar amounts of protein were separated by SDS-PAGE
and the Apo AI-GFP fusion protein was detected by immunoblotting
with an anti-GFP antibody (FIG. 38 lower panel).
[0349] Faint bands were detected in the non-transformed line,
whereas the both the ubiquitin-driven GFP (UG-14) and oleosin-GFP
(UR2) fusion are readily detected on the immunoblot by the anti-GFP
antibody. However, while a similar amount of leaf protein was
loaded on the immunoblot as can be seen by Ponceau-S staining of
the membrane (upper panel, FIG. 38), no bands can be detected for
any of the Apo AI-GFP fusion protein constructs. The faint band
detected in the background of the immunoblot may be due to
cross-reactivity with Rubisco, the most prevalent protein found in
leaf tissue (Spreitzer R J & Salvucci M E (2002) Rubisco:
structure, regulatory interactions, and possibilities for a better
enzyme. Annu Rev Plant Biol 53: 449-475.), which can be seen in the
Ponceau-S stained panel of the immunoblot by size (55 kDa)
according to the molecular weight markers.
Seed Specific Expression of Untargeted and Secreted Apo AI in
Seeds
[0350] One transgenic line was selected from each of the T,
generation Apo AI transgenic plants that showed some accumulation
of either pro- or mature ApoAI protein in total seed extracts. Of
the plant lines that were putative transgenics, only 1 line for the
following showed any protein expression: Apo21 (untargeted mature
Apo AI), Apo22 (untargeted pro-Apo AI), and Apo29 (secreted mature
Apo AI); two lines were found for Apo30 (secreted pro-Apo AI). All
transgenic plant lines that contained the constructs Apo23 and
Apo24 appeared to express and accumulate the oleosin-Apo AI fusion
protein. Similar amounts of protein were separated by SDS-PAGE and
the Apo AI protein was detected by immunoblotting with an anti-Apo
AI antibody (FIG. 39). A negative control was included for the
immunoblot by preparing a similar protein extract from a
non-transformed plant (c24). The correct size of band for Apo AI
expressed alone (expected molecular weight of 28.3 kDa for mature
Apo AI and 29.3 kDa for pro-Apo AI) was only observed in line
Apo22-3 (untargeted pro-Apo AI). The predominant band detected in
Apo21-11 (untargeted mature Apo AI) is the incorrect size, and
while only minor bands are detected in Apo29-11 (secreted mature
Apo AI) and Apo30-14 (secreted proApo AI) none are the correct
size. However, for both of the oleosin fusions, Apo23-11
(oleosin-mature Apo AI) and Apo24-6 (oleosin-pro-Apo AI), a
significant amount of Apo AI fusion protein is detected by anti-Apo
AI antibody and can also be detected in the Ponceau-S stained
immunoblot (upper panel).
Subcellular Localization of Untargeted Apo AI (Apo22) T3 Seeds
[0351] The seed-specific expression of the pro-form of the
untargeted Apo AI protein (Apo22) and its association with a
specific cellular fraction was examined in mature seeds (FIG. 40).
Seeds were homogenized and treated as described above, and the
cellular fractions were subjected to immunoblotting with an
antibody against Apo AI. Apo22-3 shows that the presence of the
native pro-sequence of Apo AI targets the Apo AI protein to the oil
body fraction, and that the protein requires high stringency washes
to remove the protein from the oil bodies. Some protein can also be
detected in the aqueous phase indicating that not all the pro-Apo
AI protein is associated with the oil bodies. Multiple lower
molecular weight bands can be detected in these fractions, which
may be degradation products.
Example 5
Confocal Microscopy for Apolipoprotein Localization
[0352] Immature embryos were dissected out of Apo AI-GFP siliques
under sterile water into a Petri dish using forceps and a
dissecting microscope. Embryos were removed from the seed coat by
gentle pressure on the immature seeds with a glass microscope slide
cover. Embryos were transferred by pipette into a 1.5 mL microfuge
tube with water added to a final volume of 1 mL. Nile Red
(Molecular Probes) was used at a final concentration of 1
.mu.g/.mu.L. Embryos in diluted Nile Red were left to incubate for
15 min in darkness at room temperature. Embryos were rinsed 3 times
in sterile water and mounted in water on glass slides for
microscopy. Leaf epidermal cells were prepared by simply cutting
small portions of leaves (0.5 cm.sup.2) with a scalpel and mounting
them in water on microscope slides with cover slips. The leaf
sections were placed so that the lower epidermis faced upwards
(less interference with autofluorescent chloroplasts).
[0353] All GFP-dependent fluorescence was analyzed from Arabidopsis
embryos and leaf epidermal cells mounted in water for microscopic
observations and examined with a Zeiss LSM 510 laser scanning
confocal microscope (Edmonton, AB). For simultaneous detection of
GFP and Nile Red a line-sequential single-tracking mode with the
AOTF-controlled excitation with 488 nm and 543 nm light was set at
20% and 100% respectively. A Plan-Appochromat 63x/1.4 Oil DIC
objective was used with a 5.times. scan zoom. The pinhole was
optimized for Channel 2 (green) at 94 .mu.m and for Channel 1 (red)
at 106 .mu.m.
[0354] The resulting micrographs can be seen in FIGS. 42 to 45.
Colocalization (indicated in yellow) between untargeted pro-Apo
AI-GFP fusions (Apo11) and Nile red stained oil bodies, is evident
in FIG. 41 (D-F). There is also colocalization (indicated in
yellow) between Apo12, mature Apo AI fused to GFP targeted to oil
bodies using oleosin, (G-I of FIG. 41) and Apo13, pro-Apo AI fused
to GFP targeted to oil bodies using oleosin (A-C of FIG. 42).
Colocalization (indicated in yellow) between untargeted pro-Apo
AI-GFP fusions (Apo18) and Nile red stained oil bodies is evident
in (D-F of FIG. 43). No colocalization between Apo AI-GFP fusion
protein (Apo19) and oil bodies is observed (G-H of FIG. 43). In
FIG. 44, no colocalization is evident in the leaves. In conclusion,
in the absence of an oil body target (i.e. oleosin),
co-localization of Apo AI is observed only in the embryos (in the
presence of neutral lipid) and only when the pro-peptide of Apo AI
is expressed in the cytoplasm (i.e. not when secreted).
Example 6
Cleavage and HPLC Analysis of Apo 25, 26 and 28 Expressing
Arabidopsis Seed
Cleavage of Apo25, Apo26 and Apo28 Recombinant Protein
[0355] The isolation of oil bodies was performed as previously
described (van Rooijen & Moloney, 1995) with the following
modifications. Briefly, 250 mg of dry mature seeds were surface
sterilized with 70% ethanol, rinsed twice with sterile water and
once with a phosphate buffer (100 mM phosphate buffer pH 8 with
0.5M NaCl). After washing, the seeds were resuspended in phosphate
buffer for analytical analysis and then ground using a sterilized
mortar and pestle. After grinding, the sample was transferred to a
centrifuge bottle and centrifuged for 15 min at 10,000 g at RT.
After centrifugation, the fat pad containing the oil bodies was
transferred to a 1.5 mL microfuge tube and resuspended in a urea
buffer (8M Urea in 100 mM Na-Carbonate buffer pH 8). The sample was
centrifuged for 15 min at 10,000 g at 4.degree. C. and the
undernatant removed. The fat pad was resuspended in sterile
ddH.sub.2O, centrifuged for 15 min at 10,000 g at 4.degree. C. and
the undernatant removed. The oil bodies were resuspended in 50
.mu.L of sterile ddH.sub.2O and stored in the dark at 4.degree. C.
The cleavage reaction was performed in a 20 pit reaction volume
containing 100 mM phosphate buffer pH 4.5, with a final ratio of
1:100 protease to oil body protein, at 37.degree. C. for 2 hrs. A
sample reaction would be as follows: 20 .mu.g of purified Apo25,
Apo26 or Apo28 was combined with 2 .mu.L 1M phosphate buffer pH 4.5
(final concentration 100 mM), 2 .mu.L, chymosin (0.1 _g/_l) with
sterile ddH.sub.2O to bring up to final volume of 20 .mu.L. After 2
hrs, the cleavage reaction was centrifuged for 15 min, and the
undernatent was removed from the fat pad and each phase was
analyzed for recombinant protein.
Purification of Apo25, Apo26 and Apo28 by Reverse Phase
Chromatography
[0356] Approximately, 1000 .mu.g of Apo25, Apo26 or Apo28 was
cleaved by chymosin for 2 hrs at 37.degree. C. After the cleavage
reaction was completed, the reaction was centrifuged for 15 min at
10,000 g at 4.degree. C. and the undernatent was recovered. The fat
pad was resupsended in a urea buffer (8M Urea in 0.1 mM
Na-Carbonate buffer pH 8), and recentrifuged for 15 min. The
undernatent or wash was recovered, and the washes were repeated for
an additional three times, with the undernatent being recovered
each time and pooled into a 15 mL Falcon tube. After the urea
washes were completed, the washes were aliquoted into 1.5 mL
microfuge tubes, and centrifuged for 15 minutes to remove any
contaminating oil body residue. The undernatents were recovered and
filtered into a new 15 mL Falcon tube using a 0.2 micron filter. A
VYDAC 214TP54 C4 silica 5 micron (Grace Vydac, Anaheim, Calif.)
reverse-phase chromatography column (0.24.times.25 cm) was
equilibrated in buffer A (10% acetonitrile and 0.1% trifluoroacetic
acid) at a flow-rate of 2 mL/min. The pooled chymosin-cleaved
Apo25, Apo26 or Apo28 urea undernatent was loaded on the column. A
linear gradient was applied to the column 0 to 60% buffer B (95%
acetonitrile, 0.1% trifluoroacetic acid) for the elution of Apo AI.
Apo AI (US Biological, Catalogue number A2299-10) was used as a
standard for comparising the cleavage products from Apo25, Apo26
and Apo28. Fractions 19.6' to 20.8' (0.2'=0.4 mL each) were
collected. Comparing the relative intensities of the DAD traces at
214, 254, 280 & 326 nm indicates that the material eluting in
the 19.5-21.0' zone most likely represents the Apo AI polypeptide
(FIG. 45A). These peaks also increased in intensity compared to a
previous injection of 0.020 mL of the same sample. To purify the
cleavage product from Apo25 (oleosin-klip8-Apo AI(met+)), chymosin
treated fractions were collected from 7 to 25' @ 1 mL each. The
major polypeptide peak is at 20.5.degree. (FIG. 45B), which is just
0.2' later than the suspected hApo AI standard. To purify the
cleavage product from Apo26 (oleosin-klip8-pro-Apo AI(met+)),
fractions were collected from 7 to 25' at 1 mL each. The major
polypeptide peak is at 18' (FIG. 45C), which is 2.4' earlier than
the suspected hApo AI standard. To purify the cleavage product from
Apo28 (oleosin-klip8-pro-Apo AI), fractions were collected from 7
to 25' at 1 mL each. The major polypeptide peak is at 18' (FIG.
45D), which is 2.4' earlier than the suspected hApo AI standard but
similar to the Apo26 run.
Mass Spectrometry
[0357] Mass spectra were acquired by Doug Olson (National Research
Council of Canada, Plant Biotechnology Institute BioAnalytical
Spectroscopy Group, Saskatoon, SK) on an Applied Biosystems
Voyager-DE STR matrix assisted laser desorption ionisation time of
flight (MALDI-TOF) mass spectrometer instrument (Applied
Biosystems, Foster City, Calif.). Samples were spotted onto a
OPI-TOF LC MALDI insert (Applied Biosystems, Foster City, Calif.)
using a matrix of sinapinic acid saturated in 30% acetonitrile/70%
water/0.1% TFA. Ions were accelerated at +20 kV and masses were
detected in linear mode, with horse heart myoglobin used as an
external calibrant. --Electrospray ionization mass spectrometry of
the purified recombinant mature Apo25 protein gave a molecular mass
of 28, 325 Da, which is 6 Da greater than calculated molecular
weight of 28, 319 Da. The difference in value from the observed
value from the expected, may be due to a case of limited sample
leading to a decreased signal to noise ratio and a decreased
accuracy. The expected molecular weight of mature Apo AI is 28, 187
Da, but due to the presence of the additional Met residue, the
cleaved recombinant mature Apo AI protein has an increased
molecular weight. The purified standard hApo AI protein was also
analyzed by mass spectrometry, and it possessed two distinct peaks
at 25, 969 Da and 22, 815 Da. Both of these observed values are
significantly lower than the expected value of 28, 187 Da; however,
these two predominant lower molecular weights were previously
observed on the immunoblots. It is likely that it is this decrease
in molecular weight that results in the slightly different elution
profile of the human and recombinant proteins as was seen by
RP-HPLC.
Example 7
Transformation of Safflower
[0358] This transformation protocol is similar to that outlined by
Orlilcowska T. K. et al. ((1995) Plant Cell, Tissue and Organ
Culture 40: 85-91), but with modifications and improvements both
for transforming S-317 and for using phosphinothricin as the
selectable marker. Decontaminate seeds from S-317 California
variety of safflower, which are not damaged, cracked or diseased,
in 0.1% HCl.sub.2 for 12 minutes followed by 4-5 rinses with
sterile distilled water. Germinate sterile seeds in the dark on MS
medium (Murashige T. & Skoog F (1962) Physiol. Plant. 15:
473-497) with 1% sucrose and 0.25% Gelrite. Initiate Agrobacterium
cultures from frozen glycerol stocks in 5 ml AB minimal liquid
media with antibiotic selection, and grow for 48 hours at
28.degree. C. Grow an aliquot of this culture grown overnight in 5
ml of Luria broth with selection for transformation. Wash 6-8 ml of
bacterial cells twice with AB media, and make up to a final cell
density of 0.4-0.5 (OD600).
[0359] Remove two-day-old cotyledons from germinated seedlings, dip
in the prepared Agrobacterium cells, and plate on MS medium with 3%
sucrose, 4 .mu.M N6-benzyladenine (BA) and 0.8 .mu.M
naphthaleneacetic acid (NAA). Incubate plates at 21.degree. C.
under dark conditions. After 3 days, transfer to the same medium
with 300 mg/L timentin. After an additional 4 days, move all
cultures to the light. After 3 days, place explants on selection
medium with phosphinothricin added at 0.5 mg/L. For continued bud
elongation, transfer explants weekly onto MS medium without
phytohormones but with twice the basal amount of KNO.sub.3. Excise
shoots that had elongated to greater than 10 mm from the initial
explant and individually grow on selection. For rooting, place
green shoots, representing putative transgenic tissue, on MS medium
with 2% sucrose, 10 .mu.M indolebutyric acid and 0.5 .mu.M NAA.
Transfer rooted shoots to a well drained soil-less mix and grow
under high humidity and 12 hours of light.
Example 8
Flax Transformation Protocol
[0360] This transformation procedure is similar to that outlined by
Dong J. and McHughen A. (Plant Cell Reports (1991) 10:555-560),
Dong J. and McHughen A. (Plant Sciences (1993) 88:61-71) and
Mlynarova et al. (Plant Cell Reports (1994) 13: 282-285).
Decontaminate flax seeds, which are not damaged, cracked or
diseased, in a 70% ethanol solution for 5 to 7 minutes, followed by
25 minutes in a 50% bleach solution with Tween 20 (3-4 drops per
100 ml) with continuous stirring. Rinse seeds 5 to 7 times with
sterile distilled water. Germinate decontaminated seeds in the
light on MS medium (Murashige T. & Skoog F (1962) Physiol.
Plant. 15: 473-497) with 2% sucrose and 0.3% Gelrite.RTM. in
Magenta jars. For transformation, grow Agrobacterium cultures
overnight in AB broth plus the appropriate antibiotic for
selection. Wash 6 to 8 ml of overnight cells twice, and
re-suspended in 5 ml of AB broth; add 2 ml of this stock to 98 ml
of induction medium (MS basal medium with 3% sucrose, 5 .mu.M
6-benzylaminopurine (BA) and 0.25 .mu.M alpha-naphthalene acetic
acid (NAA) and adjust for a final OD.sub.600 of 1.0.
[0361] Section hypocotyl explants, and inoculate in the prepared
Agrobacterium cell solution for about 4 h (stir plates gently 1-2
times during this period). After the infection period, remove
explants from the liquid inoculation medium and blot on sterile
filter paper. Plate 15-20 explants on 0.7% agar-solidified
induction medium in tissue culture plates. Seal the plates with
plastic wrap, and co-cultivate explants for 48 h under light
conditions (23-24.degree. C.). After 2 days, transfer the green,
meristematic explants to the same medium containing 300 mg/L
Timentin (pre-selection media) and wrap with plastic wrap. After 3
days, transfer the cultures to the above medium containing 10 mg/L
DL PPT (Selection 1). Wrap the plates with Parafilm.RTM. and
incubate at 24.degree. C. under light conditions. Transfer cultures
every two weeks and keep on this media for one month. For shoot
elongation, transfer the cultures every two weeks on selection
medium II (MS basal medium containing 2% sucrose, 500 mg/L MES
buffer, 300 mg/L Timentin and 10 mg/L DL PPT) in Magenta jars.
Putative transformed shoots, which survived selection, are dark
green and form vigorous roots in 7-10 days when planted
individually on selection II media. Transfer rooted shoots to
sterilized greenhouse soil mix in small pots and cover plantlets
with clear plastic cups for acclimatization. For maturation,
transfer actively growing plants to one-gallon pots with a
well-drained soil mix and grow under greenhouse conditions.
[0362] While the present invention has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the invention is not limited
to the disclosed examples. To the contrary, the invention is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims.
[0363] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
SUMMARY OF SEQUENCES
[0364] SEQ ID NO:1 and 2 set forth the nucleotide sequence and the
deduced amino acid sequence, respectively, of the human pro-Apo AI
protein.
[0365] SEQ ID NO:3 and 4 set forth the nucleotide sequence and the
deduced amino acid sequence, respectively, of the human Apo AI
Milano protein.
[0366] SEQ ID NO:5 and 6 set forth the nucleotide sequence and the
deduced amino acid sequence, respectively, of the human Apo AI
Paris protein.
[0367] SEQ ID NO:1, 7 and 8 are known human Apolipoprotein
sequences which are described in Table 1.
[0368] SEQ ID NO:9-24 are known Apolipoprotein A-I sequences which
are described in Table 1.
[0369] SEQ ID NO:25-34 are known Apolipoprotein A-IV sequences
which are described in Table 1.
[0370] SEQ ID NO:35-55 are known Apolipoprotein E sequences which
are described in Table 1.
[0371] SEQ ID NO:56 sets forth the nucleic acid sequence of an
Arabidopsis thaliana thioredoxin
[0372] SEQ ID NO:57 sets forth the nucleic acid sequence of a
soluble green fluorescent protein
[0373] SEQ ID NO:58 sets forth the amino acid sequence of a PRS
signal sequence.
[0374] SEQ ID NO:59-116 are known oleosin oil body protein
sequences which are described in Table 3.
[0375] SEQ ID NO:117-129 are known caleosin oil body protein
sequences which are described in Table 3.
[0376] SEQ ID NO:130-137 are known steroleosin oil body protein
sequences which are described in Table 3.
[0377] SEQ ID NO:138 sets forth a known Arabidopsis thaliana
oleosin oil body protein sequence.
[0378] SEQ ID NO:139 sets forth a known Brassica napus oleosin oil
body protein sequence.
[0379] SEQ ID NO:140 sets forth a known Arabidopsis thaliana
caleosin oil body protein nucleic acid sequence.
[0380] SEQ ID NO:141 sets forth a known Arabidopsis thaliana
caleosin oil body protein nucleic acid sequence.
[0381] SEQ ID NO:142 sets forth a known stereoleosin oil body
protein nucleic acid sequence.
[0382] SEQ ID NO:143 sets forth the nucleotide sequence for the
klip8 cleavage sequence.
[0383] SEQ ID NO:144 and 145 set forth the nucleotide sequence and
the deduced amino acid sequence, respectively, of the Apo10
clone.
[0384] SEQ ID NO:146 sets forth the nucleotide sequence of the
forward primer 1186 which is complementary to the 5' region of GFP
and is designed to remove the NcoI site.
[0385] SEQ ID NO:147 sets forth the nucleotide sequence of the
reverse primer 1187 which is complementary to the 3' region of GFP
and is designed to add PstI, XbaI and HindIII sites after the stop
codon.
[0386] SEQ ID NO:148 sets forth the nucleotide sequence of the
forward primer 1190 which is complementary to the 5' region of
mature Apo AI and is designed to add a NcoI site to the start of
the gene.
[0387] SEQ ID NO:149 sets forth the nucleotide sequence of the
reverse primer 1189 which is complementary to the 5' region of
mature Apo AI and is designed to remove the stop codon of the gene
and add a BamHI site to assist in creating an in-frame
translational fusion with GFP.
[0388] SEQ ID NO:150 and 151 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo11 clone.
[0389] SEQ ID NO:152 sets forth the nucleotide sequence of the
forward primer 1191 which is complementary to the 5' region of
pro-Apo AI and is designed to add a NcoI site to the start of the
gene.
[0390] SEQ ID NO:153 and 154 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo12 clone.
[0391] SEQ ID NO:155 and 156 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo13 clone.
[0392] SEQ ID NO:157 and 158 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo15 clone.
[0393] SEQ ID NO:159 and 160 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo16 clone.
[0394] SEQ ID NO:161 and 162 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo17 clone.
[0395] SEQ ID NO:163 and 164 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo18 clone.
[0396] SEQ ID NO:165 and 166 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo19 clone.
[0397] SEQ ID NO:167 sets forth the nucleotide sequence for forward
primer 1177 which is complementary to the 5' region of PRS/Apo AI
(clone Apo15) and is designed to amplify the start of the plant
presequence (PRS) which contains a BspHI site at the start
codon.
[0398] SEQ ID NO:168 sets forth the nucleotide sequence for reverse
primer 1178 which is complementant to the 3' region of Apo AI and
is designed to remove the stop codon of the gene and add a BamHI
site to assist in creating an in-frame translational fusion with
GFP.
[0399] SEQ ID NO:169 and 170 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo20 clone.
[0400] SEQ ID NO:171 and 172 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo21 clone.
[0401] SEQ ID NO:173 sets forth the nucleotide sequence of forward
primer 1203 which is complementary to the 5' region of Apo AI and
is designed to add a NcoI site to the start of mature Apo AI.
[0402] SEQ ID NO:174 sets forth the nucleotide sequence of reverse
primer 1206 which is complementary to the 3' region of Apo AI and
is designed to add a HindIII site after the stop codon.
[0403] SEQ ID NO:175 and 176 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo22 clone.
[0404] SEQ ID NO:177 sets forth the nucleotide sequence of forward
primer 1201 which is complementary to the 5' region of pro AI and
is designed to add an NcoI site to the start of pro-Apo AI.
[0405] SEQ ID NO:178 and 179 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo23 clone.
[0406] SEQ ID NO:180 and 181 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo24 clone.
[0407] SEQ ID NO:182 and 183 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo25 clone.
[0408] SEQ ID NO:184 and 185 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo26 clone.
[0409] SEQ ID NO:186 and 187 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo27 clone.
[0410] SEQ ID NO:188 sets forth the nucleotide sequence of forward
primer 1200 which is complementary to the 5' region of mature Apo
AI and is design to add an XhoI site and extra nucleotides to
facilitate in-frame cloning into the klip8 cleavage sequence to the
start of pro-Apo AI.
[0411] SEQ ID NO:189 and 190 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo27M clone.
[0412] SEQ ID NO:191 sets forth the nucleotide sequence of forward
primer 1202 which is complementary to the 5' region of the human
Apo AI and is designed to amplify the human Apo AI sequence, add a
XhoI site and extra nucleotides to facilitate in-frame cloning into
the klip8 cleavage sequence to the start of mat-Apo AI.
[0413] SEQ ID NO:192 sets forth the nucleotide sequence of forward
primer 1225 is a blunt ended primer which makes a base pair
mutation from C to T to change an Arg residue into a Cys residue
thereby creating the Apo-Milano mutation.
[0414] SEQ ID NO:193 and 194 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo28 clone.
[0415] SEQ ID NO:195 sets forth the nucleotide sequence of forward
primer 1205 which is complementary to the 5' region of pro-Apo AII
and is designed to be a blunt ended primer which adds a silent
mutation to remove the first XhoI site.
[0416] SEQ ID NO:196 and 197 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo29 clone.
[0417] SEQ ID NO:198 and 199 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo30 clone.
[0418] SEQ ID NO:200 and 201 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo31 clone.
[0419] SEQ ID NO:202 and 203 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo32 clone.
[0420] SEQ ID NO:204 and 205 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo33 clone.
[0421] SEQ ID NO:206 and 207 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo34 clone.
[0422] SEQ ID NO:208 and 209 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo35 clone.
[0423] SEQ ID NO:210 sets forth the nucleotide sequence of reverse
primer 1208 which is complementary to the 3' region of pro-Apo AI
and is designed to add a KDEL sequence before the stop codon and a
HindIII site after the stop codon.
[0424] SEQ ID NO:211 and 212 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo36 clone.
[0425] SEQ ID NO:213 and 214 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo37 clone.
[0426] SEQ ID NO:215 and 216 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo38 clone.
[0427] SEQ ID NO:217 and 218 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo39 clone.
[0428] SEQ ID NO:219 sets forth the nucleotide sequence of forward
primer 1207 which is complementary to the 5' region of the klip8
cleavage sequence and is designed to amplifies the start of the
klip8 sequence and adds a SalI site to the start codon.
[0429] SEQ ID NO:220 and 221 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo40 clone.
[0430] SEQ ID NO:222 and 223 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo41 clone.
[0431] SEQ ID NO:224 and 225 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo42 clone.
[0432] SEQ ID NO:226 and 227 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo43 clone.
[0433] SEQ ID NO:228 and 229 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo44 clone.
[0434] SEQ ID NO:230 and 231 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo45 clone.
[0435] SEQ ID NO:232 and 233 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo46 clone.
[0436] SEQ ID NO:234 and 235 set forth the nucleotide and deduced
amino acid sequences, respectively, of the Apo47 clone.
[0437] SEQ ID NO:236 sets forth the nucleotide sequence of forward
primer 1226 which is complementary to the 5' region of the maize
oleosin sequence and is designed to amplifies the maize oleosin
sequence and adds a NcoI site to the start codon.
[0438] SEQ ID NO:237 sets forth the nucleotide sequence of forward
primer 1227 which is complementary to the 3' region of the maize
oleosin sequence and is designed to amplify the maize oleosin,
remove the stop codon of the gene and add a HindIII site to assist
in creating an in-frame translational fusion with klip8/matApo
AI.
[0439] SEQ ID NO:238 sets forth the nucleotide sequence of forward
primer 1228 which is complementary to the 5' region of the Apo25
clone and is designed to amplify the Apo25 sequence and adds a SalI
site to the start codon.
[0440] SEQ ID NO:239 sets forth the nucleotide sequence of reverse
primer 1229 which is complementary to the 3' region of the Apo25
clone and is designed to amplify the Apo25 sequence and adds a
BamHI site after the stop codon.
[0441] SEQ ID NO:240 sets forth the amino acid sequence of the
single chain antibody D9scFv.
[0442] SEQ ID NOS. 241-251 are known Apolipoprotein AV sequences
which are described in Table 1.
TABLE-US-00001 TABLE 1 Examples of known Apolipoprotein sequences
SEQUENCE ID. NO. APOLIPOPROTEIN SOURCE (Accession number)
Apolipoprotein A-I 1 Human (NM 000039, BC005380, J00098, M11791,
M27875, M29068, X00566, X01038, X02162, X07496) 9 Danio rerio
(NP_571203) 10 Rattus norvegicus (P04639) 11 Bos taurus (P15497) 12
Mus musculus (Q00623) 13 Ovis aries (AAB57840) 14 Sus scrofa
(P18648) 15 Cyprinus carpio (CAC34942) 16 Gallus gallus (AAA48593)
17 Oryctolagus cuniculus (P09809) 18 Macaca fascicularis (P15568)
19 Coturnix japonica (P32918) 20 Canis familiaris (P02648) 21
Tupaia belangeri (O18759) 22 Anas platyrhynchos (O42296) 23 Papio
anubis (AAA35380) 24 Macaca mulatto (P14417) Apolipoprotein A-IV 7
Human (NM 0000482, J02758, M10373, M13654, M14566, M14642, X13629,
P0672) 25 Rattus norvegicus (AAA85909) 26 Mus musculus (P06728) 27
Mus musculus castaneus (AAA37216) 28 Sus scrofa (O46409) 29 Papio
anubis (Q28758) 30 Macaca fascicularis (P33621) 31 Pan troglodytes
(I54248) 32 Papio sp. (A47141) 33 Gillichthys mirabilis (AAG13299)
34 Oryctolagus cuniculus (AAB34783) Apolipoprotein A-V 241 Mus
musculus (NM_080434) 242 Rattus norvegicus (NM_080576) 243 Homo
sapiens (NP_443200) 244 Mus musculus (BC011198) 245 Homo sapiens
(AY555191) 246 Homo sapiens (AY422949) 247 Mus musculus (AF327059)
248 Homo sapiens (AF202890) 249 Homo sapiens (AF202889) 250 Rattus
norvegicus (AF202888) 251 Rattus norvegicus (AF202887)
Apolipoprotein E 8 Human (NM 000041, AF050154, AF261279, BC003557,
K00396, M10065, M12529, X00199, X92000, Z70760) 35 Rattus
norvegicus (P02650) 36 Danio rerio (O42364) 37 Bos Taurus (Q03247)
38 Mus musculus (P08226) 39 Canis familiaris (P18649) 40 Saimiri
sciureus (Q28995) 41 Macaca mulatto (Q28502) 42 Sus scrofa (P18650)
43 Oryctolagus cuniculus (P18287) 44 Papio anubis (P05770) 45
Macaca fascicularis (P10517) 46 Cavia porcellus (P23529) 47
Zalophus californianus (JC5566) 48 Ovis sp. (JC6549) 49 Pongo
pygmaeus (AAG28580) 50 Hylobates lar (AAG28581) 51 Gorilla gorilla
(AAG28579) 52 Pan troglodytes (AAG28578) 53 Tupaia glis (AAG21401)
54 Oncorhynchus mykiss (CAB65320) 55 Scophthalmus maximus
(CAB65356)
TABLE-US-00002 TABLE 2 Examples of known apolipoprotein mutations
and modifications Protein Mutation Reference POINT MUTATIONS Apo AI
Glu198Lys Strobl W et al., Pediatr Res. August 1988; 24(2):222-8
Apo AI Gly26Arg Vigushin DM et al. Q J Med. March 1994;
87(3):149-54 Apo AI Leu60Arg Soutar AK et al. Proc Natl Acad Sci U
S A. Aug. 15, 1992; 89(16):7389-93 Apo AI Val156Glu Cho KH and
Jonas A. J Biol Chem. Sep. 1, 2000; 275(35):26821-7 Apo AI
Baltimore Arg10Leu Ladias JA et al., Hum Genet. April 1990;
84(5):439-45 Apo AI Giessen Pro143Arg Utermann G et al., Eur J
Biochem. Oct. 15, 1984; 144(2):325-31 Apo AI Fukuoka Glu110Lys
Takada Y et al., Biochim Biophys Acta. Apr. 2, 1990;
1043(2):169-76. Apo AI Fin Arg159Leu Miettinen HE et al.
Arterioscler Thromb Vasc Biol. January 1997; 17(1):83-90 Apo AI
Milano Arg173Cys Cheung MC et al., Biochim Biophys Acta. May 2,
1988; 960(1):73-82 Apo AI Paris Arg151Cys Bruckert E et al.,
Atherosclerosis. Jan. 3, 1997; 128(1):121-8 Apo AV Val153Met
Hubacek et al. Physiol. Res. 2004. Cys185Gly 53:225-228 Apo AV
Thr131Cys Hubacek et al. Clin. Genet. 2004. 65: Ser19Trp 126-130
Apo E Arg136Cys Hubacek JA et al. Physiol Res. 2002; 51(1):107-8
Apo E*5 Gln204Lys, Scacchi R. et al. Hum Biol. Cys112Arg, or April
2003; 75(2):293-300; Feussner et al., J Glu212Lys Lipid Res. August
1996; 37(8):1632-45 Apo E1 Lys146Glu Mann WA et al., J Clin Invest.
August 1995; 96(2):1100-7 Apo E2 Arg136Cys Feussner G. et al. Eur J
Clin Invest. January 1996; 26(1):13-23 Apo E2 Arg142Leu Richard P
et al. Atherosclerosis. Jan. 6, 1995; 112(1):19-28 Apo E2 Arg25Cys
Matsunaga et al. Kidney Int. August 1999; 56(2):421-7 Apo E2
Lys146Gln Smit M et al., j Lipid Res. January 1990; 31(1):45-53 Apo
E2 Arg136Ser Wardell MR et al. J Clin Invest. Christchurch August
1987; 80(2):483-90. Apo E3 Arg136Cys Walden CC et al. J Clin
Endocrinol Metab. March 1994; 78(3):699-704 Apo E3 Arg136His
Minnich A et al. J Lipid Res. January 1995; 36(1):57-66. Apo
E5-Frankfurt Gln81Lys, Cys112Arg Ruzicka V et al. Electrophoresis.
October 1993; 14(10):1032-7 DELETION MUTATIONS Apo AI nichinan
G1u235 deletion Han et al. Arterioscler Thromb Vasc Biol. June
1999; 19(6):1447-55 Apo AI Lys 107 deletion Amarzguioui M et el.
Biochem Biophys Res Commun. Jan. 26, 1998; 242(3):534-9 FRAMESHIFT
MUTATIONS Apo AI Sasebo partial gene Moriyama K et al. Arterioscler
duplication, tandem Thromb Vasc Biol. repeat of bases 333 to
December 1996; 16(12):1416-23 355 from the 5' end of exon 4
resulting with premature termination after amino acid 207 CHEMICAL
MODIFICATIONS Apo AI sulfoxidized Met-112 Jonas A et al. Biochim
Biophys Acta. and Met-148 residues Feb. 24, 1993; 1166(2-3):202-10
and the corresponding reduced form
TABLE-US-00003 TABLE 3 Examples of known oil body protein sequences
SEQ. ID Oil Body Protein Motif (Amino Acid Sequence NO. Identifier)
{Nucleic Acid Sequence Identifier} Oleosin 59 (A84654) Arabidopsis
thaliana probable oleosin 60 (AAA87295) Arabidopsis thaliana
oleosin {Gene L40954} 61 (AAC42242) Arabidopsis thaliana oleosin
{Gene AC005395} 62 (AAF01542) Arabidopsis thaliana putative oleosin
{Gene AC009325} 63 (AAF69712) Arabidopsis thaliana F27J15.22 {Gene
AC016041} 64 (AAK96731) Arabidopsis thaliana oleosin-like protein
{Gene AY054540} 65 (AAL14385) Arabidopsis thaliana
AT5g40420/MPO12_130 oleosin isoform {Gene AY057590} 66 (AAL24418)
Arabidopsis thaliana putative oleosin {Gene AY059936} 67 (AAL47366)
Arabidopsis thaliana oleosin-like protein {Gene AY064657} 68
(AAM10217) Arabidopsis thaliana putative oleosin {Gene AY081655} 69
(AAM47319) Arabidopsis thaliana AT5g40420/MPO12_130 oleosin isoform
{Gene AY113011} 70 (AAM63098) Arabidopsis thaliana oleosin isoform
{Gene AY085886} 71 (AAO22633) Arabidopsis thaliana putative oleosin
{Gene BT002813} 72 (AAO22794) Arabidopsis thaliana putative oleosin
protein {Gene BT002985} 73 (AAO42120) Arabidopsis thaliana putative
oleosin {Gene BT004094} 74 (AAO50491) Arabidopsis thaliana putative
oleosin {Gene BT004958} 75 (AAO63989) Arabidopsis thaliana putative
oleosin {Gene BT005569} 76 (AAQ56108) Arabidopsis lyrata subsp.
Lyrata Oleosin. {Gene AY292860} 77 (BAA97384) Arabidopsis thaliana
oleosin-like {Gene AB023044} 78 (BAB02690) Arabidopsis thaliana
oleosin-like protein {Gene AB018114} 79 (BAB11599) Arabidopsis
thaliana oleosin, isoform 21K {Gene AB006702} 80 (BAC42839)
Arabidopsis thaliana putative oleosin protein {Gene AK118217} 81
(CAA44225) Arabidopsis thaliana oleosin {Gene X62353} 82 (CAA63011)
Arabidopsis thaliana oleosin, type 4 {Gene X91918} 83 (CAA63022)
Arabidopsis thaliana oleosin, type 2 {Gene X91956} 84 (CAA90877)
Arabidopsis thaliana oleosin {Gene Z54164} 85 (CAA90878)
Arabidopsis thaliana oleosin {Gene Z54165} 86 (CAB36756)
Arabidopsis thaliana oleosin, 18.5 K {Gene AL035523} 87 (CAB79423)
Arabidopsis thaliana oleosin, 18.5 K {Gene AL161562} 88 (CAB87945)
Arabidopsis thaliana oleosin-like protein {Gene AL163912} 89
(P29525) Arabidopsis thaliana oleosin 18.5 kDa {Gene X62353,
CAA44225, AL035523, CAB36756, CAB36756, CAB79423, Z17738, S22538}
90 (Q39165) Arabidopsis thaliana Oleosin 21.2 kDa (Oleosin type 2).
{Gene L40954, AAA87295, X91956, CAA63022, Z17657, AB006702,
BAB11599, AY057590, AAL14385, S71253 91 (Q42431) Arabidopsis
thaliana Oleosin 20.3 kDa (Oleosin type 4) {Gene Z54164, CAA90877,
X91918, CAA63011, AB018114, BAB02690, AY054540, AAK96731, AY064657,
AAL47366, AY085886, AAM63098, Z27260, Z29859, S71286 92 (Q43284)
Arabidopsis thaliana Oleosin 14.9 kDa. {Gene Z54165, CAA90878,
AB023044, BAA97384, Z27008, CAA81561} 93 (S22538) Arabidopsis
thaliana oleosin, 18.5 K 94 (S71253) Arabidopsis thaliana oleosin,
21 K 95 (S71286) Arabidopsis thaliana oleosin, 20 K 96 (T49895)
Arabidopsis thaliana oleosin-like protein 97 (AAB22218) Brassica
napus oleosin napII 98 (AAD24547) Brassica oleracea oleosin 99
(CAA43941) Brassica napus oleosin BN-III {Gene X63779} 100
(CAA45313) Brassica napus oleosin BN-V {Gene X63779} 101 (P29109)
Brassica napus Oleosin Bn-V (BnV) {Gene X63779, CAA45313, S25089)
102 (P29110) Brassica napus Oleosin Bn-III (BnIII) {Gene X61937,
CAA43941, S22475) 103 (P29111) Brassica napus Major oleosin NAP-II
{Gene X58000, CAA41064, S70915) 104 (S22475) Brassica napus oleosin
BN-III 105 (S50195) Brassica napus Oleosin 106 (T08134) Brassica
napus Oleosin-like 107 (AAB01098) Daucus carota oleosin 108
(T14307) carrot oleosin 109 (A35040) Zea mays oleosin 18 110
(AAA67699)Zea mays oleosin KD18 {Gene J05212} 111 (AAA68065) Zea
mays 16 kDa oleosin {Gene U13701} 112 (AAA68066) Zea mays 17 kDa
oleosin {Gene U13702} 113 (P13436) Zea mays OLEOSIN ZM-I (OLEOSIN
16 KD) (LIPID BODY-ASSOCIATED MAJOR PROTEIN) {Gene U13701,
AAA68065, M17225, AAA33481, A29788} 114 (P21641) Zea mays Oleosin
Zm-II (Oleosin 18 kDa) (Lipid body-associated protein L2) {Gene
105212, AAA67699, A35040} 115 (S52029) Zea mays oleosin 16 116
(S52030) Zea mays oleosin 17 Caleosin 117 (XP_467656) putative
caleosin [Oryza sativa (japonica cultivar- group)]. 118 (BAD16161)
putative caleosin [Oryza sativa (japonica cultivar- group)]. {Gene
AP005319} 119 (NP_973892) caleosin-related family protein
[Arabidopsis thaliana]. {Gene NM_202163} 120 (NP_564996)
caleosin-related family protein [Arabidopsis thaliana]. {Gene
NM_105736} 121 (NP_564995) caleosin-related family protein
[Arabidopsis thaliana] {Gene NM_105735} 122 (NP_200335)
caleosin-related family protein/embryo-specific protein, putative
[Arabidopsis thaliana]. {Gene NM_124906} 123 (NP_173739)
caleosin-related [Arabidopsis thaliana]. {Gene NM_102174} 124
(NP_173738) caleosin-related family protein [Arabidopsis thaliana]
{Gene NM_102173} 125 (AAQ74240) caleosin 2 [Hordeum vulgare]. {Gene
AY370892} 126 (AAQ74239) caleosin 2 [Hordeum vulgare]. {Gene
AY370891} 127 (AAQ74238) caleosin 1 [Hordeum vulgare]. {Gene
AY370890} 128 (AAQ74237) caleosin 1 [Hordeum vulgare]. {Gene
AY370889} 129 (AAF13743) caleosin [Sesamum indicum]. {Gene
AF109921} Steroleosin 130 (XP_465935) putative steroleosin [Oryza
sativa (japonica cultivar-group)]. {Gene XM_465935} 131 (XP_465933)
putative steroleosin [Oryza sativa (japonica cultivar-group)].
{Gene XM_465933} 132 (AAT77030) putative steroleosin-B [Oryza
sativa (japonica cultivar-group)]. {Gene AC096856} 133 (BAD23084)
putative steroleosin [Oryza sativa (japonica cultivar-group)] {Gene
AP004861} 134 (BAD23082) putative steroleosin [Oryza sativa
(japonica cultivar-group)] {Gene AP004861} 135 (AAM46847)
steroleosin-B [Sesamum indicum]. {Gene AF498264} 136 (AAL13315)
steroleosin [Sesamuin indicum]. {Gene AF421889} 137 (AAL09328)
steroleosin [Sesamum indicum]. {Gene AF302806}
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120059150A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120059150A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References