U.S. patent application number 10/260562 was filed with the patent office on 2003-05-22 for oil bodies and associated proteins as affinity matrices.
This patent application is currently assigned to SemBioSys Genetics Inc.. Invention is credited to Boothe, Joseph, Moloney, Maurice, Van Rooijen, Gijs.
Application Number | 20030096320 10/260562 |
Document ID | / |
Family ID | 25078275 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030096320 |
Kind Code |
A1 |
Moloney, Maurice ; et
al. |
May 22, 2003 |
Oil bodies and associated proteins as affinity matrices
Abstract
A method for the separation of a target molecule from a mixture
is described. The method employs oil bodies and their associated
proteins as affinity matrices for the selective, non-covalent
binding of desired target molecules. The oil body proteins may be
genetically fused to a ligand having specificity for the desired
target molecule. Native oil body proteins can also be used in
conjunction with an oil body protein specific ligand such as an
antibody or an oil body binding protein. The method allows the
separation and recovery of the desired target molecules due to the
difference in densities between oil bodies and aqueous
solutions.
Inventors: |
Moloney, Maurice; (Calgary,
CA) ; Boothe, Joseph; (Calgary, CA) ; Van
Rooijen, Gijs; (Calgary, CA) |
Correspondence
Address: |
BERESKIN AND PARR
SCOTIA PLAZA
40 KING STREET WEST-SUITE 4000 BOX 401
TORONTO
ON
M5H 3Y2
CA
|
Assignee: |
SemBioSys Genetics Inc.
Bay # 110, 2985-23rd Avenue N.E.
Calgary
CA
T1Y 7L3
|
Family ID: |
25078275 |
Appl. No.: |
10/260562 |
Filed: |
October 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10260562 |
Oct 1, 2002 |
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09319275 |
Aug 27, 1999 |
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6509453 |
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09319275 |
Aug 27, 1999 |
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PCT/CA97/00951 |
Dec 5, 1997 |
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PCT/CA97/00951 |
Dec 5, 1997 |
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08767026 |
Dec 16, 1996 |
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Current U.S.
Class: |
435/7.5 ;
530/370; 530/400 |
Current CPC
Class: |
C01G 11/003 20130101;
C12Y 304/21005 20130101; C07K 1/22 20130101; C07K 16/065 20130101;
B01D 15/08 20130101; C07H 21/00 20130101; B01D 15/3804 20130101;
C07K 14/415 20130101; C08B 1/00 20130101; B01D 15/3809 20130101;
C12N 9/6429 20130101 |
Class at
Publication: |
435/7.5 ;
530/370; 530/400 |
International
Class: |
G01N 033/53; C07K
014/415 |
Claims
We claim:
1. A method for the separation of a target molecule from a sample
comprising: 1) contacting (i) oil bodies with (ii) a sample
containing the target molecule to allow the target molecule to
associate with the oil bodies; and 2) separating the oil bodies
associated with the target molecule from the sample.
2. A method according to claim 1 further comprising a ligand
molecule that associates with the oil bodies and the target
molecule.
3. A method according to claim 2 wherein the ligand molecule is
covalently attached to the oil bodies.
4. A method according to claim 3 wherein the ligand molecule is
covalently attached to an oil body protein in the oil bodies.
5. A method according to claim 2 wherein the ligand is comprised of
two molecules, a first molecule that associates with the oil bodies
and a second molecule that associates with the target, wherein the
first molecule and the second molecule associate with each
other.
6. A method according to claim 5 wherein the first and second
ligand molecules are conjugated to each other.
7. A method according to claim 4 wherein the ligand molecule is a
protein.
8. A method according to claim 7 wherein the protein ligand is a
fusion protein with the oil body protein.
9. A method according to any one of claims 1 to 8 wherein the
target molecule is selected from the group consisting of proteins,
peptides, organic molecules, lipids, carbohydrates, nucleic acids,
cells, cell organelles, cell components, viruses, metals, metal
ions and ions.
10. A method according to claim 8 wherein the ligand molecule is
hirudin and the target molecule is thrombin.
11. A method according to claim 8 wherein the ligand molecule is
protein A and the target molecule an immunoglobulin.
12. A method according to claim 8 wherein the ligand molecule is
metallothionein and the target molecule is cadmium.
13. A method according to claim 8 wherein the ligand molecule is a
cellulose binding protein and the target molecule is cellulose.
14. A method according to claim 8 wherein the ligand molecule is a
nucleic acid binding protein and the target molecule is a nucleic
acid.
15. A method according to claim 14 wherein the ligand is a single
stranded DNA binding protein or an RNA binding protein and the
target is a single stranded nucleic acid molecule.
16. A method according to claim 2 wherein the ligand is an antibody
that binds to the oil body or an oil body protein.
17. A method according to claim 16 wherein the target is a cell,
cell organelle or cell component capable of binding the ligand
antibody.
18. A method according to claim 16 wherein the cell is
Staphylococcus aureus.
19. A method according to claim 16 wherein the ligand is a bivalent
antibody that binds to both the oil body and the target.
20. A method according to claim 6 wherein the ligand is an antibody
conjugated to avidin and the target molecule is biotin.
21. A method according to any one of claims 4, 7, 8, 9, 10, 11, 12,
13, 14, 15 wherein the oil body protein is an oleosin.
22. A method according to claim 21 wherein the oleosin is derived
from a plant selected from the group consisting of rapeseed
(Brassica spp.), soybean (Glycine max), sunflower (Helianthus
annuus), oil palm (Elaeis guineeis), coconut (Cocus nucifera),
castor (Ricinus communis), safflower (Carthamus tinctorius),
mustard (Brassica spp. and Sinapis alba), coriander (Coriandrum
sativum) linseed/flax (Linum usitatissimum), thale cress
(Arabidopsis thaliana) and maize (Zea mays).
23. A method according to any one of claims 1 to 22 wherein in step
1) the oil bodies and the sample are mixed and then incubated for
about 1 minute to about 24 hours.
24. A method according to claim 23 wherein the mixed oil bodies and
sample are incubated at a temperature range from about 4.degree. C.
to about room temperature.
25. A method according to any one of claims 1 to 24 wherein the oil
bodies associated with the target molecule are separated from the
sample in step (2) by centrifugation, floatation or size
exclusion.
26. A method according to any one of claims 1 to 25, further
comprising 3) separating the target molecule from the oil
bodies.
27. A method according to claim 26 wherein the target molecule is
separated by elution under appropriate conditions.
28. A method according to any one of claims 1 to 27 wherein the oil
bodies are obtained from the group of plants consisting of rapeseed
(Brassica spp.), soybean (Glycine max), sunflower (Helianthus
annuus), oil palm (Elaeis guineeis), coconut (Cocus nucifera),
castor (Ricinus communis), safflower (Carthamus tinctorius),
mustard (Brassica spp. and Sinapis alba), coriander (Coriandrum
sativum) linseed/flax (Linum usitatissimum), thale cress
(Arabidopsis thaliana) and maize (Zea mays).
29. A composition comprising oil bodies associated with a
molecule.
30. A composition according to claim 29, wherein the molecule is a
target molecule selected from the group consisting of organic
molecules, lipids, carbohydrates, nucleic acids, cells, cell
organelles, cell components, viruses, metals, metal ions and
ions.
31. A composition according to claim 30 further comprising a ligand
molecule that associates with the oil bodies and the target
molecule.
32. A composition according to claim 29, wherein the molecule is a
ligand molecule.
33. A composition according to claim 31 or 32 wherein the ligand
molecule is covalently attached to the oil bodies.
34. A composition according to claim 33 wherein the ligand is
biotin.
35. An affinity matrix for use in separating a target molecule from
a sample comprising an oil body that can associate with the target
molecule.
36. An affinity matrix for use in separating a target molecule from
a sample comprising (a) an oil body and (b) a ligand molecule
associated with the oil body, wherein the ligand molecule is
capable of associating with the target molecule.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the use of oil bodies and their
associated proteins as affinity matrices for the separation and
purification of target molecules from samples.
BACKGROUND OF THE INVENTION
[0002] Within the general field of biotechnology, the ability to
effectively separate and purify molecules from complex sources,
such as living cells, blood serum, or fermentation broth, is of
critical importance. Applications in industry and research
laboratories (where, for example, purified or partly purified
proteins are used) are numerous and well documented in prior
literature. See, for example, R. Meadon and G. Walsh in
Biotechnological Advances 1994, 12: pp 635-646.
[0003] The majority of currently employed techniques for the
separation of molecules capitalizes on the innate physical and
chemical properties of the molecule of interest. Affinity-based
purification technologies are unique in that they exploit the
highly specific biological recognition between two molecular
species which form an affinity pair. Binding of the two entities of
the affinity pair occurs in almost all instances as a result of
relatively weak chemical interactions, known as non-covalent bonds.
Some art-recognized and commonly used affinity pairs include
antibodies and their binding antigenic substances, nucleic acid
binding proteins and nucleic acids, lipid binding proteins and
lipids, lectins and carbohydrates, streptavidin/biotin complexes,
protein A/immunoglobulin G complexes, and receptors and their
binding molecules.
[0004] In general, affinity-based purification processes require
that one member of the affinity pair is immobilized on a solid
substrate or matrix that is insoluble in the fluid in which the
other member of the pair resides. The molecular species of the
affinity pair bound to the matrix is generally referred to as the
ligand, while the liquid soluble member is generally referred to as
the target member. However, it is important to note that these
definitions do not impose any restrictions in a strict chemical
sense. The vast majority of current ligand immobilization
techniques rely on physical or chemical approaches. Physical ligand
immobilization involves adsorption or entrapment of the ligand to a
suitable support, while the chemical mode of immobilization is
characterized by the formation of strong crosslinks or covalent
attachments between the ligand and the matrix. It is a requirement
that immobilization is accomplished in such a fashion that the
capacity of the members of the affinity pair to recognize each
other is not adversely affected by the immobilization
procedure.
[0005] It is a disadvantage of the currently available physical and
chemical techniques for immobilizing ligands that production
processes are frequently time consuming and expensive. This is
mainly due to the fact that immobilization techniques require the
separate production of matrix material and ligands, which in a
subsequent step must be coupled. An alternative mode of
immobilizing proteins is described in U.S. Pat. No. 5,474,925 which
documents a biological production system for the immobilization of
enzymes in the fibre of cotton plants. This patent discloses what
is believed to be the first biologically produced enzyme
immobilization system and allows a one step production of matrix
and ligand.
[0006] Subsequent to immobilization of the ligand on the matrix, a
variety of affinity based purification techniques may be employed
to accomplish selective binding between the affinity immobilized
ligand and the target member. Affinity based purification
techniques known in the prior art include perfusion affinity
chromatography, affinity repulsion chromatography, hyperdiffusion
affinity chromatography, affinity precipitation, membrane affinity
partitioning, affinity cross-flow ultrafiltration and affinity
precipitation. In the most widely used affinity based purification
technique, affinity chromatography, a matrix containing a ligand is
coated to, or packed on, the inside of a chromatographic column. A
complex mixture containing the target member is then applied to the
chromatographic column. Ideally, only the target molecules that
specifically recognize the ligand bind in a non-covalent fashion to
the chromatographic column, while all other molecular species
present in the sample pass through the column.
[0007] In affinity partitioning, two solutions of substantially
different densities are employed. The complex solution containing
the target member is mixed with a solution of a different density
containing the affinity ligand. Subsequent to mixing, the solutions
are left to settle in order to permit the formation of two separate
phases. Molecules tend to partition differentially between phases
depending on their size, charge and specific interactions with the
phase-forming solutions. Ligand-bound target protein selectively
partitions to the phase containing the affinity ligand. For
example, Coughlin and Baclaski in Biotechnology Progress, 1990 6:
307-309 reported the use of the biotin containing organic solution
isooctane to transfer avidin from an aqueous solution to the
isooctane solution. However, so far applications of affinity
partitioning have been limited mainly due to the current lack of
availability of suitable affinity matrix substances which can be
employed in specific partitioning in two phase systems.
[0008] An important factor for the commercial development of
biotechnology is the purification of bioproducts, which typically
accounts for 50% or more of the total costs (Labrou, N. and Clonis,
Y. D. in the Journal of Biotechnology 36: 95-119 (1994)). Many
protein purification steps rely on column type separation
procedures. In particular, large scale high-separation techniques
such as column chromatography or batch-type based protein
purification techniques are costly. In addition, crude material is
less suitable for either column chromatography or batch
separations, as contaminants may foul up sedimented resins and plug
columns. Thus, affinity matrices are often only employed in a later
stage of purification processes where substantial purity is
critical, where the proteins are present in extremely dilute
concentrations, or where high value proteins are required, for
example in diagnostic and therapeutic proteins. These and other
topics related to the use of affinity technology in
biotechnological processes have been reviewed by Labrou, N. and
Clonis, Y. D. in the Journal of Biotechnology 36: 95-119
(1994).
[0009] There is a need in the art to develop novel and economical
methods for separating and purifying biological products from
complex mixtures. The present inventors have found that subcellular
oil storage structures, known as oil bodies, and their associated
proteins are useful in this regard.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a novel versatile
biological system for the production of affinity matrices. The
present inventors have found that oil bodies and their associated
proteins can be used as affinity matrices for the separation of a
wide variety of target molecules such as proteins, carbohydrates,
lipids, organic molecules, nucleic acids, metals, cells and cell
fractions from a sample.
[0011] In accordance with the invention, there is provided a method
for the separation of a target molecule from a sample comprising:
1) contacting (i) oil bodies that can associate, either directly or
indirectly, with the target molecule with (ii) a sample containing
the target molecule; and 2) separating the oil bodies associated
with the target molecule from the sample. The oil bodies and the
sample containing the target molecule are brought into contact in a
manner sufficient to allow the oil bodies to associate with the
target. Preferably, oil bodies are mixed with the target. If
desired, the target molecule may be further separated from the oil
bodies.
[0012] In one aspect, the target molecule has affinity for, or
binds directly to, the oil bodies or oil body protein. Examples of
such targets include antibodies or other proteins that bind to oil
bodies.
[0013] In another aspect, a ligand molecule may be used to
associate the target molecule with the oil bodies.
[0014] In one embodiment, the ligand has natural affinity for the
oil bodies or oil body protein. In a specific embodiment, the
ligand is an antibody that binds the oil body protein. Such an
antibody can be used to separate targets having affinity for the
ligand antibody such as anti-IgG antibodies or protein A. A
bivalent antibody may also be prepared having binding specificities
for both the oil body protein and the target. The antibody against
the oil body protein may also be fused to a second ligand having
affinity for the target.
[0015] In another embodiment, the ligand is covalently attached to
the oil bodies or oil body protein. In one embodiment, the ligand
is a protein that is chemically conjugated or produced as a fusion
protein with the oil body protein (as described in WO 96/21029). In
the latter case, the fusion protein is targeted to and expressed on
the oil bodies. In one example, the ligand fused to the oil body
protein may be hirudin and can be used to purify thrombin. In
another example, the ligand fused to the oil body protein may be
metallothionein and can be used to separate cadmium from a sample.
In a further example, the ligand fused to the oil body protein may
be protein A and can be used to separate immunoglobulins. In yet
another example, the ligand fused to the oil body protein may be
cellulose binding protein and can be used to separate cellulose
from a sample.
[0016] In another embodiment, the ligand may be covalently attached
to the oil bodies. For example, the ligand may be a small organic
molecule such as biotin. Biotinylated oil bodies can be used to
separate avidin from a sample.
[0017] The present invention also includes modified oil bodies for
use as an affinity matrix. Accordingly, the present invention
includes a composition comprising oil bodies associated with a
molecule, such as a ligand molecule or a target molecule. In one
embodiment, the composition comprises oil bodies covalently
attached to a ligand molecule, such as biotin.
[0018] The present invention also includes an affinity matrix for
use in separating a target molecule from a sample, comprising oil
bodies that can associate with the target molecule. The affinity
matrix may additionally include a ligand molecule associated with
the oil bodies, wherein the ligand molecule is capable of
associating with the target molecule.
[0019] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description and attached drawings. It should be understood,
however, that the detailed description and associated examples are
given by way of illustration only, and various changes and
modifications thereto falling within the scope of the invention
will become apparent to those skilled in the art. In addition,
reference is made herein to various publications, patents and
patent applications which are hereby incorporated by reference in
their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. The nucleotide and deduced amino acid sequence of
the 18 KDa oleosin from Arabidopsis thaliana as shown in
SEQ.ID.NO:1 and SEQ.ID.NO:2.
[0021] FIG. 2. Sequence of an Arabidopsis oleosin-hirudin fusion.
Indicated are a portion of the oleosin genomic sequence (from base
1-1620 as reported in van Rooijen et al 1992, Plant Mol. Biol. 18:
1177-1179), a spacer sequence (base 1621-1635, underlined) and the
synthetic DNA sequence encoding the mature hirudin variant-2
isoform (base 1636-1833, italicized) This gene fusion is regulated
by the 5' upstream region of the Arabidopsis oleosin (bases 1-861)
and the nopaline synthase termination sequence (base 1855-2109).
The sequence is also shown in SEQ.ID.NO:3 and SEQ.ID.NO:4.
[0022] FIG. 3. Outline of the steps employed in the construction of
pCGOBHIRT, containing the entire oleosin-hirudin construct.
[0023] FIG. 4. Schematic diagram illustrating the configuration of
the oleosin-hirudin fusion protein on the oil body and the binding
of thrombin.
[0024] FIG. 5. NaCl elution profiles of thrombin from wild type and
4A4 oil body matrices transformed with a construct expressing an
oleosin-hirudin fusion.
[0025] FIG. 6. Purification of a horseradish peroxidase conjugated
anti-IgG antibody using an anti-oleosin antibody as a ligand.
Schematic diagram illustrating the configuration of the
oleosin/anti-oleosin/anti-I- gG sandwich complex bound to an oil
body.
[0026] FIG. 7. Illustrates specific binding of anti-IgG antibodies
to wild type oil bodies complexed with primary anti-oleosin
antibodies as a ligand (left) and binding of anti-IgG antibodies to
oil bodies which were not complexed with primary antibodies prior
to binding with the secondary antibodies (right).
[0027] FIG. 8. Sequence of an oleosin metallothionein fusion.
Indicated are the coding sequence of a B. napus oleosin cDNA (bases
1092-1652, van Rooijen, 1993, Ph.D. Thesis, University of Calgary),
a spacer sequence (bases 1653-1670, underlined) and the human
metallothionein gene mt-II (bases 1671-1876, Varshney and Gedamu,
1984, Gene, 31: 135-145)). The gene fusion is regulated by an
Arabidopsis oleosin promoter (bases 1-1072) and ubiquitin
termination sequence (bases 1870-2361, ubi3'; Kawalleck et al.,
1993, Plant Mol. Biol. 21: 673-684). The sequence is also shown in
SEQ.ID.NO:6 and SEQ.ID.NO:7.
[0028] FIG. 9. Outline of the steps employed in the construction of
pBIOOM3' containing the entire oleosin-metallothionein
construct.
[0029] FIG. 10. Schematic diagram illustrating the configuration of
the oleosin-metallothionein fusion protein on the oil body and
binding of cadmium ions.
[0030] FIG. 11. Illustrates the binding (A) and elution (B) of
cadmium to an oil body matrix from wildtype B. carinata seeds and
B. carinata seeds transformed with a construct expressing oleosin
metallothionein gene fusion. Shown is the percentage cadmium bound
to the oil body fraction of an oil body fraction harvested from
transgenic and untransformed seeds. Bars represent average values
of 5 replicate experiments (binding) and 3 replicates
(elution).
[0031] FIG. 12. Illustrates the binding of protein A expressing S.
aureus cells to oil bodies treated with varying amounts of
anti-oleosin IgGs. Bars represent OD.sub.600 readings obtained
following the procedures as described in Example 5 and using
varying amounts of IgGs (0 .mu.l, 3, .mu.l, 30 .mu.l, 100 .mu.l of
added IgG).
[0032] FIG. 13. Oligonucleotide primers used to amplify the
sequence of the S. aureus protein A (The sequence is also shown in
SEQ.ID.NO:8; The protein sequence is also shown in SEQ.ID.NO:9).
Primer BK266, 5'C TCC ATG GAT CAA CGC AAT GGT TTA TC 3'
(SEQ.ID.NO:10), a NcoI site (italicized) and a sequence identical
to a portion of the protein A gene as contained within vector
pRITZ2T (Pharmacia) (underlined) are indicated. Primer BK267, 5' GC
AAG CTT CTA ATT TGT TAT CTG CAG GTC 3' (SEQ.ID.NO:11), a HindIII
site (italicized), a stop codon (bold) and a sequence complementary
to a portion of the protein A gene as contained within pRIT2T
(Pharmacia) (underlined) are indicated. The PCR product was
digested with NcoI and HindIII and ligated into pCGNOBPGUSA (Van
Rooijen and Moloney, 1995, Plant Physiol. 109: 1353-1361) from
which the NcoI-GUS-HindIII fragment had been removed.
[0033] FIG. 14. Sequence of an Arabidopsis oleosin-protein A fusion
(The sequence is also shown in SEQ.ID.NO:12 and the protein
sequence is also shown in SEQ.ID.NO:13 and 14). Indicated are a
portion of the oleosin genomic sequence (from base 1-1626, as
reported in van Rooijen et al., 1992 Plant Mol. Biol. 18:
1177-1179), a spacer sequence encoding a thrombin cleavage site
(base 1627-1647, underlined) and the DNA sequence encoding protein
A (base 1648-2437, italicized). Expression is regulated by the
Arabidopsis 5' upstream region of the Arabidopsis oleosin (base
1-867) and the nopaline synthase terminator region (base
2437-2700).
[0034] FIG. 15. Schematic diagram illustrating the configuration of
the oleosin-protein A fusion protein on the oil body and binding of
the immunoglobulin.
[0035] FIG. 16. A western blot illustrating the binding of
HRP-conjugated mouse anti-rabbit antibodies to oil body protein
extracts obtained from transgenic B. napus lines expressing
oleosin-protein A fusion proteins. Shown on a Western blot probed
with an HRP-conjugated antibody are oil body protein extracts from
transgenic lines, opa 30 (lane 3), opa 31 (lane 4), opa 34 (lane
5), opa 36 (lane 6), opa 47 (lane 7), opa 93 (lane 8), all
expressing an oleosin-protein A fusion protein and a control
untransformed B. napus line (lane 9), as well as lysates of E. coli
DH5.alpha. transformed with pRIT2T expressing protein A (lane 2)
and untransformed E. coli DH5.alpha. (lane 1).
[0036] FIG. 17 illustrates binding and elution of IgGs to oil
bodies isolated from wildtype B. napus (bn wt) and a transgenic B.
napus line, expressing an oleosin protein A fusions. Error bars
represent the results from 4 independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0037] As hereinbefore mentioned, the present invention relates to
a novel biological affinity matrix system that employs oil bodies
and their associated proteins. The affinity matrix is suitable for
the highly-efficient separation of specific targets, including
proteins, carbohydrates, lipids, nucleic acids, cells and
subcellular organelles, metals and ions, from a sample.
[0038] The present invention provides a method for the separation
of a target molecule from a sample comprising: 1) contacting (i)
oil bodies that can associate either directly or indirectly with
the target molecule with (ii) a sample containing the target
molecule; and 2) separating the oil bodies associated with the
target molecule from the sample. The oil bodies and the sample
containing the target molecule are brought into contact in a manner
sufficient to allow the oil bodies to associate with the target.
Preferably, the oil bodies are mixed with the target. Indirect
association of the oil bodies with the target can be effected using
a ligand molecule that can associate with both the oil bodies and
the target molecule. The ligand therefore serves to bridge or join
the oil bodies with the target molecule. If desired, the target
molecule may be further separated from the oil bodies and the
ligand, if present.
[0039] Each of the components of the affinity matrix are discussed
in turn below.
[0040] Targets
[0041] The term "target" as used herein denotes a desired molecule
that one wants to purify, isolate or separate from a sample such as
a biological mixture. This technology is amenable for use with
virtually any target for which a ligand can be obtained or any
target that can directly associate with or bind to an oil body or
oil body protein. Possible ligand/target pairs include but are not
limited to: protein subunit/subunit associations,
antibodies/antigens, receptor protein/signal molecules, nucleic
acid binding proteins/nucleic acids; lectins/carbohydrates; lipid
binding proteins/lipids; ion binding proteins/ions; and ligands to
surface epitopes/cells or subcellular organelles. The target may be
obtained from any natural source or may be synthesized chemically.
If the target is a macromolecule such as a protein or nucleic acid
it may also be produced in recombinant form using any suitable
expression system such as bacteria, yeast, plant, insect,
mammalian, etc.
[0042] Ligands
[0043] The term "ligand" used herein denotes a molecule that is
capable of associating with both the target molecule and the oil
bodies or oil body protein (discussed below). The term "associating
with" as used herein includes both covalent and non-covalent
binding of the ligand to the oil bodies or the target molecule. For
example, the ligand molecule may be covalently attached to the oil
bodies and non-covalently associate with the target (and
vice-versa), or the ligand may non-covalently associate with both
the oil bodies and the target molecule. The ligand may be any
molecule that can bridge the oil bodies or oil body protein and the
target molecule and can include a protein, nucleic acid,
carbohydrate or small organic molecule. The ligand may be comprised
of two molecules, a first molecule that associates with the oil
bodies and a second molecule that associates with the target,
wherein the first molecule and the second molecule associate with
each other.
[0044] The affinity ligand proteins used for this methodology may
be derived from naturally-occurring, known ligand pairs such as
those listed above. Alternatively, the ligand may be obtained by
screening proteins extracted from cells or organisms, synthesized
chemically or produced in libraries comprised of combinatorial
peptide sequences, antibodies, or expressed DNA sequences.
[0045] In one embodiment, the ligand has natural affinity for the
oil bodies or the oil body protein. For example, the ligand may be
a protein such as an antibody, that has affinity for the oil body
protein. The ligand may also be a molecule other than a protein
which has natural affinity for the oil body or oil body protein.
Such ligands, capable of binding to the oil bodies or oil body
protein, may be associated with a second molecule that can bind the
target molecule. For example, the ligand molecule may be an
antibody conjugated to avidin and can be used to purify biotin from
a sample.
[0046] In another embodiment, the ligand is covalently linked to
the oil bodies or oil body protein by chemical or recombinant
means. Chemical means for preparing fusions or conjugates are known
in the art and can be used to prepare a ligand-oil body protein
fusion. The method used to conjugate the ligand and oil body must
be capable of joining the ligand with the oil body protein without
interfering with the ability of the ligand to bind to the target
molecule. In one example, the ligand may be a small organic
molecule such as biotin that is covalently attached to the oil
bodies. Biotinylated oil bodies can be used to separate avidin from
a sample. The present invention also includes modified oil bodies
such as biotinylated oil bodies for use as an affinity matrix.
Accordingly, the present invention includes a composition
comprising oil bodies attached to a molecule, such as a ligand or a
target molecule.
[0047] In a preferred embodiment, the ligand is a protein and can
be conjugated to the oil body protein using techniques well known
in the art. There are several hundred crosslinkers available that
can conjugate two proteins. (See for example "Chemistry of Protein
Conjugation and Crosslinking". 1991, Shans Wong, CRC Press, Ann
Arbor). The crosslinker is generally chosen based on the reactive
functional groups available or inserted on the ligand. In addition,
if there are no reactive groups a photoactivatible crosslinker can
be used. In certain instances, it may be desirable to include a
spacer between the ligand and the oil-body protein. Crosslinking
agents known to the art include the homobifunctional agents:
glutaraldehyde, dimethyladipimidate and Bis(diazobenzidine) and the
heterobifunctional agents: m-Maleimidobenzoyl-N-Hydroxysuccinimide
and Sulfo-m-Maleimidobenzoyl-N-Hy- droxysuccinimide.
[0048] A ligand protein-oil body protein fusion may also be
prepared using recombinant DNA techniques. In such a case a DNA
sequence encoding the ligand is fused to a DNA sequence encoding
the oil body protein, resulting in a chimeric DNA molecule that
expresses a ligand-oil body protein fusion protein (discussed in
greater detail below). In order to prepare a recombinant fusion
protein, the sequence of the DNA encoding the ligand must be known
or be obtainable. By obtainable it is meant that a DNA sequence
sufficient to encode the protein ligand may be deduced from the
known amino acid sequence. It is not necessary that the entire gene
sequence of the ligand be used provided that a subsequence encoding
the binding domain of the protein ligand is known. Therefore, the
ligand can include the complete sequence of, or the binding domain
from, the specific ligand protein in question.
[0049] If the DNA sequence of the desired ligand is known, the gene
may be synthesized chemically using an oligonucleotide synthesizer.
Alternatively, the clone carrying the ligand gene may be obtained
from either cDNA or genomic libraries containing the gene by
probing with a labelled complementary DNA sequence. The gene may
also be specifically amplified from the library using gene-specific
oligonucleotide primers and the PCR. If the DNA sequence of the
desired ligand is not known, then a partial amino acid sequence may
be obtained through N-terminal sequencing of the protein
(Matsudaira 1987; J. Biol. Chem. 262: 10035-10038). Labelled probes
may be synthesized based upon the DNA sequences deduced from this
amino acid sequence and used to screen cDNA or genomic libraries as
described above. The clone carrying the gene may also be identified
from a cDNA expression library by probing either with antibodies
raised against the protein ligand, or with the target protein.
[0050] Ligands may also be uncovered by probing mixtures of
proteins with the target. The target can be immobilized on a
support matrix and used to screen proteins extracted from cells and
tissues or synthesized chemically. Following binding between the
ligand protein and the immobilized target, the matrix is separated
from the solution and washed. The protein ligand is subsequently
eluted from the matrix and the sequence determined as described
above. Alternatively, recombinant protein libraries produced by
phage display, such as those comprised of combinatorial peptide
sequences (Smith, 1985; Science 228:1315-1317) or antibody
repertoires (Griffiths et al., 1994, EMBO J. 13: 3245-3260, Nissim
et al., 1994, EMBO J. 13: 692-698) can be screened with the
immobilized target. In this case, binding between the protein
ligand and the target would enable separation and recovery of the
phage expressing the ligand from the large, complex population of
phage encoding non-binding proteins. A two-hybrid system such as
that in yeast (Fields and Sternglanz, 1994; Trends Genet. 10:
286-292) might also be used to identify a ligand from an expressed
cDNA library. Here, a gene fusion is constructed between the
sequence encoding the target protein and that of a DNA binding
protein. Cells containing this construct are transformed with
constructs from a cDNA library where the sequences have been fused
to that of a transcriptional activator. Binding between ligands
derived from the cDNA library with the target protein allows
transcription of a reporter gene to occur. Clones expressing the
ligand are then recovered.
[0051] To specifically uncover a ligand to oil bodies, a complete
or partial oleosin protein may be used as target in any of the
above methods. Alternatively, it may be possible to employ intact
oil bodies for screening protein extracts, synthetic peptides or
phage display libraries. In this case, the oil body would serve
both as target and immobilization matrix. Using this approach, a
wider variety of ligands may be uncovered; that exhibit affinity
not only to oleosins, but to other epitopes present on oil
bodies.
[0052] Oil bodies and Oil Body Proteins
[0053] Oil bodies are small, spherical, subcellular organelles
encapsulating stored triacylglycerides, an energy reserve used by
many plants. Although they are found in most plants and in
different tissues, they are particularly abundant in the seeds of
oilseeds where they range in size from under one micron to a few
microns in diameter. Oil bodies are comprised of the
triacylglycerides surrounded by a half-unit membrane of
phospholipids and embedded with a unique type of protein known as
an oil body protein. The term "oil body" or "oil bodies" as used
herein includes any or all of the triacylglyceride, phospholipid or
protein components present in the complete structure. The term "oil
body protein" as used herein means a protein that is naturally
present in an oil body. In plants, the predominant oil body
proteins are termed "oleosins". Oleosins have been cloned and
sequenced from many plant sources including corn, rapeseed, carrot
and cotton. The oleosin protein appears to be comprised of three
domains; the two ends of the protein, N- and C-termini, are largely
hydrophilic and reside on the surface of the oil body exposed to
the cytosol while the highly hydrophobic central core of the
oleosin is firmly anchored within the membrane and
triacylglyceride. Oleosins from different species represent a small
family of proteins showing considerable amino acid sequence
conservation, particularly in the central region of protein. Within
an individual species, a small number of different isoforms may
exist.
[0054] Oil bodies from individual species exhibit a roughly uniform
size and density which is dependent in part upon the precise
protein/phospholipid/triacylglyceride composition. As a result,
they may be simply and rapidly separated from liquids of different
densities in which they are suspended. For example, in aqueous
media where the density is greater than that of the oil bodies,
they will float under the influence of gravity or applied
centrifugal force. In 95% ethanol where the density is less than
that of the oil bodies, they will sediment under the same
conditions. Oil bodies may also be separated from liquids and other
solids present in solutions or suspensions by methods that
fractionate on the basis of size. For example, the oil bodies from
B. napus are minimal, approximately 0.5 m in diameter, and thus may
be separated from smaller components using a membrane filter with a
pore size less than this diameter.
[0055] The oil bodies of the subject invention are preferably
obtained from a seed plant and more preferably from the group of
plant species comprising: thale cress (Arabidopsis thaliana),
rapeseed (Brassica spp.), soybean (Glycine max), sunflower
(Helianthus annuus), oil palm (Elaeis guineeis), cottonseed
(Gossypium spp.), groundnut (Arachis hypogaea), coconut (Cocus
nucifera), castor (Ricinus communis), safflower (Carthamus
tinctorius), mustard (Brassica spp. and Sinapis alba), coriander
(Coriandrum sativum) linseed/flax (Linum usitatissimum), and maize
(Zea mays). Plants are grown and allowed to set seed using
agricultural cultivation practises well known to a person skilled
in the art. After harvesting the seed and removal of foreign
material such as stones or seed hulls, for by example sieving,
seeds are preferably dried and subsequently processed by mechanical
pressing, grinding or crushing. The oil body fraction may be
obtained from the crushed seed fraction by capitalization on
separation techniques which exploit differences in density between
the oil body fraction and the aqueous fraction, such as
centrifugation, or using size exclusion-based separation
techniques, such as membrane filtration, or a combination of both
of these. Typically, seeds are thoroughly ground in five volumes of
a cold aqueous buffer. A wide variety of buffer compositions may be
employed, provided that they do not contain high concentrations of
strong organic solvents such as acetone or diethyl ether, as these
solvents may disrupt the oil bodies. The solution density of the
grinding buffer may be increased with the addition of 0.4-0.6 M
sucrose, in order to facilitate washing as described below. The
grinding buffer will also typically contain 0.5 M NaCl to help
remove soluble proteins that are not integrally bound to the oil
body surface.
[0056] Following grinding, the homogenate is centrifuged resulting
in a pellet of particulate and insoluble matter, an aqueous phase
containing soluble components of the seed, and a surface layer
comprised of oil bodies with their associated proteins. The oil
body layer is skimmed from the surface and thoroughly resuspended
in one volume of fresh grinding buffer. It is important that
aggregates of oil bodies are dissociated as thoroughly as possible
in order to ensure efficient removal of contaminants in the
subsequent washing steps. The resuspended oil body preparation is
layered under a floatation solution of lower density (e.g. water,
aqueous buffer) and centrifuged, again, separating oil body and
aqueous phases. This washing procedure is typically repeated at
least three times, after which the oil bodies are deemed to be
sufficiently free of contaminating soluble proteins as determined
by gel electrophoresis. It is not necessary to remove all of the
aqueous phase and to the final preparation water or 50 mM Tris-HCl
pH 7.5 may be added and if so desired the pH may be lowered to pH 2
or raised to pH 10. Protocols for isolating oil bodies from oil
seeds are available in Murphy, D. J. and Cummins I., 1989,
Phytochemistry, 28: 2063-2069; and in: Jacks, T. J. et al., 1990,
JAOCS, 67: 353-361. A preferred protocol is detailed in example 1
of the present specification.
[0057] Oil bodies other than those derived from plants may also be
used in the present invention. A system functionally equivalent to
plant oil bodies and oleosins has been described in bacteria
(Pieper-Furst et al., 1994, J. Bacteriol. 176: 4328), algae
(Rossler, P. G., 1988, J. Physiol. (London), 24: 394-400) and fungi
(Ting, J. T. et al., 1997, J. Biol Chem. 272: 3699-3706). Oil
bodies from these organisms, as well as those that may be
discovered in other living cells by a person skilled in the art,
may also be employed according to the subject invention.
[0058] Affinity Matrices
[0059] As hereinbefore mentioned, the present invention provides a
novel affinity matrix system for the purification of a target
molecule from a sample. In one embodiment, the affinity matrix
comprises oil bodies that can bind a target molecule in a sample.
In such an embodiment, the target molecule may be an antibody that
can bind an oil body protein. In another embodiment, the affinity
matrix comprises oil bodies or oil body proteins and a ligand that
is associated with the oil bodies or oil body proteins and has
affinity for a target molecule. In such an embodiment, the ligand
may be non-covalently or covalently attached to the oil bodies or
oil body protein (as described above).
[0060] It is an advantage of the present invention that target
substances can be purified or removed from samples through
non-covalent association with oil bodies followed by oil body
separation. A number of different oil body-ligand configurations
are possible. Targets with inherent affinity for a specific ligand
proteins such as hirudin to thrombin or heavy metals to
metallothionein, may be purified or separated with oil bodies
containing that ligand fused to an oleosin. Alternatively, a
protein target may also be purified or separated with an oil body
affinity matrix by fusing the target to an oil body-specific ligand
or to a ligand complimentary to that fused to an oleosin. If
desired, a protease recognition site or chemical cleavage site may
be engineered between the ligand and the target protein to enable
proteolytic removal of the ligand from the target protein in the
course of purification. A multivalent ligand may also be
constructed, such as a bivalent single-chain antibody, in which one
domain of the ligand has an affinity for an oil body and the other
domain(s) exhibits affinity for the target. In this case, neither
the oil body nor the target molecule need to be covalently fused to
a ligand. Also, concatamers of ligands may be used to increase the
affinity of a matrix for a target, or the sequence of a ligand may
be mutated to modulate the affinity for a target when such
conditions are desirable. Further, mixtures of different ligands
may be fused to recover/remove different types of targets
simultaneously. Fusions between different ligands may also be
constructed to form bridges between different types of targets or
between targets and the oil body affinity matrix. Binding to the
affinity matrix may also be achieved by forming bridges between
ligand or ligand and target sequences, such as Zn.sup.++ ions
bridging between polyhistidine sequences.
[0061] There are several advantages associated with the use of oil
body affinity matrices that make them attractive as purification
tools. The flexibility in design that is possible through the
different configurations described above, enables a matrix to be
constructed to best meet the requirements for a specific target.
Also, production of the matrix as part of a natural biological
process in seeds is extremely cost-effective, since purification
and immobilization of the ligand are not necessary. In the case of
oleosin-ligand fusions, the ligand is immobilized on the oil body
as a result of oleosin targeting within the cell, while oil
body-specific ligands will naturally associate with the matrix
while present in complex mixtures. Natural immobilization of the
ligand on the matrix may also be advantageous in that it eliminates
the requirement for chemical cross-linking that may compromise the
affinity of the ligand for the target. Finally, oil body affinity
matrices offer a unique and attractive purification option
particularly for large scale operations. The ability to separate
the matrix through floatation as a loose suspension enables it to
be employed with crude material containing what might otherwise be
prohibitive amounts of particulate contaminants. The presence of
these contaminants will often foul and block conventional solid
matrices applied in columns or batch suspensions limiting their use
at early stages in the purification process.
[0062] As mentioned previously, in one embodiment of the invention,
ligand protein sequences are genetically fused to the oil body
protein. In order to prepare such genetic fusions, a chimeric DNA
sequence is prepared that encodes an oil body protein-ligand fusion
protein and consists of (a) a DNA sequence encoding a sufficient
portion of an oil body protein to provide targeting of the fusion
protein to the oil bodies and (b) a DNA sequence encoding a
sufficient portion of the ligand protein to provide binding of the
target. The inventors have determined that, in general, the
N-terminus and the hydrophobic core of an oil body protein are
sufficient to provide targeting of the fusion protein to the oil
bodies. In particular, for oleosins derived from the plant
Arabidopsis thaliana amino acids 2 through 123 (as shown in
SEQ.ID.NO:1) are sufficient in this regard.
[0063] The ligand may be fused to either the N- and/or C-terminal
end of the oleosin. It may also be possible to construct an
internal fusion between the ligand and oleosin or to fuse the
ligand between two oleosin proteins. The chimeric DNA sequence
encoding an oil body protein fused to a ligand may be transfected
into a suitable vector and used to transform a plant. Two types of
vectors are routinely employed. The first type of vector is used
for the genetic-engineering and assembly of constructs and
typically consists of a backbone such as found in the pUC family of
vectors, enabling replication in easily-manipulated and maintained
gram negative bacteria such as E. coli. The second type of vector
typified by the Ti and Ri plasmids, specify DNA transfer functions
and are used when it is desired that the constructs be introduced
into the plant and stably integrated into its genome via
Agrobacterium-mediated transformation.
[0064] A typical construct consists, in the 5' to 3' direction, of
a regulatory region complete with a promoter capable of directing
expression in plants (preferably seed-specific expression), a
protein coding region, and a sequence containing a transcriptional
termination signal functional in plants. The sequences comprising
the construct may be either natural or synthetic or any combination
thereof.
[0065] Both non-seed specific promoters, such as the 35-S CaMV
promoter (Rothstein et al., 1987; Gene 53: 153-161) and
seed-specific promoters such as the phaseolin promoter
(Sengupta-Gopalan et al., 1985; PNAS USA 82: 3320-3324) or the
Arabidopsis 18 kDa oleosin (Van Rooijen et al., 1992; Plant Mol.
Biol. 18: 1177-1179) promoters may be used. In addition to the
promoter, the regulatory region contains a ribosome binding site
enabling translation of the transcripts in plants and may also
contain one or more enhancer sequences, such as the AMV leader
(Jobling and Gehrke 1987; Nature 325: 622-625), to increase the
expression of product.
[0066] The coding region of the construct will typically be
comprised of sequences encoding a ligand fused in frame to an
oleosin and ending with a translational termination codon. The
sequence for the oleosin may be comprised of any DNA sequence, or
part thereof, natural or synthetic, sufficient to encode a protein
that can be correctly targeted to, and stably expressed on, an oil
body. A detailed description of the characteristics of such a
sequence has been reported previously in Moloney, 1993; PCT Patent
Appl. WO 93/21320 which is hereby incorporated by reference. The
sequence may also include introns. The ligand-encoding region may
in turn be comprised of any individual, or combination of, ligand
sequences identified as described above. If desired, a protease or
chemical recognition site may be engineered between the ligand and
the target protein to enable proteolytic removal of the ligand from
the target protein in the course of purification.
[0067] The region containing the transcriptional termination signal
may comprise any such sequence functional in plants such as the
nopaline synthase termination sequence and additionally may include
enhancer sequences to increase the expression of product.
[0068] The various components of the construct are ligated together
using conventional methods, typically into a pUC-based vector. This
construct may then be introduced into an Agrobacterium vector and
subsequently into host plants, using one of the transformation
procedures outlined below.
[0069] A variety of techniques are available for the introduction
of DNA into host cells. For example, the chimeric DNA constructs
may be introduced into host cells obtained from dicotyledonous
plants, such as tobacco, and oleaginous species, such as B. napus
using standard Agrobacterium vectors; by a transformation protocol
such as that described by Moloney et al., 1989, (Plant Cell Rep.,
8: 238-242) or Hinchee et al., 1988, (Bio/Technol., 6: 915-922); or
other techniques known to those skilled in the art. For example,
the use of T-DNA for transformation of plant cells has received
extensive study and is amply described in EPA Serial No. 120,516;
Hoekema et al., 1985, (Chapter V, In: The Binary Plant Vector
System Offset-drukkerij Kanters B. V., Alblasserdam); Knauf, et
al., 1983, (Genetic Analysis of Host Range Expression by
Agrobacterium, p. 245, In Molecular Genetics of the Bacteria-Plant
Interaction, Puhler, A. ed., Springer-Verlag, NY); and An et al.,
1985, (EMBO J., 4: 277-284). Conveniently, explants may be
cultivated with A. tumefaciens or A. rhizogenes to allow for
transfer of the transcription construct to the plant cells.
Following transformation using Agrobacterium the plant cells are
dispersed in an appropriate medium for selection, subsequently
callus, shoots and eventually plantlets are recovered. The
Agrobacterium host will harbour a plasmid comprising the vir genes
necessary for transfer of the T-DNA to the plant cells. For
injection and electroporation, (see below) disarmed Ti-plasmids
(lacking the tumour genes, particularly the T-DNA region) may be
introduced into the plant cell.
[0070] The use of non-Agrobacterium techniques permits the use of
the constructs described herein to obtain transformation and
expression in a wide variety of monocotyledonous and dicotyledonous
plants and other organisms. These techniques are especially useful
for species that are intractable in an Agrobacterium transformation
system. Other techniques for gene transfer include biolistics
(Sanford, 1988, Trends in Biotech., 6: 299-302), electroporation
(Fromm et al., 1985, Proc. Natl. Acad. Sci. USA, 82: 5824-5828;
Riggs and Bates, 1986, Proc. Natl. Acad. Sci. USA 83: 5602-5606) or
PEG-mediated DNA uptake (Potrykus et al., 1985, Mol. Gen. Genet.,
199: 169-177).
[0071] In a specific application, such as to B. napus, the host
cells targeted to receive recombinant DNA constructs typically will
be derived from cotyledonary petioles as described by Moloney et
al., (1989, Plant Cell Rep., 8: 238-242). Other examples using
commercial oil seeds include cotyledon transformation in soybean
explants (Hinchee et al., 1988. Bio/Technology, 6: 915-922) and
stem transformation of cotton (Umbeck et al., 1981, Bio/Technology,
5: 263-266).
[0072] Following transformation, the cells, for example as leaf
discs, are grown in selective medium. Once shoots begin to emerge,
they are excised and placed onto rooting medium. After sufficient
roots have formed, the plants are transferred to soil. Putative
transformed plants are then tested for presence of a marker.
Southern blotting is performed on genomic DNA using an appropriate
probe, for example an A. thaliana oleosin gene, to show that
integration of the desired sequences into the host cell genome has
occurred.
[0073] The expression cassette will normally be joined to a marker
for selection in plant cells. Conveniently, the marker may be
resistance to a herbicide, e.g. phosphinothricin or glyphosate, or
more particularly an antibiotic, such as kanamycin, G418,
bleomycin, hygromycin, chloramphenicol, or the like. The particular
marker employed will be one which will allow for selection of
transformed cells compared with cells lacking the introduced
recombinant DNA.
[0074] The fusion peptide in the expression cassette constructed as
described above, expresses at least preferentially in developing
seeds. Accordingly, transformed plants grown in accordance with
conventional ways, are allowed to set seed. See, for example,
McCormick et al. (1986, Plant Cell Reports, 5: 81-84). Northern
blotting can be carried out using an appropriate gene probe with
RNA isolated from tissue in which transcription is expected to
occur, such as a seed embryo. The size of the transcripts can then
be compared with the predicted size for the fusion protein
transcript.
[0075] Oil body proteins are then isolated from the seed and
analyses performed to determine that the fusion peptide has been
expressed. Analyses can be for example by SDS-PAGE. The fusion
peptide can be detected using an antibody to the oleosin portion of
the fusion peptide. The size of the fusion peptide obtained can
then be compared with predicted size of the fusion protein.
[0076] Two or more generations of transgenic plants may be grown
and either crossed or selfed to allow identification of plants and
strains with desired phenotypic characteristics including
production of recombinant proteins. It may be desirable to ensure
homozygosity of the plants, strains or lines producing recombinant
proteins to assure continued inheritance of the recombinant trait.
Methods of selecting homozygous plants are well know to those
skilled in the art of plant breeding and include recurrent selfing
and selection and anther and microspore culture. Homozygous plants
may also be obtained by transformation of haploid cells or tissues
followed by regeneration of haploid plantlets subsequently
converted to diploid plants by any number of known means, (e.g.:
treatment with colchicine or other microtubule disrupting
agents).
[0077] Method of Separating Target Molecules Using the Affinity
Matrices
[0078] As hereinbefore mentioned, the present invention relates to
a method of separating a target molecule from a sample using the
above described oil body proteins and in some cases, ligands. In
the method of the invention, oil bodies are mixed with a sample
containing the desired target and the interaction between the
ligand and target results in the non-covalent association of the
target with the oil body. Following centrifugation, the oil bodies
and affinity-bound target are separated from the aqueous phase,
effectively purifying the target from any contaminants present in
the original sample. Repeating the washing step ensures that any
remaining contaminants are removed.
[0079] Following their attachment to oil bodies, targets may be
eluted under conditions determined empirically for each individual
ligand-target pair. Treatment of the bound matrix with the
appropriate eluent and centrifugation enables recovery of the
purified target in the aqueous phase. If the target is a
ligand-protein fusion containing a protease recognition site, then
it may be treated with the appropriate protease to remove the
ligand. The free ligand may then be separated from the target
protein by re-application of the oil body affinity matrix or
through conventional protein purification methods.
[0080] The chemical and physical properties of the affinity matrix
may be varied in at least two ways. Firstly, different plant
species contain oil bodies with different oil compositions. For
example, coconut is rich in lauric oils (C12), while erucic acid
oils (C22) are abundantly present in some Brassica spp.
Furthermore, proteins associated with the oil bodies will vary
between species. Secondly, the relative amounts of oils may be
modified within a particular plant species by applying breeding and
genetic engineering techniques or a combination of these known to
the skilled artisan. These techniques aim at altering the relative
activities of enzymes controlling the metabolic pathways involved
in oil synthesis. Through the application of these techniques,
seeds with a sophisticated set of different oils are obtainable.
For example, breeding efforts have resulted in the development of a
rapeseed with a low erucic acid content (Canola) (Bestor, T. H.,
1994, Dev. Genet. 15: 458) and plant lines with oils with
alterations in the position and number of double bonds, variation
in fatty acid chain length and the introduction of desirable
functional groups have all been generated through genetic
engineering (Topfer et al., 1995, Science, 268: 681-685). Using
similar approaches a person skilled in the art will be able to
further expand on the presently available sources of oil bodies.
Variant oil compositions will result in variant physical and
chemical properties of the oil body fraction. Thus by selecting
oilseeds or mixtures thereof from different species or plant lines
as a source for oil bodies, a broad repertoire of oil body matrices
with different textures and viscosities may be acquired.
[0081] Applications of Oil Body Affinity Matrices
[0082] Given that it is possible to engineer oil body affinity
matrices for several classes of proteins, multiple uses for oil
body based affinity matrices are envisioned. Bacteria, fungi,
plants and animals all contain proteins which are able to
specifically interact with agents such as ions, metals, nucleic
acids, sugars, lipids and other proteins. These agents may be
immobilized using oil body technology.
[0083] The oil body protein affinity matrices can be used to
isolate any target molecule that can bind to the oil body protein,
either directly or indirectly through a ligand molecule Examples of
target molecules that may be isolated from a sample using the
methodology of the present invention include proteins, peptides,
organic molecules, lipids, carbohydrates, nucleic acids, cells,
cell fragments, viruses and metals. In particular, the inventors
have shown that the affinity matrix of the present invention can be
used to separate therapeutic proteins (such as thrombin),
antibodies, metals (such as cadmium), carbohydrates (such as
cellulose), organic molecules (such as biotin) and cells (such as
bacterial cells).
[0084] Oil body affinity matrices may also be used to separate
cells of industrial or medical interest from a mixed population of
cells. For example haematopoietic stem cells, which are a
subpopulation of blood cells and are used in bone marrow
transplantations and in stem cell gene therapies, may be separated
from other blood cells using oil body based affinity technology. In
recombinant DNA technology it is often required that cells in which
recombinant DNA has been successfully introduced, known as
transformed cells, are distinguished and separated from cells which
failed to acquire recombinant DNA. Provided that part of the
recombinant DNA expresses a cell surface protein which is
complementary to a oil body based affinity ligand, it is possible
to utilize oil bodies to separate transformed cells from
untransformed cells. Oil body affinity technology may also be used
to separate cellular organelles such as chloroplasts and
mitochondria from other cellular material. Viral particles may also
be separated from complex mixtures.
[0085] It is also possible to immobilize a class of proteins known
as metalloproteins, which contain prosthetic groups that
specifically bind ions. Examples of metalloproteins are
haemoglobin, which binds iron, parvalbumbin which binds calcium and
metallothionein a protein which binds zinc and other metal ions. It
is envisioned that oil bodies could be used to scavenge metals from
streams of flowing material, which might be water contaminated with
the waste of metals from laboratories and industrial processes.
Example 4 given below further illustrates this application. Other
examples where proteins may be bioimmobilized and employed in a
bioremediation strategy include the removal of phosphates, nitrates
and phenols from waste streams. In part this approach may overcome
the real or perceived limitations of bacterial bioremediation. In
certain instances it may not be practical or necessary to rely on
affinity partitioning technology to separate the oil body matrix
from the target compound. In these instances, it is envisioned that
oil bodies may be immobilized on a solid inert surface which could
be a flat surface or the surface of a column. A solution containing
the affinity ligand may then be passed over the surface coated with
immobilized oil bodies whereupon selective affinity binding occurs.
It is envisioned that immobilized oil bodies may be used in pipes
and in ponds to assist in bioremediation.
[0086] The following examples illustrate various systems in which
oil bodies can be used as affinity matrices. It is understood that
the examples given below are intended to be illustrative rather
than limiting.
EXAMPLES
Example 1
[0087] Purification of Thrombin
[0088] The following example demonstrates the utility of an oil
body affinity matrix for the purification of thrombin. Thrombin is
a serine protease which plays a central role in blood coagulation.
It cleaves fibrinogen to produce fibrin monomers which polymerize
to form the basis of a blood clot (Fenton 1981; Ann. N.Y. Acad.
Sci. 370: 468-495). Alfa-thrombin consists of two polypeptide
chains of 36 (A-chain) and 259 (B-chain) residues linked by a
disulphide bridge. Degen et al. 1983; Biochemistry 22: 2087-2097).
Hirudin, which is found in the salivary glands of the medicinal
leech Hirudo medicinalis, is a very specific and potent inhibitor
of thrombin. This inhibition is a result of the non-covalent
binding of hirudin to specific parts of the alfa-thrombin chain.
(Stone and Hofsteenge 1986; Biochemistry 25: 4622-4628).
[0089] The immobilized ligand is comprised of an isoform of hirudin
fused to the 18 kDa Arabidopsis oleosin (oil body protein) (Van
Rooijen et al., 1992; Plant Mol. Biol. 18: 1177-1179). Expression
of the construct is regulated by the Arabidopsis 18 kDa oleosin
promoter (Van Rooijen et al., 1994; Plant Mol. Biol. 18:
1177-1179). The sequence of the oleosin-hirudin fusion is shown in
FIG. 2 and in SEQ.ID.NO:3.
[0090] Oleosin-Hirudin Construct
[0091] Oligonucleotide primers were designed based upon the
reported sequence for a Brassica napus oleosin gene (Murphy et al.
1991, Biochim. Biophys. Acta 1088: 86-94) and used to amplify a
fragment from B. napus genomic DNA through PCR. Using this fragment
as a probe, a clone carrying a 15 kbp insert was identified and
isolated from a EMBL3 Arabidopsis genomic library. Oligonucleotide
primers were used to amplify a fragment from this insert containing
the entire oleosin coding sequence and intron together with 840
basepairs of the 5' upstream region. The primers were designed so
as to eliminate the translational stop codon and to introduce a
PstI restriction endonuclease recognition site at the 5' end and a
SalI followed by a PvuI site at the 3' end of the fragment. The
fragment was end-filled and ligated into the SmaI site of the
plasmid vector pUC19. A SalI-EcoRI fragment from plasmid pBI121
(Clontech) comprising the nopaline synthetase terminator sequence
was then inserted to generate pOBILT.
[0092] A synthetic hirudin variant 2 (HV2) sequence was synthesized
based upon reported sequence information (Harvey et al. 1986, Proc.
Natl. Acad. Sci. USA 83: 1084-1088) but employing B. napus and
Arabidopsis codon usage. The sequence was amplified using four
overlapping oligonucleotide primers designed such that the
resulting fragment possessed PvuI and SalI sites at the 5' and 3'
ends respectively. This fragment was ligated into the SmaI site of
the pUC19 plasmid vector to generate pHIR. The PvuI-SalI fragment
from pHIR was then inserted into pUCOBILT between the oleosin and
terminator sequences to form an in-frame fusion with the oleosin
coding region giving pUCOBHIRT. The entire construct was subcloned
into pBluescript KS+ (pBIOBHIRT) and then into the PstI site of
pCGN1559 plasmid (McBride and Summerfelt, 1990, Plant Mol. Biol.
14: 269-276) carrying a neomycin phosphotransferase gene under
control of the 35-S CaMV promoter (pCGOBHIRT). This plasmid was
introduced into Agrobacterium tumefaciens. The preparation of this
plasmid is shown in FIG. 3.
[0093] Transformation and Regeneration
[0094] Procedures for the transformation of Agrobacterium and
plants have been described previously. Agrobacterium tumefaciens
was transformed with the above construct through electroporation
(Dower et al., 1988; Nucl. Acids Res. 16: 6127-6145). The
transformed bacteria were then used to transform cotyledonary
explants of Brassica napus, followed by plant regeneration
according to the methods of Moloney et al. (1989; Plant Cell
Reports 8: 238-242). Transgenic plant were initially identified
using a neomycin phosphotransferase assay and subsequently
confirmed by expression of the oleosin-hirudin fusion as determined
through northern and immunoblot analysis.
[0095] Preparation of Oil Bodies
[0096] Seed from either control (non-transgenic) plants or
transgenic plants expressing the oleosin-hirudin fusion were
homogenized in five volumes of cold grinding buffer (50 mM
Tris-HCl, pH 7.5, 0.4 M sucrose and 0.5 M NaCi) using a polytron
operating at high-speed. The homogenate was centrifuged at
approximately 10.times.g for 30 min. to remove particulate matter
and to separate oil bodies from the aqueous phase containing the
bulk of soluble seed protein. Oil bodies were skimmed from the
surface of the supernatant with a metal spatula and placed in one
volume of fresh grinding buffer. To achieve efficient washing in
subsequent steps, it was important to ensure that the oil bodies
were thoroughly redispersed. This was accomplished by gently
re-homogenising the oil bodies in grinding buffer with the polytron
operating at low-speed. Using a syringe, the resuspended oil bodies
were carefully layered underneath five volumes of cold 50 mM
Tris-HCl, pH 7.5 and centrifuged as above. Following
centrifugation, the oil bodies were again removed and the washing
procedure repeated three times to remove residual contaminating
soluble seed proteins. The final washed oil body preparation was
resuspended in one volume of cold 50 mM Tris-HCl pH 7.5,
redispersed with the polytron, and was then ready for use as an
affinity matrix.
[0097] Affinity Purification of Thrombin
[0098] The purification of thrombin using the oleosin-hirudin
fusion protein is shown schematically in FIG. 4. In order to
evaluate the binding of thrombin, affinity matrices were prepared
from transgenic Brassica napus seeds expressing the oleosin-hirudin
fusion protein (4A4 seeds) (Parmenter et al. Plant Molecular
Biology (1995) 29: 1167-1180) and from wild type Brassica napus cv
Westar seeds. Binding of thrombin to both matrices was evaluated.
Procedures for the preparation of washed oil bodies from seeds were
the same as those described above. Solutions containing a range of
thrombin activities between 0 and 1 units were mixed with 10 .mu.l
of a fixed amount of affinity matrix (prepared from a total of 10
mg of dried seeds; corresponding to approximately 100 .mu.g of
total oil body protein) in 500 .mu.l binding buffer (50 mM Tris-HCl
(pH 7.5); 0.1% (w/v) BSA). The oil body suspension was then
incubated for 30 minutes on ice and centrifuged at 14,000 rpm for
15 minutes at 4.degree. C. The buffer under the oil bodies (termed
`unternatant`) containing the unbound, free thrombin was recovered
using an hypodermic needle and assayed for thrombin activity as
follows. A total of 250 .mu.l of unternatant was added to 700 .mu.l
binding buffer and prewarmed to 37.degree. C. Following the
addition of 50 .mu.l of 1 mM thrombin substrate
N-p-tosyl-gly-pro-arg-p-nitroanilide (Sigma) to the unternatant,
the change in optical density at 405 nanometers was monitored
spectrophotometrically for 3 minutes. The concentration of thrombin
in the assay mixture was determined employing a standard curve
which was constructed using a set of thrombin samples containing
known concentrations of thrombin. The values obtained from these
assays were used to calculate the concentration bound thrombin
assuming:
[bound thrombin]=[total thrombin]-[free thrombin]
[0099] The ratio of the concentration of bound over the
concentration of free thrombin was plotted as a function of the
concentration of bound thrombin (Scatchard plot). From these plots
the dissociation constants of the affinity matrix were calculated
following standard procedures (Scatchard, G. Ann. N.Y. Acad. Sci.
(1949) 57: 660-672) and assuming: K.sub.a=1/K.sub.d. The
dissociation constants of the affinity matrices were
3.22.times.10.sup.-7 m for wild type and 2.60.times.10.sup.-8 m for
4A4 oil bodies.
[0100] In order to evaluate the recovery of bound thrombin from the
matrices a NaCl gradient was employed. The elution profile of
thrombin bound to oleosin-hirudin oil body matrices was compared
with the profile from thrombin bound to wildtype oil body matrices.
Procedures for preparation of wild type oil bodies from wild type
Brassica napus cv Westar seeds and for the preparation of
oleosin-hirudin oil bodies from Brassica napus 4A4 seeds (Parmenter
et al. Plant Molecular Biology (1995) 29: 1167-1180) were identical
to those described above. Procedures for binding of thrombin to the
matrices were as described above, except 100 .mu.l aliquots of oil
bodies were used to bind 0.5 units of thrombin. Oil body
suspensions were left on ice for 30 minutes prior to centrifugation
for 15 minutes at 4.degree. C. and 14,000 rpm. The unternatant was
assayed for (unbound) thrombin activity. The oil body matrix was
then resuspended in binding buffer to which NaCl was added to a
final concentration of 0.05 M. Starting with the 30 minutes
incubation of the oil body suspension on ice, the procedure was
repeated five times increasing the NaCl concentration in a stepwise
fashion. The final NaCl concentrations used were 0.05 M, 0.1 M, 0.2
M, 0.3 M, 0.4 M and 0.6 M. The NaCl concentrations in the thrombin
assay were kept constant at 150 mM. FIG. 5 shows the elution
profiles obtained when wildtype oil bodies and 4A4 oil bodies were
used.
Example 2
[0101] Use of Antibodies as Bivalent Ligands
[0102] Antibodies may be used as bivalent ligands by virtue of
their affinity both for specific epitopes and for other antibodies
or proteins (for example the Staphylococcus aureus protein A) which
have affinity for immunoglobulins (IgGs). In this example,
polyclonal anti-oleosin antibodies serve as a bivalent ligand and
antibodies raised in rabbits against the anti-oleosin antibodies
serve as the target. This example is illustrated schematically in
FIG. 6.
[0103] Oil bodies were prepared from 5 g of wild type Brassica
napus cv Westar seeds following the procedure described in Example
1. Subsequently, oil bodies were washed twice with 100 mM glycine
(pH 2.5), neutralized through two washes in binding buffer (50 mM
Tris-HCl, pH 7.5) and resuspended in 5 ml of binding buffer. A 150
.mu.l aliquot of the washed oil body preparation was combined with
500 .mu.l of rabbit serum containing anti-oleosin antibodies
(ligand antibodies), diluted 1:10 with binding buffer. The oil body
suspension was mixed thoroughly and incubated for 1 h at 4.degree.
C. with agitation. Following incubation, unbound ligand antibodies
were removed from the oil body suspension through three washes with
1 ml of binding buffer. Oil bodies were then combined with 500
.mu.l of serum diluted 1:500 in binding buffer and containing
anti-rabbit IgG antibodies (the target antibodies) conjugated with
horseradish peroxidase (HRP) as a detection label (Sigma). This
suspension was mixed and incubated under conditions identical to
those used for the anti-oleosin antibody binding. As a control,
target antibodies were incubated with oil bodies which had not been
previously bound to ligand antibodies. Both samples were
subsequently washed four times with 1 ml of binding buffer to
remove unbound antibodies. Using binding buffer, the samples were
equalized with respect to concentration of oil bodies as determined
by measuring sample turbidity spectrophotometrically at 600 nm. To
assay for bound target antibody, samples containing 5 .mu.l of oil
bodies were mixed with 1 ml of the HRP calorimetric substrate
tetramethylbenzidine in 0.01% hydrogen peroxide and reacted for 10
minutes at room temperature. Reactions were stopped by the addition
of 500 .mu.l of 1 M H.sub.2SO.sub.4 and the absorbance at 450 nm
was determined. Corrections for the presence of residual, unbound
target antibody remaining after washing were made by assaying 5
.mu.l of the final wash fraction. The results obtained for control
and ligand bound oil body preparations are set forth in FIG. 7.
Example 3
[0104] Use of Oleosin-Specific Ligands
[0105] The use of an oleosin-specific ligand represents an
alternative to the use of an antibody or genetically-engineered
oleosin fusion proteins for the purification of recombinant target
proteins. In this case, the target protein is fused to the
oleosin-specific ligand and the endogenous oleosins present on the
oil bodies of non-transgenic seeds serve as the complementary
ligand-affinity matrix. In addition to eliminating the requirement
for a transgenic line expressing an oleosin fusion, this approach
increases the overall capacity of the affinity matrix, since all of
the endogenous oleosins may now participate in binding.
[0106] Oleosin-specific ligands may be identified and isolated from
a peptide phage display library screened with oleosin protein.
Since the extreme hydrophobicity of the oleosin central domain can
result in aggregation and precipitation of the protein when removed
from oil bodies, a mutant protein lacking this domain may be used
for screening. This has little effect on the efficacy of the
ligand, as only the hydrophillic portions of the oleosin are
exposed to the cytoplasm (i.e. the N- and C-termini). Hence, these
are the only regions available for binding to a ligand. Once
isolated, the ligand may be fused to a common reporter protein,
green fluorescent protein (GFP) (Prasher, 1995, Trends Genet.
11:320-323), to demonstrate purification.
[0107] Removal of the Oleosin Central Domain
[0108] Oligonucleotide primers specific for the Arabidopsis oleosin
gene described above can be used to amplify an oleosin gene from a
B. napus cDNA library (van Rooijen 1993, Ph.D. Thesis, University
of Calgary). Primers flanking sequences encoding the N-terminal 62
amino acids and the C-terminal 55 amino acids, may be used to
amplify sequences for the respective N- and C-terminal oleosin
domains in separate reactions. Additionally, the primer for the 5'
end of the N-terminal domain contains a sequence for a thrombin
recognition site to enable cleavage of the fusion protein as
described below. The resulting fragment was ligated into the SmaI
site of the bacterial expression vector pEZZ 18 (Pharmacia). This
vector contains sequences encoding a signal peptide for protein
secretion into the periplasm, and synthetic IgG binding domains
derived from protein A to facilitate protein purification,
downstream of the multiple cloning site.
[0109] Expression and Purification of the Oleosin Deletion
Construct
[0110] The vector carrying the deletion mutant construct is
introduced into E. coli using standard methods and transformants
selected. A culture of the transformed bacteria can be induced to
express the synthetic protein A-mutant oleosin fusion protein by
addition of 1 mM IPTG. Induced cells may be pelleted and
resuspended in 5 mM MgSO.sub.4 causing lysis of the periplasmic
membrane through osmotic shock. The lysed cells are centrifuged and
the supernatant containing the secreted protein is loaded on to a
column containing IgG-coupled sepharose. After washing to remove
unbound protein, the column is loaded with a buffer containing 50
mM Tris-HCl, pH 7.5, 150 mM NaCl and 1.0 U/ml of purified Bovine
thrombin (Sigma) to cleave the mutant oleosin from the synthetic
protein A. Following incubation at 37.degree. C. for 4 h, the
column is drained and the eluate passed through a column of
heparin-coupled sepharose to remove thrombin. The eluate from this
column, containing the mutant oleosin protein, is recovered and
purity of the protein examined through gel electrophoresis followed
by staining with Coomassie blue R250.
[0111] Generation of a Peptide Combinatorial Library
[0112] A random peptide combinatorial library may be generated
according to the methods of Scott and Smith (1990; Science 249:
386-390). Briefly, the PCR is used to amplify a synthetic DNA
fragment containing the degenerate sequence (NNK).sub.6; where `N`
represents an equal mixture of deoxynucleotides G, A, T, and C, and
K represents an equal mixture of deoxynucleotides G and T. The
degenerate sequence encodes for hexameric peptides among which are
represented every possible combination of the 20 amino acids and
amber stop codon. The PCR product is ligated into the gene III
sequence of the filamentous bacteriophage fUSE and the resulting
phagemid introduced into E. coli through electroporation.
[0113] Identification and Isolation of Oleosin-Specific Ligands
[0114] The peptide phage display libraries are amplified,
concentrated and stored in aliquots of 10.sup.12 tdu/ml. Purified
mutant oleosin protein is biotinylated using a thiol-cleavable
linker (S-S biotin, Pierce) and purified by size exclusion
chromatography. Aliquots of the peptide phage display library
containing 5.times.10.sup.11 tdu in two ml are screened with the
biotinylated protein at a concentration of 50 nM. Phage binding the
mutant oleosin protein are recovered using streptavidin-coated
paramagnetic beads. Following washing, the phage are eluted through
the addition of 50 mM dithiothreitol which cleaves the disulphide
bond. The eluted phage are then incubated with an excess of
log-phase F+ E. coli. Aliquots of the infected cells are plated to
determine the phage titre and the remaining cells used in
successive rounds of amplification and screening. Following
enrichment of the eluted phage by 3-4 orders of magnitude,
individual phage are selected and tested for binding to mutant
oleosin by direct ELISA. Binding by phage is detected using
anti-phage antibodies (Crosby and Schorr, 1995, In Annual Review of
Cell Biology). Single stranded DNA is isolated from phage
exhibiting binding and the peptide-encoding sequence
determined.
[0115] Affinity Purification with Oleosin-Specific Ligands
[0116] The sequence for an oleosin ligand isolated as described
above is fused in-frame upstream the sequence for gfp10 (Prasher et
al., 1992, Gene 111: 229-233) encoding GFP and the construct
ligated into the bacterial expression vector pKK233 (Pharmacia).
Soluble protein is extracted through sonication of cells induced to
express the ligand-GFP fusion, and adjusted to a concentration of
10 mg/ml in 50 mM Tris-HCl, pH 7.5.
[0117] Twenty ml of the protein solution is mixed with 2 ml of oil
bodies prepared as described above, from seeds of non-transgenic
plants. The mixture is incubated at 4.degree. C. for 30 min with
agitation to allow binding and then centrifuged to separate the oil
bodies and soluble fraction. The amount of GFP remaining in the
soluble fraction after removal of oil bodies is determined by
fluorescence spectrofluorometry at a wavelength of 508 nm and
compared with that in the original bacterial extract. The amount of
bound GFP is calculated to determine the capacity of the
matrix.
[0118] The oil bodies are washed twice in 20 ml of 50 mM Tris-HCl,
pH 7.5, resuspended in 2 ml of the same buffer and divided into 20
aliquots of 100 .mu.l. Conditions for the elution of ligand-GFP
fusion protein are determined by adding 1 ml of solutions ranging
in pH from 2-10 and in NaCl concentration from 0-1 M to different
aliquots. After mixing and incubation at 4.degree. C. for 30 min,
the oil bodies are removed and the soluble fractions collected. The
amount of ligand-GFP fusion protein in the soluble fraction is
determined by fluorescence spectrophotometry.
Example 4
[0119] Removal of Heavy Metal Ions
[0120] The following example demonstrates the utility of oil body
affinity matrices for the recovery/removal of non-protein targets
from complex solutions. For the purpose of this example the
metallothionein/Cd.sup.++ ligand pair was used. However other metal
binding proteins such as phytochelatins (Rauser, 1990; Ann. Rev.
Biochem; 59: 61-86) and metal ions including Cu.sup.++ and
Zn.sup.++ could also be used.
[0121] Oleosin-Metallothionein Fusion
[0122] An oleosin gene from a B. napus cDNA library (van Rooijen
1993, Ph.D. Thesis, University of Calgary) was amplified through
PCR with oligonucleotide primers designed so as to create NotI and
NcoI sites at the 5' and 3' ends of the gene respectively. The
resulting fragment was digested and placed into the NotI/NcoI sites
of pGN to yield plasmid poleGN. The human metallothionein gene,
mt-II (Varshney and Gedamu, 1984, Gene, 31: 135-145) was amplified
using oligonucleotide primers designed to create a unique NotI site
at the 3'-end of the gene. The resulting PCR product was subcloned
into the blunt-end EcoRV site of pBluescript KS+ to form pBSMTC.
The mt-II gene was then excised from this plasmid and subcloned
into the NcoI/KpnI sites of poleGN replacing the GUS-NOS region to
generate pOLEMTC. The 773 base oleosin-MT fusion of pOLEMTC was
excised with NotI digestion and inserted into the unique NotI site
of polePN3' between the oleosin promoter (oleP; Van Rooijen et al.,
1992, Plant Mol. Biol. 18: 1177-1179) and the P. crispum ubi4-2
gene terminator (ubi3'; Kawalleck et al., 1993, Plant Mol. Biol.
21: 673-684.) to generate pOOM3'. After the fusion was determined
to be in the correct orientation, pOOM3' was digested with KpnI to
release the oleP-oleMT-ubi3' insert. This expression cassette was
inserted at the KpnI site of the binary vector pCGN1559 to yield
the final construct pBIOOM3'. The sequence of the
oleosin-metallothionein fusion is shown in FIG. 8 and SEQ.ID.NO.6.
The construction of plasmid pB100M3' is shown in FIG. 9.
[0123] Transformation and Regeneration
[0124] Transgenic B. carinata plants expressing the
oleosin-metallothionein fusion were created using transformation
and regeneration protocols as described in Example 1.
[0125] Oil Body Preparation
[0126] Washed oil bodies were prepared from B. carinata seeds of
transgenic and control plants as described in Example 1.
[0127] Removal of Cd++ From Solution Using an Oil Body Affinity
Matrix
[0128] The use of the oleosin-metallothionein fusion to bind
cadmium ions in solution is shown schematically in FIG. 10.
[0129] A solution of 10 .mu.M CdCl.sub.2 in 10 mM Tris-HCl, pH 7.2
containing 0.01 .mu.Ci/ml .sup.109Cd was prepared. A 1 ml aliquot
of this CdCl.sub.2 solution was thoroughly mixed with 100 .mu.l of
washed oil bodies (1.6 mg oil body protein) prepared from seeds
expressing the oleosin-metallothionein fusion protein and incubated
at 22.degree. C. for 1 hr. Following centrifugation for 5' at
10,000.times.g to separate the oil bodies from the aqueous phase
and 2 washes in 1 ml of 10 mM Tris-Cl, pH 7.2, the amount of
.sup.109Cd.sup.++ remaining bound to oil body fraction was
determined using a gamma-counter (Cobra auto-gamma, Canberra
Packard, Canada). An identical experiment was performed with oil
bodies from non-transgenic seeds to detect and correct for
non-specific binding of Cd ions to the matrix.
[0130] Cd.sup.++ ions were eluted from the oil body metallothionein
affinity matrix by mixing of the oil body fraction with 1 ml of 100
mM glycine (pH=3.0) buffer (Pazirandeh et al., 1995; Appl.
Microbiol. Biotechn. 43: 1112-1117). Following centrifugation for 5
min. At 10,000.times.g, the oil body fraction was removed and
assayed for bound Cd.sup.++ ions as above. FIG. 11 shows Cd binding
and elution from the affinity matrix.
Example 5
[0131] Separation of Whole Cells
[0132] The following example illustrates the capacity of oil bodies
to immobilize whole cells. One potential for the use of bacterial
cell separation lies in the utility for diagnostics. It is also
desirable to separate unique eukaryotic cells such as lymphocytes
and stem cells from complex mixtures of cells where the cell type
of interest is present in relatively low numbers.
[0133] Binding of Staphylococcus aureus to Oil Bodies via Protein
A
[0134] For the purpose of this example, S. aureus cells, which
express protein A as a surface antigen were mixed with oil bodies
with varying amounts of polyclonal anti-oleosin antibodies.
[0135] Preparation of Oil Bodies
[0136] Seeds of B. napus cv Westar were surface sterilized in
bleach, rinsed and ground with a mortar and pestle in grinding
buffer (50 mM Tris pH 7.5, 0.4 M sucrose and 100 mM glycine). The
homogenate was filtered through Miracloth into sterile 15 ml Corex
tubes. The filtered homogenate was then centrifuged for at
4.degree. C. for 10 min at 10,000.times.g. The oil body fraction
was removed and resuspended in 50 mM Tris pH 7.5 and 0.4 M sucrose
and washed two times using the same buffer. Aliquots of 1 ml oil
bodies were transferred to 1.5 ml Eppendorf tubes and centrifuged
at room temperature for 10 min at 16,000.times.g. The oil bodies
were washed in 50 mM Tris pH 7.5 and 0.4 M sucrose 5-6 more times
until no visible pellet was observed.
[0137] Binding of S. aureus Cells to Anti-Oleosin Coated Oil
Bodies
[0138] Formalin fixed S. aureus cells (Sigma, P-7155) were washed
3-4 times in 50 mM Tris-Cl pH 7.5. and resuspended. Washed oil
bodies (300 .mu.l) and S. aureus cells (were mixed with varying
amounts of anti-oleosin IgGs (50 .mu.l). After mixing and
incubating at room temperature for 2 hrs, the mixtures were
centrifuged at room temp at 16,000.times.g for 5 min. The oil body
fraction and unternatant were carefully removed and the cell pellet
was washed twice in 1 ml 50 mM Tris-Cl pH 7.5. The walls of the
tube were wiped with a tissue to remove traces of oil. Subsequently
the drained cell pellets were resuspended in 1 ml of water and the
OD.sub.600 were determined. FIG. 12 is a representative experiment
showing the decrease in the amount of cells present in the cell
pellet as the concentration of anti-IgGs present in the oil-body S.
aureus mixture increases.
[0139] Differential Binding of Two Strains of Staphylococcus
aureus.
[0140] In this experiment an oil body affinity matrix is employed
to demonstrate differential binding of two strains of
Staphylococcus aureus. Formalin fixed S. aureus strains, one
expressing the IgG binding surface antigen protein A and one
lacking protein A, are commercially available from Sigma. Dilute
aliquots of both S. aureus strains of equal OD.sub.550 could be
prepared. To each of these aliquots control oil bodies from
untransformed plants or oil bodies mixed with anti-oleosin
antibodies could be added. Following incubation for an appropriate
length of time at an appropriate temperature, the samples could be
centrifuged to pellet unbound bacterial cells and to separate the
oil body fraction. The oil bodies could be decanted, vortexed and
the OD.sub.550 could be determined. The pellets could be
resuspended and the OD.sub.550 of the unternatant could be
determined. It is anticipated that only in the sample containing
the S. aureus strain expressing protein A and the oil body
complexed with anti-oleosin antibodies, fractionation of these
cells to the oil body fraction will be observed. Binding of the
cells to the oil body could be further demonstrated by lowering of
the pH of the oil body fraction. Subsequent to centrifugation the
release of cells from the oil bodies could be evidenced by the
presence of a pellet and/or an increase in OD.sub.550 upon
resuspension of the pellet.
[0141] Separation of Staphylococcus aureus from E. coli
[0142] A viable S. aureus strain could be mixed with varying
quantities of cells of an E. coli strain having a specific
antibiotic resistance. The mixed bacterial sample could be vortexed
with control antibodies and with oil bodies which have been
complexed with anti-oleosin antibodies. After incubation for an
appropriate length of time and at an appropriate temperature oil
bodies could be washed and the unternatant and oil bodies could be
directly titrated and selectively plated on blood agar for S.
aureus growth and on LB plates for E. coli growth. The enrichment
or actual separation obtained could be determine by an estimate of
colony forming units.
[0143] Identification of Pathogens Present in Low Concentrations in
a Complex Mixture
[0144] For diagnostic purposes it is often desirable to concentrate
bacterial or viral pathogens which invade human or animal tissues
in low numbers. An oil body affinity matrix could be used to enrich
for these pathogens, so that they could subsequently be identified
and characterized.
[0145] Pathogens often specifically bind to human or animal cells
through the interaction with a receptor or surface protein. Oleosin
could be fused to the human or animal protein ligand and
recombinant oil bodies could be employed to immobilize the
pathogens. Examples of the formation of protein complexes formed
between proteins of human and pathogenic origins known to the prior
art include: human fibrinogen or fibrin specific domains which bind
to S. aureus protein clumping factor A (clf-A) (McDevitt et al.
1995; Mol. Microbiol. 16; 895-907); human decay accelerating factor
(DAF) to which urinary and intestinal tract pathogenic E. Coli bind
(Nowicki et al. 1993: J. Of Experim. Med. 178: 2115-2121); a human
cell ligand which is expressed in the carcinoma cell line Caco-2
and which binds uniquely to the 28 kD Klebsiella pneumoniae fimbria
protein KPF-28 (Di Maretino et al., 1996; Infect. and Immun. 64:
2263-2266) and human cell extracellular matrix fibronectin specific
domains which complex specifically with Streptococcus pyrogenes
adhesin (protein F) (Ozeri et al., 1996; EMBO J. 15: 989-998).
Example 6
[0146] Separation of Small Organic Molecules
[0147] This example describes how an oil body affinity matrix may
be used for the recovery/removal of small organic molecules from
solution. By way of example, the small organic molecule, biotin, is
purified using avidin as a ligand.
[0148] Construction of Avidin Ligands
[0149] Avidin is a protein synthesized by avian species and
exhibits an extremely high affinity for biotin, a natural co-factor
for many carboxylases. Preparations of purified avidin
(commercially available from Sigma) can be conjugated chemically to
anti-oleosin antibodies using standard procedures known to those
skilled in the art. This approach would yield a bivalent avidin
ligand suitable to demonstrate affinity based removal of biotin.
Alternatively, an oleosin-avidin gene fusion may be utilized. The
gene encoding avidin in chicken (Gallus gallus) has been identified
and its sequence has been determined (Beattie et al., 1987, Nucl
Acids Res. 15: 3595-3606). Based on the sequence the gene for
avidin could be synthesized chemically or through the PCR and fused
to the B. napus oleosin (van Rooijen, 1993, Ph.D. Thesis,
University of Calgary) as described in example 4. Streptavidin, an
analogous bacterial biotin binding protein, could also be
employed.
[0150] Oil Body Preparation
[0151] Washed oil bodies would be prepared from seeds of transgenic
plants and/or control plants as described in example 1.
[0152] Binding of Bivalent Avidin-Oleosin Ligand
[0153] Binding of anti-oleosin antibodies and removal of unbound
ligand will be as detailed in example 3.
[0154] Removal of Biotin from Solution
[0155] Solutions containing known concentrations of biotin could be
combined with a fixed amount of oil bodies complexed with an
anti-oleosin antibodies conjugated with avidin. Following binding,
the mixture would be centrifuged to separate oil body and aqueous
fraction. The amount of biotin remaining in the aqueous fraction is
determined by competitive ELISA using anti-biotin antibodies
conjugated to horse radish peroxidase (HRP). The amount of bound
biotin may be calculated assuming:
[bound biotin]=[total biotin]-[free biotin]
[0156] From the obtained values, the dissociation constants can be
determined as described in example 2. As a control, an identical
experiment could be performed with oil bodies bound to anti-oleosin
antibodies which have not been conjugated with avidin. If desired,
biotin could be released from the oil body-avidin matrix through
competitive elution using an excess of 2-(4'-hydroxybenzene)
benzoic acid (HABA). Elution may also be aided by employing a
genetically engineered mutant of avidin which exhibits a lower
affinity for biotin. Such mutants have been described for the
analogous biotin binding protein from bacteria, streptavidin
(Chilkoti et al., 1995; Bio/Technol. 13: 1198-1204).
Example 7
[0157] Separation of Carbohydrates
[0158] The following example describes the utility of oil body
matrices for the recovery of carbohydrates from complex biological
mixtures. In this example the inventors demonstrate that an oil
body immobilized cellulase is capable of binding cellulose.
[0159] Oleosin-Cellulose Binding Domain Fusion
[0160] Several of the cellulases produced by the bacterium
Cellulomonas fimi contain discrete cellulose binding domains
(CBDs). These CBDs independently bind to cellulose even when they
are separated by proteolytic cleavage or genetic manipulation from
the catalytic domain of the enzyme. Plasmid pUC18-CBDPT contains a
fragment coding for the CBD of the beta-1,4-glucanase (Gilkes et
al., 1992, Journal of Biol. Chem. 267: 6743-6749) and could be used
to construct an oleosin-CBD gene fusion. A DNA fragment encoding
the CBD domain could be isolated from pUC18-CBDPT using appropriate
restriction enzymes or using the PCR. Alternatively, the CBDs of
other cellulases from C. Fimi or cellulases from other sources
could be used. An oleosin gene from B. Napus isolated from a cDNA
library (van Rooijen, 1993, Ph.D. Thesis, University of Calgary)
was cloned in pGN using the PCR and yielding plasmid pOLEGN as
described in example 4. An in-frame gene fusion between the oleosin
gene and the CBD gene could be generated using standard molecular
techniques known to those skilled in the art. The final construct
would comprise the CBD domain translationally fused immediately
downstream of the oleosin.
[0161] Transformation and Regeneration
[0162] In order to introduce the fusion gene construct in plants,
it would be subcloned in a binary vector, such as pCGN1559.
Transgenic plants which express the oleosin-CBD fusion could be
generated as described in example 1.
[0163] Oil Body Preparation
[0164] Washed oil bodies could be prepared from the seeds of
transgenic and control wild type plants as described in example
1.
[0165] Removal of Cellulose from Solution Using an Oil Body
Affinity Matrix
[0166] In order to evaluate binding of cellulose to the oil body
affinity matrix, the binding capacities of oil bodies of wild type
and transgenic plants are compared. Oil bodies could be mixed with
appropriately buffered solutions containing a range of cellulose
concentrations. The oil body suspension could then be incubated for
an appropriate length of time and at an appropriate temperature.
Upon centrifugation, the unternatant could be recovered and assayed
for cellulose concentrations. The concentrations bound cellulose
and free cellulose could be calculated assuming:
[bound cellulose]=[total cellulose]-[free cellulose]
[0167] The ratio of the concentration bound over the concentration
free cellulose could be plotted as a function of the concentration
of bound cellulose. From these plots dissociation constants could
be calculated following standard procedures (Scatchard, G. Ann.
N.Y. Acad. Sci. (1949) 57: 660-672) and as detailed in example
2.
Example 8
[0168] Separation of Nucleic Acids
[0169] The following example describes a method in which oil bodies
are employed to bind single stranded (SS) nucleic acids.
[0170] Isolation of Single Stranded Nucleic Acids
[0171] A method for capturing SS nucleic acids may be used in
diagnostics, such as plant viral disease, or in research
applications where non-reannealed SS nucleic acids need to be
selectively removed from solutions such as in hybridization
reactions for differential screening of expressed genes. Oleosins
could be fused with SS DNA or RNA binding proteins or specific
domains thereof and could be used to trap SS nucleic acids for
identification or further amplification. Well characterized SS
nucleic acid binding proteins include: Agrobacterial Ti plasmid Vir
E2 protein (Zupan et al., 1995, Plant Physiol. 107: 1041-1047);
Tobacco Mosaic Virus (TMV) movement protein P30 (Citovsky et al.,
1990; Cell 60: 637-647; Waigmann et al., 1994 Proc Natl. Acad. Sci
(USA) 91: 1433-1437); Cauliflower Mosaic Virus coat protein
(Thompson et al., 1993; J. Gen. Virol 74: 1141-1148) and E. Coli
RecA and single stranded binding (SSB) proteins (Radding, 1991 J.
Biol. Chem. 266: 5355-5358).
Example 9
[0172] Separation of Recombinant Proteins
[0173] The following example further demonstrates the utility of an
oil body affinity matrix for the purification of recombinant target
proteins. For the purpose of this example, the IgG/protein A ligand
pair has been chosen. The construct employed consists of a protein
A domain which was fused to the 18 kDa Arabidopsis oleosin (Van
Rooijen et al., 1992; Plant Mol. Biol. 18: 1177-1179). Oil bodies
containing oleosin-protein A fusion proteins were isolated and used
to demonstrate specific binding of rabbit-anti-mouse IgGs
conjugated to Horse Raddish Peroxidase (HRP). The configuration of
the oleosin-protein A fusion on the oil body and binding of IgG to
the fusion is shown in FIG. 15.
[0174] The Oleosin-Protein A Fusion
[0175] A synthetic protein A sequence encoding a protein capable of
binding to IgG was synthesized based on reported sequence
information (pRIT2T, protein A gene fusion vector; Pharmacia) and
was amplified through the PCR. Each primer used in the PCR
contained restriction sites 5' to the protein A-specific sequence
in order to facilitate cloning. The reverse primer (i.e. the primer
in the antisense direction) also contained a translational stop
codon following the coding sequence. FIG. 13 shows the position of
the PCR primers relative to the protein A sequence. (The protein A
sequence and the primer sequences are also separately shown in
SEQ.ID.NO:8, SEQ.ID.NO:10 and SEQ.ID.NO:11 respectively). The
resulting fragment was ligated into a pUC19 plasmid carrying the
Arabidopsis oleosin gene comprised of an 867 bp upstream promoter
region followed by the coding region (with its associated intron)
from which the translational stop codon had been removed. The 3'
end of the construct contains the nopaline synthase transcriptional
terminator. A spacer sequence encoding a recognition sequence for
the endoprotease thrombin was incorporated immediately downstream
of the oleosin coding sequence. The protein A gene sequence was
introduced between this spacer sequence and the terminator
sequence. In the final expression construct the oleosin and protein
A coding regions were fused in the same reading frame. The entire
construct (FIG. 14 and SEQ.ID.NO:12) was then excised from the
pUC19 plasmid and subcloned into the plant transformation vector
pCGN1559 (McBride and Summerfelt, 1990, Plant Mol. Biol. 14:
269-276) carrying a neomycin phosphotransferase gene under the
control of the 35S CaMV promoter. The resulting plasmid was
introduced in Agrobacterium (strain EHA101).
[0176] Transformation and Regeneration
[0177] Plants were transformed and regenerated as described in
example 1. Transgenic plants were initially identified using a
neomycin phosphotransferase assay and subsequently confirmed by
expression of protein A fusions through immunoblot analysis.
[0178] Preparation of Oil Bodies
[0179] Oil bodies from the transgenic B. napus and B. carinata
lines expressing the oleosin-protein A fusion were prepared
following the procedure described in example 1.
[0180] Binding of Oleosin-Protein A Fusions to IgG
[0181] Oil body protein extracts (20 .mu.g/aliquot) from various
transgenic B. napus lines expressing oleosin-protein A fusion
proteins were subjected to polyacrylamide gelelectrophoresis and
subsequently transferred to a PVDF membrane following standard
procedures. The membrane was then probed with a HRP-conjugated
mouse anti-rabbit antibody and visualised following the procedure
as outlined in Antibodies, a laboratory manual (Harlow and Lane,
1988, Cold Spring Harbor). In FIG. 16 the stained PVDF membrane is
shown. A 50 kDa protein (predicted molecular mass of the
oleosin-protein A fusion protein: 48,801 Da) was specifically
detected in the protein extracts of all of the six transgenic B.
napus lines tested. Untransformed control plants did not exhibit
HRP activity, while the a 30 kDa protein (predicted molecular mass
29,652 Da) was present in a bacterial lysate transformed with
pRIT2T encoding protein A and undetectable in the untransformed
lysate.
[0182] Binding and Elution of IgGs to Oil Bodies Containing
Oleosin-Protein A Fusion Proteins
[0183] Washed oil bodies (10 mg/ml protein) were prepared from
wildtype B. napus and a transgenic B. napus line transformed with a
construct expressing an oleosin-protein A fusion protein as
described in example 1 and suspended in 10 mM Tris-Cl pH 8.0. A
volume of 2 .mu.l (+34 .mu.g) of HRP-conjugated rabbit anti-mouse
antibodies (Sigma, cat no A9044) was added to 500 .mu.l of the
washed oil body preparation and the suspension was incubated for 1
hr at room temperature or overnight at 4.degree. C. The samples
were then centrifuged for 15 min. at 16,000.times.g and the
undernatant was removed. Subsequently, the oil bodies were
thoroughly resuspended in 500 .mu.l 10 mM Tris-Cl pH 8.0 using a
pestle. This washing step in Tris-Cl was repeated 4 times
(henceforth termed washed oil body preparation). A 5 .mu.l aliquot
from the washed oil body preparation was washed a fifth time and
then assayed for HRP activity.
[0184] HRP assays were carried out by adding 1 .mu.l of the washed
oil body preparation to 1 ml of HRP assay mix (9.8 ml of 0.1 M
NaOAc, 0.2 ml of 2.5 mg/ml Trimethylbenzidine in DMSO, 4 .mu.l
H.sub.2O.sub.2) and incubating the mixture for 5 min at room
temperature. The reaction was then stopped by adding 0.5 ml 1M
H.sub.2SO.sub.4. The samples were filtered through a 0.22 .mu.m
filter and subsequently the OD.sub.450's were determined
spectrophotometrically.
[0185] In order to elute the IgGs from the oil bodies, the washed
oil body preparation was resuspended in 100 mM glycine pH 3.0 and
centrifuged for 15 min at 16,000.times.g and incubated for 30' at
room temperature. Following neutralization in 500 .mu.l 100 mM
Tris-Cl pH 8.0, both the oil body fraction and the eluate were
assayed for HRP activity as above. The binding and elution of IgGs
to oil bodies from weld type B. napus and transgenic B. napus
expressing an oleosin protein A fusion, are illustrated in FIG.
17.
[0186] 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.
Sequence CWU 1
1
14 1 522 DNA Arabidopsis Thaliana CDS (1)..(522) 1 atg gcg gat aca
gct aga gga acc cat cac gat atc atc ggc aga gac 48 Met Ala Asp Thr
Ala Arg Gly Thr His His Asp Ile Ile Gly Arg Asp 1 5 10 15 cag tac
ccg atg atg ggc cga gac cga gac cag tac cag atg tcc gga 96 Gln Tyr
Pro Met Met Gly Arg Asp Arg Asp Gln Tyr Gln Met Ser Gly 20 25 30
cga gga tct gac tac tcc aag tct agg cag att gct aaa gct gca act 144
Arg Gly Ser Asp Tyr Ser Lys Ser Arg Gln Ile Ala Lys Ala Ala Thr 35
40 45 gct gtc aca gct ggt ggt tcc ctc ctt gtt ctc tcc agc ctt acc
ctt 192 Ala Val Thr Ala Gly Gly Ser Leu Leu Val Leu Ser Ser Leu Thr
Leu 50 55 60 gtt gga act gtc ata gct ttg act gtt gca aca cct ctg
ctc gtt atc 240 Val Gly Thr Val Ile Ala Leu Thr Val Ala Thr Pro Leu
Leu Val Ile 65 70 75 80 ttc agc cca atc ctt gtc ccg gct ctc atc aca
gtt gca ctc ctc atc 288 Phe Ser Pro Ile Leu Val Pro Ala Leu Ile Thr
Val Ala Leu Leu Ile 85 90 95 acc ggt ttt ctt tcc tct gga ggg ttt
ggc att gcc gct ata acc gtt 336 Thr Gly Phe Leu Ser Ser Gly Gly Phe
Gly Ile Ala Ala Ile Thr Val 100 105 110 ttc tct tgg att tac aag tac
gca acg gga gag cac cca cag gga tca 384 Phe Ser Trp Ile Tyr Lys Tyr
Ala Thr Gly Glu His Pro Gln Gly Ser 115 120 125 gac aag ttg gac agt
gca agg atg aag ttg gga agc aaa gct cag gat 432 Asp Lys Leu Asp Ser
Ala Arg Met Lys Leu Gly Ser Lys Ala Gln Asp 130 135 140 ctg aaa gac
aga gct cag tac tac gga cag caa cat act ggt ggg gaa 480 Leu Lys Asp
Arg Ala Gln Tyr Tyr Gly Gln Gln His Thr Gly Gly Glu 145 150 155 160
cat gac cgt gac cgt act cgt ggt ggc cag cac act act taa 522 His Asp
Arg Asp Arg Thr Arg Gly Gly Gln His Thr Thr 165 170 2 173 PRT
Arabidopsis Thaliana 2 Met Ala Asp Thr Ala Arg Gly Thr His His Asp
Ile Ile Gly Arg Asp 1 5 10 15 Gln Tyr Pro Met Met Gly Arg Asp Arg
Asp Gln Tyr Gln Met Ser Gly 20 25 30 Arg Gly Ser Asp Tyr Ser Lys
Ser Arg Gln Ile Ala Lys Ala Ala Thr 35 40 45 Ala Val Thr Ala Gly
Gly Ser Leu Leu Val Leu Ser Ser Leu Thr Leu 50 55 60 Val Gly Thr
Val Ile Ala Leu Thr Val Ala Thr Pro Leu Leu Val Ile 65 70 75 80 Phe
Ser Pro Ile Leu Val Pro Ala Leu Ile Thr Val Ala Leu Leu Ile 85 90
95 Thr Gly Phe Leu Ser Ser Gly Gly Phe Gly Ile Ala Ala Ile Thr Val
100 105 110 Phe Ser Trp Ile Tyr Lys Tyr Ala Thr Gly Glu His Pro Gln
Gly Ser 115 120 125 Asp Lys Leu Asp Ser Ala Arg Met Lys Leu Gly Ser
Lys Ala Gln Asp 130 135 140 Leu Lys Asp Arg Ala Gln Tyr Tyr Gly Gln
Gln His Thr Gly Gly Glu 145 150 155 160 His Asp Arg Asp Arg Thr Arg
Gly Gly Gln His Thr Thr 165 170 3 2115 DNA Artificial Sequence
Oleosin - Hirudin Fusion 3 ctatacccaa cctcggtctt ggtcacacca
ggaactctct ggtaagctag ctccactccc 60 cagaaacaac cggcgccaaa
ttgccggaat tgctgacctg aagacggaac atcatcgtcg 120 ggtccttggg
cgattgcggc ggaagatggg tcagcttggg cttgaggacg agacccgaat 180
cgagtctgtt gaaaggttgt tcattgggat ttgtatacgg agattggtcg tcgagaggtt
240 tgagggaaag gacaaatggg tttggctctg gagaaagaga gtgcggcttt
agagagagaa 300 ttgagaggtt tagagagaga tgcggcggcg atgacgggag
gagagacgac gaggacctgc 360 attatcaaag cagtgacgtg gtgaaatttg
gaacttttaa gaggcagata gatttattat 420 ttgtatccat tttcttcatt
gttctagaat gtcgcggaac aaattttaaa actaaatcct 480 aaatttttct
aattttgttg ccaatagtgg atatgtgggc cgtatagaag gaatctattg 540
aaggcccaaa cccatactga cgagcccaaa ggttcgtttt gcgttttatg tttcggttcg
600 atgccaacgc cacattctga gctaggcaaa aaacaaacgt gtctttgaat
agactcctct 660 cgttaacaca tgcagcggct gcatggtgac gccattaaca
cgtggcctac aattgcatga 720 tgtctccatt gacacgtgac ttctcgtctc
ctttcttaat atatctaaca aacactccta 780 cctcttccaa aatatataca
catctttttg atcaatctct cattcaaaat ctcattctct 840 ctagtaaaca
agaacaaaaa a atg gcg gat aca gct aga gga acc cat cac 891 Met Ala
Asp Thr Ala Arg Gly Thr His His 1 5 10 gat atc atc ggc aga gac cag
tac ccg atg atg ggc cga gac cga gac 939 Asp Ile Ile Gly Arg Asp Gln
Tyr Pro Met Met Gly Arg Asp Arg Asp 15 20 25 cag tac cag atg tcc
gga cga gga tct gac tac tcc aag tct agg cag 987 Gln Tyr Gln Met Ser
Gly Arg Gly Ser Asp Tyr Ser Lys Ser Arg Gln 30 35 40 att gct aaa
gct gca act gct gtc aca gct ggt ggt tcc ctc ctt gtt 1035 Ile Ala
Lys Ala Ala Thr Ala Val Thr Ala Gly Gly Ser Leu Leu Val 45 50 55
ctc tcc agc ctt acc ctt gtt gga act gtc ata gct ttg act gtt gca
1083 Leu Ser Ser Leu Thr Leu Val Gly Thr Val Ile Ala Leu Thr Val
Ala 60 65 70 aca cct ctg ctc gtt atc ttc agc cca atc ctt gtc ccg
gct ctc atc 1131 Thr Pro Leu Leu Val Ile Phe Ser Pro Ile Leu Val
Pro Ala Leu Ile 75 80 85 90 aca gtt gca ctc ctc atc acc ggt ttt ctt
tcc tct gga ggg ttt ggc 1179 Thr Val Ala Leu Leu Ile Thr Gly Phe
Leu Ser Ser Gly Gly Phe Gly 95 100 105 att gcc gct ata acc gtt ttc
tct tgg att tac aag taagcacaca 1225 Ile Ala Ala Ile Thr Val Phe Ser
Trp Ile Tyr Lys 110 115 tttatcatct tacttcataa ttttgtgcaa tatgtgcatg
catgtgttga gccagtagct 1285 ttggatcaat ttttttggtc gaataacaaa
tgtaacaata agaaattgca aattctaggg 1345 aacatttggt taactaaata
cgaaatttga cctagctagc ttgaatgtgt ctgtgtatat 1405 catctatata
ggtaaaatgc ttggtatgat acctattgat tgtgaatagg tac gca 1461 Tyr Ala
120 acg gga gag cac cca cag gga tca gac aag ttg gac agt gca agg atg
1509 Thr Gly Glu His Pro Gln Gly Ser Asp Lys Leu Asp Ser Ala Arg
Met 125 130 135 aag ttg gga agc aaa gct cag gat ctg aaa gac aga gct
cag tac tac 1557 Lys Leu Gly Ser Lys Ala Gln Asp Leu Lys Asp Arg
Ala Gln Tyr Tyr 140 145 150 gga cag caa cat act ggt tgg gaa cat gac
cgt gac cgt act cgt ggt 1605 Gly Gln Gln His Thr Gly Trp Glu His
Asp Arg Asp Arg Thr Arg Gly 155 160 165 ggc cag cac act act gcg atc
gaa ggg aga atc act tac act gac tgt 1653 Gly Gln His Thr Thr Ala
Ile Glu Gly Arg Ile Thr Tyr Thr Asp Cys 170 175 180 act gaa tct gga
cag aac ctc tgt ctc tgt gaa gga tct aac gtt tgt 1701 Thr Glu Ser
Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys 185 190 195 200
gga aag gga aac aag tgt atc ctc gga tct aac gga aag gga aac cag
1749 Gly Lys Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Lys Gly Asn
Gln 205 210 215 tgt gtt act gga gaa gga act cca aac cca gaa tct cac
aac aac gga 1797 Cys Val Thr Gly Glu Gly Thr Pro Asn Pro Glu Ser
His Asn Asn Gly 220 225 230 gac ttc gaa gaa atc cct gaa gaa tac ctc
cag taa gtcgactcta 1843 Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln
235 240 gacggatctc ccgatcgttc aaacatttgg caataaagtt tcttaagatt
gaatcctgtt 1903 gccggtcttg cgatgattat catataattt ctgttgaatt
acgttaagca tgtaataatt 1963 aacatgtaat gcatgacgtt atttatgaga
tgggttttta tgattagagt cccgcaatta 2023 tacatttaat acgcgataga
aaacaaaata tagcgcgcaa actaggataa attatcgcgc 2083 gcggtgtcat
ctatgttact agatcggaat tc 2115 4 118 PRT Artificial Sequence Oleosin
- Hirudin Fusion 4 Met Ala Asp Thr Ala Arg Gly Thr His His Asp Ile
Ile Gly Arg Asp 1 5 10 15 Gln Tyr Pro Met Met Gly Arg Asp Arg Asp
Gln Tyr Gln Met Ser Gly 20 25 30 Arg Gly Ser Asp Tyr Ser Lys Ser
Arg Gln Ile Ala Lys Ala Ala Thr 35 40 45 Ala Val Thr Ala Gly Gly
Ser Leu Leu Val Leu Ser Ser Leu Thr Leu 50 55 60 Val Gly Thr Val
Ile Ala Leu Thr Val Ala Thr Pro Leu Leu Val Ile 65 70 75 80 Phe Ser
Pro Ile Leu Val Pro Ala Leu Ile Thr Val Ala Leu Leu Ile 85 90 95
Thr Gly Phe Leu Ser Ser Gly Gly Phe Gly Ile Ala Ala Ile Thr Val 100
105 110 Phe Ser Trp Ile Tyr Lys 115 5 125 PRT Artificial Sequence
Oleosin - Hirudin Fusion 5 Tyr Ala Thr Gly Glu His Pro Gln Gly Ser
Asp Lys Leu Asp Ser Ala 1 5 10 15 Arg Met Lys Leu Gly Ser Lys Ala
Gln Asp Leu Lys Asp Arg Ala Gln 20 25 30 Tyr Tyr Gly Gln Gln His
Thr Gly Trp Glu His Asp Arg Asp Arg Thr 35 40 45 Arg Gly Gly Gln
His Thr Thr Ala Ile Glu Gly Arg Ile Thr Tyr Thr 50 55 60 Asp Cys
Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn 65 70 75 80
Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Lys Gly 85
90 95 Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Asn Pro Glu Ser His
Asn 100 105 110 Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln
115 120 125 6 2366 DNA Artificial Sequence Oleosin -
Metallothionein Fusion 6 gagctcaaat acgatctgat actgataacg
tctagatttt tagggttaaa gcaatcaatc 60 acctgacgat tcaaggtggt
tggatcatga cgattccaga aaacatcaag caagctctca 120 aagctacact
ctttgggatc atactgaact ctaacaacct cgttatgtcc cgtagtgcca 180
gtacagacat cctcgtaact cggattatgc acgatgccat ggctataccc aacctcggtc
240 ttggtcacac caggaactct ctggtaagct agctccactc cccagaaaca
accggcgcca 300 aattgccgga attgctgacc tgaagacgga acatcatcgt
cgggtccttg ggcgattgcg 360 gcggaagatg ggtcagcttg ggcttgagga
cgagacccga atcgagtctg ttgaaaggtt 420 gttcattggg atttgtatac
ggagattggt cgtcgagagg tttgagggaa aggacaaatg 480 ggtttggctc
tggagaaaga gagtgcggct ttagagagag aattgagagg tttagagaga 540
gatgcggcgg cgatgacggg aggagagacg acgaggacct gcattatcaa agcagtgacg
600 tggtgaaatt tggaactttt aagaggcaga tagatttatt atttgtatcc
attttcttca 660 ttgttctaga atgtcgcgga acaaatttta aaactaaatc
ctaaattttt ctaattttgt 720 tgccaatagt ggatatgtgg gccgtataga
aggaatctat tgaaggccca aacccatact 780 gacgagccca aaggttcgtt
ttgcgtttta tgtttcggtt cgatgccaac gccacattct 840 gagctaggca
aaaaacaaac gtgtctttga atagactcct ctcgttaaca catgcagcgg 900
ctgcatggtg acgccattaa cacgtggcct acaattgcat gatgtctcca ttgacacgtg
960 acttctcgtc tcctttctta atatatctaa caaacactcc tacctcttcc
aaaatatata 1020 cacatctttt tgatcaatct ctcattcaaa atctcattct
ctctagtaaa caggatcccc 1080 ctcgcggccg c atg gcg gat aca gct aga acc
cat cac gat gtc aca agt 1130 Met Ala Asp Thr Ala Arg Thr His His
Asp Val Thr Ser 1 5 10 cga gat cag tat ccc cga gac cga gac cag tat
tct atg atc ggt cga 1178 Arg Asp Gln Tyr Pro Arg Asp Arg Asp Gln
Tyr Ser Met Ile Gly Arg 15 20 25 gac cgt gac cag tac tct atg atg
ggc cga gac cga gac cag tac aac 1226 Asp Arg Asp Gln Tyr Ser Met
Met Gly Arg Asp Arg Asp Gln Tyr Asn 30 35 40 45 atg tat ggt cga gac
tac tcc aag tct aga cag att gct aag gct gtt 1274 Met Tyr Gly Arg
Asp Tyr Ser Lys Ser Arg Gln Ile Ala Lys Ala Val 50 55 60 acc gca
gtc acg gcg ggt ggg tcc ctc ctt gtc ctc tcc agt ctc acc 1322 Thr
Ala Val Thr Ala Gly Gly Ser Leu Leu Val Leu Ser Ser Leu Thr 65 70
75 ctt gtt ggt act gtc att gct ttg act gtt gcc act cca ctc ctc gtt
1370 Leu Val Gly Thr Val Ile Ala Leu Thr Val Ala Thr Pro Leu Leu
Val 80 85 90 atc ttt agc cca atc ctc gtg ccg gct ctc atc acc gta
gca ctt ctc 1418 Ile Phe Ser Pro Ile Leu Val Pro Ala Leu Ile Thr
Val Ala Leu Leu 95 100 105 atc act ggc ttt ctc tcc tct ggt ggg ttt
gcc att gca gct ata acc 1466 Ile Thr Gly Phe Leu Ser Ser Gly Gly
Phe Ala Ile Ala Ala Ile Thr 110 115 120 125 gtc ttc tcc tgg atc tat
aag tac gca acg gga gag cac cca cag ggg 1514 Val Phe Ser Trp Ile
Tyr Lys Tyr Ala Thr Gly Glu His Pro Gln Gly 130 135 140 tca gat aag
ttg gac agt gca agg atg aag ctg gga acc aaa gct cag 1562 Ser Asp
Lys Leu Asp Ser Ala Arg Met Lys Leu Gly Thr Lys Ala Gln 145 150 155
gat att aaa gac aga gct caa tac tac gga cag caa cat aca ggt ggt
1610 Asp Ile Lys Asp Arg Ala Gln Tyr Tyr Gly Gln Gln His Thr Gly
Gly 160 165 170 gag cat gac cgt gac cgt act cgt ggt ggc cag cac act
act ctc gtt 1658 Glu His Asp Arg Asp Arg Thr Arg Gly Gly Gln His
Thr Thr Leu Val 175 180 185 cca cga gga tcc atg gat ccc aac tgc tcc
tgt gcc gcc agt gac tcc 1706 Pro Arg Gly Ser Met Asp Pro Asn Cys
Ser Cys Ala Ala Ser Asp Ser 190 195 200 205 tgc acc tgc gcc ggc tcc
tgc aag tgc aaa gag tgc aaa tgc acc tcc 1754 Cys Thr Cys Ala Gly
Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser 210 215 220 tgc aag aaa
agc tgc tgc tcc tgc tgt cct gtg ggc tgt gcc aag tgt 1802 Cys Lys
Lys Ser Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys 225 230 235
gcc cag ggc tgc atc tgc aaa ggg gcg tcg gac aag tgc agc tgc tgt
1850 Ala Gln Gly Cys Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys
Cys 240 245 250 gcc tga gcggccgcga gggctgcaga atgagttcca agatggtttg
tgacgaagtt 1906 Ala agttggttgt ttttatggaa ctttgtttaa gcttgtaatg
tggaaagaac gtgtggcttt 1966 gtggttttta aatgttggtg aataaagatg
tttcctttgg attaactagt atttttccta 2026 ttggtttcat ggttttagca
cacaacattt taaatatgct gttagatgat atgctgcctg 2086 ctttattatt
tacttacccc tcaccttcag tttcaaagtt gttgcaatga ctctgtgtag 2146
tttaagatcg agtgaaagta gattttgtct atatttatta ggggtatttg atatgctaat
2206 ggtaaacatg gtttatgaca gcgtactttt ttggttatgg tgttgacgtt
tccttttaaa 2266 cattatagta gcgtccttgg tctgtgttca ttggttgaac
aaaggcacac tcacttggag 2326 atgccgtctc cactgatatt tgaacaaaga
attcggtacc 2366 7 254 PRT Artificial Sequence Oleosin -
Metallothionein Fusion 7 Met Ala Asp Thr Ala Arg Thr His His Asp
Val Thr Ser Arg Asp Gln 1 5 10 15 Tyr Pro Arg Asp Arg Asp Gln Tyr
Ser Met Ile Gly Arg Asp Arg Asp 20 25 30 Gln Tyr Ser Met Met Gly
Arg Asp Arg Asp Gln Tyr Asn Met Tyr Gly 35 40 45 Arg Asp Tyr Ser
Lys Ser Arg Gln Ile Ala Lys Ala Val Thr Ala Val 50 55 60 Thr Ala
Gly Gly Ser Leu Leu Val Leu Ser Ser Leu Thr Leu Val Gly 65 70 75 80
Thr Val Ile Ala Leu Thr Val Ala Thr Pro Leu Leu Val Ile Phe Ser 85
90 95 Pro Ile Leu Val Pro Ala Leu Ile Thr Val Ala Leu Leu Ile Thr
Gly 100 105 110 Phe Leu Ser Ser Gly Gly Phe Ala Ile Ala Ala Ile Thr
Val Phe Ser 115 120 125 Trp Ile Tyr Lys Tyr Ala Thr Gly Glu His Pro
Gln Gly Ser Asp Lys 130 135 140 Leu Asp Ser Ala Arg Met Lys Leu Gly
Thr Lys Ala Gln Asp Ile Lys 145 150 155 160 Asp Arg Ala Gln Tyr Tyr
Gly Gln Gln His Thr Gly Gly Glu His Asp 165 170 175 Arg Asp Arg Thr
Arg Gly Gly Gln His Thr Thr Leu Val Pro Arg Gly 180 185 190 Ser Met
Asp Pro Asn Cys Ser Cys Ala Ala Ser Asp Ser Cys Thr Cys 195 200 205
Ala Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys 210
215 220 Ser Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln
Gly 225 230 235 240 Cys Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys
Cys Ala 245 250 8 804 DNA Artificial Sequence Protein A Primers 8
ctcc atg gat caa cgc aat ggt ttt atc caa agc ctt aaa gat gat cca 49
Met Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro 1 5 10
15 agc caa agt gct aac gtt tta ggt gaa gct caa aaa ctt aat gac tct
97 Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser
20 25 30 caa gct cca aaa gct gat gcg caa caa aat aac ttc aac aaa
gat caa 145 Gln Ala Pro Lys Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys
Asp Gln 35 40 45 caa agc gcc ttc tat gaa atc ttg aac atg cct aac
tta aac gaa gcg 193 Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn
Leu Asn Glu Ala 50 55 60 caa cgt aac ggc ttc att caa agt ctt aaa
gac gac cca agc caa agc 241 Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
Asp Asp Pro Ser
Gln Ser 65 70 75 act aac gtt tta ggt gaa gct aaa aaa tta aac gaa
tct caa gca ccg 289 Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu
Ser Gln Ala Pro 80 85 90 95 aaa gct gat aac aat ttc aac aaa gaa caa
caa aat gct ttc tat gaa 337 Lys Ala Asp Asn Asn Phe Asn Lys Glu Gln
Gln Asn Ala Phe Tyr Glu 100 105 110 atc ttg aat atg cct aac tta aac
gaa gaa caa cgc aat ggt ttc atc 385 Ile Leu Asn Met Pro Asn Leu Asn
Glu Glu Gln Arg Asn Gly Phe Ile 115 120 125 caa agc tta aaa gat gac
cca agc caa agt gct aac cta ttg tca gaa 433 Gln Ser Leu Lys Asp Asp
Pro Ser Gln Ser Ala Asn Leu Leu Ser Glu 130 135 140 gct aaa aag tta
aat gaa tct caa gca ccg aaa gcg gat aac aaa ttc 481 Ala Lys Lys Leu
Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Lys Phe 145 150 155 aac aaa
gaa caa caa aat gct ttc tat gaa atc tta cat tta cct aac 529 Asn Lys
Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn 160 165 170
175 tta aac gaa gaa caa cgc aat ggt ttc atc caa agc cta aaa gat gac
577 Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp
180 185 190 cca agc caa agc gct aac ctt tta gca gaa gct aaa aag cta
aat gat 625 Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu
Asn Asp 195 200 205 gct caa gca cca aaa gct gac aac aaa ttc aac aaa
gaa caa caa aat 673 Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys
Glu Gln Gln Asn 210 215 220 gct ttc tat gaa att tta cat tta cct aac
tta act gaa gaa caa cgt 721 Ala Phe Tyr Glu Ile Leu His Leu Pro Asn
Leu Thr Glu Glu Gln Arg 225 230 235 aac ggc ttc atc caa agc ctt aaa
gac gat ccg ggg aat tcc cgg gga 769 Asn Gly Phe Ile Gln Ser Leu Lys
Asp Asp Pro Gly Asn Ser Arg Gly 240 245 250 255 tcc gtc gac ctg cag
ata aca aat tag aagcttgc 804 Ser Val Asp Leu Gln Ile Thr Asn 260 9
263 PRT Artificial Sequence Protein A Primers 9 Met Asp Gln Arg Asn
Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser 1 5 10 15 Gln Ser Ala
Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln 20 25 30 Ala
Pro Lys Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln 35 40
45 Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln
50 55 60 Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln
Ser Thr 65 70 75 80 Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser
Gln Ala Pro Lys 85 90 95 Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln
Asn Ala Phe Tyr Glu Ile 100 105 110 Leu Asn Met Pro Asn Leu Asn Glu
Glu Gln Arg Asn Gly Phe Ile Gln 115 120 125 Ser Leu Lys Asp Asp Pro
Ser Gln Ser Ala Asn Leu Leu Ser Glu Ala 130 135 140 Lys Lys Leu Asn
Glu Ser Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn 145 150 155 160 Lys
Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu 165 170
175 Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro
180 185 190 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn
Asp Ala 195 200 205 Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu
Gln Gln Asn Ala 210 215 220 Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
Thr Glu Glu Gln Arg Asn 225 230 235 240 Gly Phe Ile Gln Ser Leu Lys
Asp Asp Pro Gly Asn Ser Arg Gly Ser 245 250 255 Val Asp Leu Gln Ile
Thr Asn 260 10 26 DNA Artificial Sequence Primer Bk 266 10
tccatggatc aacgcaatgg tttatc 26 11 29 DNA Artificial Sequence
Primer Bk 267 11 gcaagcttct aatttgttat ctgcaggtc 29 12 2709 DNA
Artificial Sequence Oleosin - Protein A Fusion 12 ccatggctat
acccaacctc ggtcttggtc acaccaggaa ctctctggta agctagctcc 60
actccccaga aacaaccggc gccaaattgc cggaattgct gacctgaaga cggaacatca
120 tcgtcgggtc cttgggcgat tgcggcggaa gatgggtcag cttgggcttg
aggacgagac 180 ccgaatcgag tctgttgaaa ggttgttcat tgggatttgt
atacggagat tggtcgtcga 240 gaggtttgag ggaaaggaca aatgggtttg
gctctggaga aagagagtgc ggctttagag 300 agagaattga gaggtttaga
gagagatgcg gcggcgatga cgggaggaga gacgacgagg 360 acctgcatta
tcaaagcagt gacgtggtga aatttggaac ttttaagagg cagatagatt 420
tattatttgt atccattttc ttcattgttc tagaatgtcg cggaacaaat tttaaaacta
480 aatcctaaat ttttctaatt ttgttgccaa tagtggatat gtgggccgta
tagaaggaat 540 ctattgaagg cccaaaccca tactgacgag cccaaaggtt
cgttttgcgt tttatgtttc 600 ggttcgatgc caacgccaca ttctgagcta
ggcaaaaaac aaacgtgtct ttgaatagac 660 tcctctcgtt aacacatgca
gcggctgcat ggtgacgcca ttaacacgtg gcctacaatt 720 gcatgatgtc
tccattgaca cgtgacttct cgtctccttt cttaatatat ctaacaaaca 780
ctcctacctc ttccaaaata tatacacatc tttttgatca atctctcatt caaaatctca
840 ttctctctag taaacaagaa caaaaaa atg gcg gat aca gct aga gga acc
cat 894 Met Ala Asp Thr Ala Arg Gly Thr His 1 5 cac gat atc atc ggc
aga gac cag tac ccg atg atg ggc cga gac cga 942 His Asp Ile Ile Gly
Arg Asp Gln Tyr Pro Met Met Gly Arg Asp Arg 10 15 20 25 gac cag tac
cag atg tcc gga cga gga tct gac tac tcc aag tct agg 990 Asp Gln Tyr
Gln Met Ser Gly Arg Gly Ser Asp Tyr Ser Lys Ser Arg 30 35 40 cag
att gct aaa gct gca act gct gtc aca gct ggt ggt tcc ctc ctt 1038
Gln Ile Ala Lys Ala Ala Thr Ala Val Thr Ala Gly Gly Ser Leu Leu 45
50 55 gtt ctc tcc agc ctt acc ctt gtt gga act gtc ata gct ttg act
gtt 1086 Val Leu Ser Ser Leu Thr Leu Val Gly Thr Val Ile Ala Leu
Thr Val 60 65 70 gca aca cct ctg ctc gtt atc ttc agc cca atc ctt
gtc ccg gct ctc 1134 Ala Thr Pro Leu Leu Val Ile Phe Ser Pro Ile
Leu Val Pro Ala Leu 75 80 85 atc aca gtt gca ctc ctc atc acc ggt
ttt ctt tcc tct gga ggg ttt 1182 Ile Thr Val Ala Leu Leu Ile Thr
Gly Phe Leu Ser Ser Gly Gly Phe 90 95 100 105 ggc att gcc gct ata
acc gtt ttc tct tgg att tac aagtaagcac 1228 Gly Ile Ala Ala Ile Thr
Val Phe Ser Trp Ile Tyr 110 115 acatttatca tcttacttca taattttgtg
caatatgtgc atgcatgtgt tgagccagta 1288 gctttggatc aatttttttg
gtcgaataac aaatgtaaca ataagaaatt gcaaattcta 1348 gggaacattt
ggttaactaa atacgaaatt tgacctagct agcttgaatg tgtctgtgta 1408
tatcatctat ataggtaaaa tgcttggtat gatacctatt gattgtgaat agg tac 1464
Tyr gca acg gga gag cac cca cag gga tca gac aag ttg gac agt gca agg
1512 Ala Thr Gly Glu His Pro Gln Gly Ser Asp Lys Leu Asp Ser Ala
Arg 120 125 130 atg aag ttg gga agc aaa gct cag gat ctg aaa gac aga
gct cag tac 1560 Met Lys Leu Gly Ser Lys Ala Gln Asp Leu Lys Asp
Arg Ala Gln Tyr 135 140 145 150 tac gga cag caa cat act ggt ggg gaa
cat gac cgt gac cgt act cgt 1608 Tyr Gly Gln Gln His Thr Gly Gly
Glu His Asp Arg Asp Arg Thr Arg 155 160 165 ggt ggc cag cac act act
ctc gtt cca cga gga tcc atg gat caa cgc 1656 Gly Gly Gln His Thr
Thr Leu Val Pro Arg Gly Ser Met Asp Gln Arg 170 175 180 aat ggt ttt
atc caa agc ctt aaa gat gat cca agc caa agt gct aac 1704 Asn Gly
Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn 185 190 195
gtt tta ggt gaa gct caa aaa ctt aat gac tct caa gct cca aaa gct
1752 Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys
Ala 200 205 210 gat gcg caa caa aat aac ttc aac aaa gat caa caa agc
gcc ttc tat 1800 Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln
Ser Ala Phe Tyr 215 220 225 230 gaa atc ttg aac atg cct aac tta aac
gaa gcg caa cgt aac ggc ttc 1848 Glu Ile Leu Asn Met Pro Asn Leu
Asn Glu Ala Gln Arg Asn Gly Phe 235 240 245 att caa agt ctt aaa gac
gac cca agc caa agc act aac gtt tta ggt 1896 Ile Gln Ser Leu Lys
Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly 250 255 260 gaa gct aaa
aaa tta aac gaa tct caa gca ccg aaa gct gat aac aat 1944 Glu Ala
Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn 265 270 275
ttc aac aaa gaa caa caa aat gct ttc tat gaa atc ttg aat atg cct
1992 Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met
Pro 280 285 290 aac tta aac gaa gaa caa cgc aat ggt ttc atc caa agc
tta aaa gat 2040 Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln
Ser Leu Lys Asp 295 300 305 310 gac cca agc caa agt gct aac cta ttg
tca gaa gct aaa aag tta aat 2088 Asp Pro Ser Gln Ser Ala Asn Leu
Leu Ser Glu Ala Lys Lys Leu Asn 315 320 325 gaa tct caa gca ccg aaa
gcg gat aac aaa ttc aac aaa gaa caa caa 2136 Glu Ser Gln Ala Pro
Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln 330 335 340 aat gct ttc
tat gaa atc tta cat tta cct aac tta aac gaa gaa caa 2184 Asn Ala
Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln 345 350 355
cgc aat ggt ttc atc caa agc cta aaa gat gac cca agc caa agc gct
2232 Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser
Ala 360 365 370 aac ctt tta gca gaa gct aaa aag cta aat gat gct caa
gca cca aaa 2280 Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
Gln Ala Pro Lys 375 380 385 390 gct gac aac aaa ttc aac aaa gaa caa
caa aat gct ttc tat gaa att 2328 Ala Asp Asn Lys Phe Asn Lys Glu
Gln Gln Asn Ala Phe Tyr Glu Ile 395 400 405 tta cat tta cct aac tta
act gaa gaa caa cgt aac ggc ttc atc caa 2376 Leu His Leu Pro Asn
Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln 410 415 420 agc ctt aaa
gac gat ccg ggg aat tcc cgg gga tcc gtc gac ctg cag 2424 Ser Leu
Lys Asp Asp Pro Gly Asn Ser Arg Gly Ser Val Asp Leu Gln 425 430 435
ata aca aat tagaagcttg catgcctgca ggtcgatcgt tcaaacattt 2473 Ile
Thr Asn 440 ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt
atcatataat 2533 ttctgttgaa ttacgttaag catgtaataa ttaacatgta
atgcatgacg ttatttatga 2593 gatgggtttt tatgattaga gtcccgcaat
tatacattta atacgcgata gaaaacaaaa 2653 tatagcgcgc aaactaggat
aaattatcgc gcgcggtgtc atctatgtta ctagat 2709 13 117 PRT Artificial
Sequence Oleosin - Protein A Fusion 13 Met Ala Asp Thr Ala Arg Gly
Thr His His Asp Ile Ile Gly Arg Asp 1 5 10 15 Gln Tyr Pro Met Met
Gly Arg Asp Arg Asp Gln Tyr Gln Met Ser Gly 20 25 30 Arg Gly Ser
Asp Tyr Ser Lys Ser Arg Gln Ile Ala Lys Ala Ala Thr 35 40 45 Ala
Val Thr Ala Gly Gly Ser Leu Leu Val Leu Ser Ser Leu Thr Leu 50 55
60 Val Gly Thr Val Ile Ala Leu Thr Val Ala Thr Pro Leu Leu Val Ile
65 70 75 80 Phe Ser Pro Ile Leu Val Pro Ala Leu Ile Thr Val Ala Leu
Leu Ile 85 90 95 Thr Gly Phe Leu Ser Ser Gly Gly Phe Gly Ile Ala
Ala Ile Thr Val 100 105 110 Phe Ser Trp Ile Tyr 115 14 324 PRT
Artificial Sequence Oleosin - Protein A Fusion 14 Tyr Ala Thr Gly
Glu His Pro Gln Gly Ser Asp Lys Leu Asp Ser Ala 1 5 10 15 Arg Met
Lys Leu Gly Ser Lys Ala Gln Asp Leu Lys Asp Arg Ala Gln 20 25 30
Tyr Tyr Gly Gln Gln His Thr Gly Gly Glu His Asp Arg Asp Arg Thr 35
40 45 Arg Gly Gly Gln His Thr Thr Leu Val Pro Arg Gly Ser Met Asp
Gln 50 55 60 Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser
Gln Ser Ala 65 70 75 80 Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp
Ser Gln Ala Pro Lys 85 90 95 Ala Asp Ala Gln Gln Asn Asn Phe Asn
Lys Asp Gln Gln Ser Ala Phe 100 105 110 Tyr Glu Ile Leu Asn Met Pro
Asn Leu Asn Glu Ala Gln Arg Asn Gly 115 120 125 Phe Ile Gln Ser Leu
Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu 130 135 140 Gly Glu Ala
Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn 145 150 155 160
Asn Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met 165
170 175 Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu
Lys 180 185 190 Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ser Glu Ala
Lys Lys Leu 195 200 205 Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Lys
Phe Asn Lys Glu Gln 210 215 220 Gln Asn Ala Phe Tyr Glu Ile Leu His
Leu Pro Asn Leu Asn Glu Glu 225 230 235 240 Gln Arg Asn Gly Phe Ile
Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser 245 250 255 Ala Asn Leu Leu
Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro 260 265 270 Lys Ala
Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu 275 280 285
Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile 290
295 300 Gln Ser Leu Lys Asp Asp Pro Gly Asn Ser Arg Gly Ser Val Asp
Leu 305 310 315 320 Gln Ile Thr Asn
* * * * *