U.S. patent application number 13/166579 was filed with the patent office on 2012-01-26 for production of biologically active proteins.
This patent application is currently assigned to ERA BIOTECH, S.A.. Invention is credited to Miriam BASTIDA VIRGILI, Roser Pallisse BERGWERF, Peter Bernard HEIFETZ, Blanca LLOMPART ROYO, M Immaculada LLOP TOUS, M Dolores LUDEVID M GICA, Pablo MARZ BAL LUNA, Kevin James O'CONNER, Margarita TORRENT QUETGLAS.
Application Number | 20120020992 13/166579 |
Document ID | / |
Family ID | 38134862 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120020992 |
Kind Code |
A1 |
HEIFETZ; Peter Bernard ; et
al. |
January 26, 2012 |
Production of Biologically Active Proteins
Abstract
A fusion protein that is expressed in a recombinant protein
body-like assembly (RPBLA) in host eukaryotic cells and organisms
is disclosed. More particularly, a biologically active polypeptide
fused to a protein sequence that mediates the induction of RPBLA
formation is expressed and accumulated in host cells after
transformation with an appropriate vector. The eukaryotic host cell
does not produce protein bodies in the absence of the fusion
protein. Methods for preparing and using the RPBLAs and the fusion
protein are also disclosed, as are nucleic acid molecules that
encode the fusion proteins.
Inventors: |
HEIFETZ; Peter Bernard; (San
Diego, CA) ; LLOMPART ROYO; Blanca; (Barcelona,
ES) ; MARZ BAL LUNA; Pablo; (Barcelona, ES) ;
BASTIDA VIRGILI; Miriam; (Molins de Rei, ES) ;
LUDEVID M GICA; M Dolores; (Sant Just Desvern, ES) ;
TORRENT QUETGLAS; Margarita; (Barcelona, ES) ;
O'CONNER; Kevin James; (El Prat de Llobregat, ES) ;
BERGWERF; Roser Pallisse; (Valldoreix, ES) ; LLOP
TOUS; M Immaculada; (St. Feliu de Llobregat, ES) |
Assignee: |
ERA BIOTECH, S.A.
BARCELONA
ES
|
Family ID: |
38134862 |
Appl. No.: |
13/166579 |
Filed: |
June 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11709527 |
Feb 22, 2007 |
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13166579 |
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60776391 |
Feb 23, 2006 |
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Current U.S.
Class: |
424/192.1 ;
435/375; 530/350 |
Current CPC
Class: |
C12N 9/6475 20130101;
A61K 9/0053 20130101; A61K 9/0019 20130101; C07K 14/43504 20130101;
C12N 9/6424 20130101; C12N 15/62 20130101; C07K 14/425 20130101;
A61P 37/04 20180101; C12N 2799/026 20130101; C07K 14/61 20130101;
C07K 14/415 20130101; C07K 14/485 20130101; A61K 39/00 20130101;
C12N 15/8257 20130101; C12Y 304/21009 20130101; C12P 21/02
20130101 |
Class at
Publication: |
424/192.1 ;
530/350; 435/375 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 37/04 20060101 A61P037/04; C12N 5/0783 20100101
C12N005/0783; C12N 5/0786 20100101 C12N005/0786; C07K 14/00
20060101 C07K014/00; C12N 5/0781 20100101 C12N005/0781 |
Claims
1-15. (canceled)
16. A recombinant protein body-like assembly (RPBLA) that comprises
a membrane-enclosed fusion protein, said fusion protein comprising
two sequences linked together in which one sequence is a protein
body-inducing sequence (PBIS) and the other is a biologically
active polypeptide.
17-26. (canceled)
27. A vaccine or inoculum comprising an immunogenic effective
amount of recombinant protein body-like assemblies (RPBLAs) that
comprise a recombinant fusion protein dissolved or dispersed in a
pharmaceutically acceptable diluent, said recombinant fusion
protein comprising two sequences linked together in which one
sequence is a protein body-inducing sequence (PBIS) and the other
is an immunogenic polypeptide to which an immunological response is
to be induced by said vaccine or inoculum.
28. The vaccine or inoculum according to claim 27 wherein said
fusion protein further includes a linker sequence between the
protein body-inducing sequence and the sequence of the immunogenic
polypeptide.
29. The vaccine or inoculum according to claim 27 wherein the PBIS
comprises a prolamin sequence.
30. The vaccine or inoculum according to claim 29 wherein the
prolamin sequence is gamma-zein, alpha-zein, gamma-gliadin, or rice
prolamin.
31. The vaccine or inoculum according to claim 30 wherein the
prolamin sequence is the gamma-zein RX3 sequence.
32. The vaccine or inoculum according to claim 27 wherein said
RPBLAs improve antigen delivery to antigen-presenting cells.
33. The vaccine or inoculum according to claim 27 wherein said
RPBLAs improve antigen processing and presentation to
antigen-presenting cells.
34. The vaccine or inoculum according to claim 27 wherein the
vaccine or inoculum is for oral delivery.
35. The vaccine or inoculum according to claim 27 wherein the
vaccine or inoculum is for parenteral delivery.
36. An immunogenic composition comprising a recombinant protein
body-like assembly (RBPLA) or a membrane-less RPBLA and a
pharmaceutically acceptable diluent.
37. The immunogenic composition of claim 36, wherein the RPBLA or
the membrane-less RPBLA comprises a recombinant fusion protein, and
the recombinant fusion protein comprises a protein body-inducing
sequence and an adjuvant.
38. The immunogenic composition of claim 36, wherein the RPBLA or
the membrane-less RPBLA comprises a recombinant fusion protein, and
the recombinant fusion protein comprises a protein body-inducing
sequence- and an antigen.
39. A method of making the immunogenic composition of claim 36
comprising dissolving or dispersing an RPBLA or a membrane-less
RPBLA in a pharmaceutically acceptable diluent.
40. A method of inoculating an animal comprising administering the
immunogenic composition of claim 36 to the animal.
41. The method of claim 36, further comprising administering an
antigen.
42. A method of stimulating a B-cell or T-cell comprising
contacting the cell with the RPBLA of claim 16.
43. A method for promoting cellular uptake comprising contacting an
antigen presenting cell with the RPBLA of claim 16.
44. The method of claim 43, wherein said cellular uptake occurs via
phagocytosis.
45. The method of claim 43, wherein the antigen-presenting cell is
a macrophage or dendritic cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of provisional application
Ser. No. 60/776,391 that was filed on Feb. 23, 2006.
TECHNICAL FIELD
[0002] The present invention contemplates the production of
biologically active recombinant peptides and proteins, collectively
referred to as polypeptides, in eukaryotic cells and organisms as
host systems. More particularly, a biologically active polypeptide
is fused to a protein body-inducing sequence (PBIS) that mediates
the induction of recombinant protein body-like assemblies (RPBLA)
to form a fusion protein that is stably expressed and accumulated
in the host system as an RPBLA after transformation of the host
cells with an appropriate vector.
BACKGROUND ART
[0003] The production of recombinant proteins for therapeutic,
nutraceutical or industrial uses has enjoyed great success over the
past several decades. Introduction of heterologous genes having
desired nucleotide sequences into a variety of expression hosts is
now routine. This process nearly always leads to expression of
polypeptides or proteins having the correct predicted primary amino
acid residue sequence (primary structure) encoded by the introduced
nucleotides. In many instances, however, the protein or polypeptide
that is ultimately produced can possess the correct primary amino
acid residue sequence of the naturally-produced molecule, but lack
the biological activity expected of that material.
[0004] Biological activity, given the proper primary structure of
the expressed product, can be a function of the protein's
secondary, tertiary or quaternary structure. These structural
features include having the proper folding and internal hydrogen,
Van der Waals, ionic and disulfide bonding patterns, appropriate
intermolecular and intramolecular subunit interactions, and also
having the proper post-translational modifications, as for instance
glycosylation. For example, disulfide bond formation occurs
spontaneously in the lumen of the endoplasmic reticulum (ER) of
eukaryotic cells, but not in the reducing environment of the
cytosol of prokaryotes, which makes bacterial cells such as
Escherichia coli poor hosts for the synthesis of correctly-folded
mammalian proteins that are normally stabilized by disulfide bonds.
Disulfide bond formation can occur in the periplasmic space of E.
coli where certain prokaryotic chaperonins, foldases and PDI-like
proteins are functional (Fernandez, et al., 2001. Mol. Microbiol.
April 40(2):332-346). However, even in this compartment the
bacterial oxi-redox system is not very efficient for eukaryotic
proteins.
[0005] A particular case in point relates to erythropoietin (EPO),
a protein that stimulates red blood cell production. Recombinant
EPO is disclosed in Lin, U.S. Pat. No. 4,703,008, which describes
activities for heterologous human EPO protein expressed from E.
coli, S. cerevisiae, and mammalian Chinese hamster ovary (CHO) and
African green monkey kidney (COS-1) cells. Although EPO expressed
by each cell type had the correct primary amino acid sequence and
was cross-reactive with anti-EPO antisera, only the proteins
expressed from mammalian cells exhibited the expected levels of
biological activity as determined by in vitro and in vivo assays.
These observed differences in biological activity were determined
to be a function of improper glycosylation in the prokaryotic and
lower eukaryotic host cells. E. coli, a prokaryote, does not
perform the eukaryotic enzymatic steps of N-linked glycosylation.
Yeast cells are eukaryotes and capable of N-linked glycosylation,
but their glycosylation enzymes differ from that of animals and
plants and consequently result in a different pattern of terminal
glycosylation for secreted proteins. On the other hand, the CHO and
COS-1 cells used to provide proteins of substantially correct
biological activity were mammalian, and the proteins expressed
therefrom were consequently useful. Published studies of
glycosylated and aglycosylated EPO indicate that glycosylation
plays a critical role in stabilizing erythropoietin under
denaturing conditions (Narhi et al., (1991) J. Biol. Chem.
266(34):23022-23026). In addition, it has been reported that in
vivo life time and activity of EPO can be related to the
glycosylation state of the molecule, and correct interaction with
the erythrogenic EPO receptor is also affected by EPO glycosylation
pattern.
[0006] Eukaryotic cells are therefore greatly preferred for
recombinant production of therapeutic, industrial and other useful
proteins of eukaryotic origin. Consequently, many different types
of eukaryotic cells and organisms have been shown to be capable of
producing biologically active recombinant proteins. Unfortunately,
many such eukaryotic expression systems are inefficient with
respect to protein product yield and cost of manufacture, even when
proteins are secreted extracellularly. The high costs frequently
derived from low recombinant protein production levels and/or from
complicated downstream protein isolation and purification
procedures can invalidate a protein's commercial application.
Active research is thus being done to improve both production
levels and purification procedures.
[0007] One way of improving the efficiency of recombinant protein
isolation is by means of intracellular concentration. One of these
approaches is the random aggregation of recombinant proteins into
non-secreted inclusion bodies which can be separated from lysed
cells by density-based purification techniques. Insoluble inclusion
bodies are amorphous protein deposits found in bacteria expressing
complex recombinant proteins (such as those of eukaryotic origin).
The absence of specialized eukaryotic molecular chaperones in
prokaryotic cells results in random folding of eukaryotic proteins.
Structural characterization studies have shown that the insoluble
nature of inclusion bodies may be due to the random hydrophobic
intermolecular interactions of proteins which are not correctly
folded (Seshadri et al., 1999, Methods Enzymol, 309:559-576). The
general strategy used to recover active proteins from inclusion
bodies subsequent to their separation from cell material requires
the complete solubilization of the recombinant protein to disrupt
the random aggregates followed by one or more chemical refolding
steps. This is an important issue because the efficiency of protein
renaturation is highly limiting, particularly if the protein
contains disulfide bonds (Clarc, Ed., April 2001 Curr. Opin.
Biotechnol. 12(2):202-207).
[0008] More particularly, high concentrations of strong denaturants
and chaotropic agents (e.g. detergents, urea and guanidinium
hydrochloride) are required for solubilization of the aggregated
and unfolded proteins in inclusion bodies. These agents must be
dialyzed away completely in order to later refold the proteins into
their correct and biologically active conformations. As a
consequence the yield of correctly refolded recombinant proteins
from inclusion bodies is extremely low, and moreover the biological
activities of such refolded proteins are typically much less than
that of the native-formed proteins.
[0009] Protein bodies (PBs) are naturally-occurring structures in
certain plant seeds that have evolved to concentrate storage
proteins intracellularly in eukaryotic cells while retaining
correct folding and biological activity. PBs share some of the
characteristics of the inclusion bodies from bacteria. They are
dense, and contain a high quantity of proteins that are tightly
packed by hydrophobic interactions [Momany et al., 2006 J. Agric.
Food Chem. January 25; 54(2):543-547 and Garrat, et al., 1993
Proteins January; 15(1):88-99]. However, in contrast to the
randomly-aggregated proteins in bacterial inclusion bodies, the
proteins in PBs are thought to be aggregated in a non-random
(assembled) manner.
[0010] A new technology for creation of synthetic PBs based on the
fusion of a plant seed storage protein domain with a heterologous
protein of interest (WO 2004/003207) has been developed to increase
the stability and accumulation of recombinant proteins in higher
plants. These storage proteins are specific to plant seeds wherein
they accumulate stably in natural PBs (Galili et al., 1993, Trends
Cell Biol 3:437-442) following insertion into the lumen of the ER
via a signal peptide and assembly into ER-derived protein bodies
(ER-PBs) (Okita et al., 1996 Annu. Rev. Plant Physiol Mol. Biol.
47:327-350; Herman et al., 1999 Plant Cell 11:601-613; Sanderfoot
et al., 1999 Plant Cell 11:629-642). Full-length recombinant
storage proteins have also been observed to assemble into PB-like
organelles in non-plant host systems as Xenopus oocytes following
injection of the corresponding mRNAs. This system has been used as
a model to study the targeting properties of these storage proteins
(Simon et al., 1990, Plant Cell 2:941-950; Altschuler et al., 1993,
Plant Cell 5:443-450; Torrent et al., 1994, Planta 192:512-518) and
to test the possibility of modifying the 19 kDa .alpha.-zein, a
maize prolamin, by introducing the essential amino acids lysine and
tryptophan into its sequence, without altering its stability
(Wallace et al, 1988, Science 240:662-664).
[0011] Zeins, the complex group of maize prolamins, have also been
produced recombinantly in yeast. Coraggio et al. (1988, Eur J Cell
Biol 47:165-172), expressed native and modified .alpha.-zeins in
yeast to study targeting determinants of this protein. Kim et al.,
2002, Plant Cell 14: 655-672, studied the possible .alpha.-,
.beta.-, .gamma.- and .delta.-zein interactions that could lead to
protein body formation. To address this question, they transformed
yeast cells with cDNAs encoding these proteins. In addition, those
authors constructed zein-GFP fusion proteins to determine the
subcellular localization of zein proteins in yeast cells but did
not observe formation of dense, concentrated structures
characteristic of bona fide PBs. It is worth to noting that Kim et
al. (2002, Plant Cell 14: 655-672) concluded that yeast is a poor
model for the study of zein interactions because zeins accumulated
very poorly in transformed yeast. Yeast has also been used as a
model to study the mechanisms that control the transport and
deposition of gliadin storage proteins in wheat (Rosenberg et al.,
1993, Plant Physiol 102:61-69).
[0012] These results in yeast as well as the similarities between
bacterial inclusion bodies and PBs suggested that proteins
accumulated in synthetic PBs would not be active unless
renaturation steps were performed. Moreover, the presence of
disulfide bonds in some natural PB-assembling protein domains, as
for instance RX3, [Ludevid et al., 1984 Plant Mol. Biol. 3:227-234
and Kawagoe et al., 2005 Plant Cell April 17(4):1141-1153], which
are probably involved in PB formation and stabilization, could
represent an additional difficulty for production of a biologically
active, native-folded protein in PBs. This would be particularly
relevant for a recombinant protein that contains its own cysteine
residues that might interact inappropriately with cysteines in the
PB-assembling domain. The observation of biological activity
without the need for refolding and renaturation of a wide variety
of proteins produced in synthetic PBs in non-yeast eukaryotic hosts
was therefore unexpected.
[0013] Biological activity is particularly relevant for vaccines,
which must induce a correct immune response in an immunized human
or other animal, Several new vaccines are composed of synthetic,
recombinant, or highly purified subunit immunogens (antigens) that
are thought to be safer than whole-inactivated or live-attenuated
vaccines. However, the absence of immunomodulatory components
having adjuvant properties associated with attenuated or killed
vaccines often results in weaker immunogenicity for such
vaccines.
[0014] Immunologic adjuvants are agents that enhance specific
immune responses. An immunologic adjuvant can be defined as any
substance or formulation that, when incorporated into a vaccine,
acts generally to accelerate, prolong, or enhance the quality of
specific immune responses to vaccine antigens. The word adjuvant is
derived from the Latin verb adjuvare, which means to help or aid.
Adjuvant mechanisms of action include the following: (1) increasing
the biological or immunologic half-life of vaccine immunogens; (2)
improving antigen delivery to antigen-presenting cells (APCs), as
well as antigen processing and presentation by the APCs; and (3)
inducing the production of immunomodulatory cytokines.
[0015] Phagocytosis involves the entry of large particles, such as
apoptotic cells or whole microbes. The capacity of the cells to
engulf large particles likely appeared as a nutritional function in
unicellular organisms; however complex organisms have taken
advantage of the phagocytic machinery to fulfill additional
functions. For instance, the phagocytosis of antigens undertaken by
the macrophages, the B-cells or the dendritic cells represents a
key process in innate and adaptive immunity. Indeed, phagocytosis
and the subsequent killing of microbes in phagosomes form the basis
of an organism's innate defense against intracellular pathogens.
Furthermore, the degradation of pathogens in the phagosome lumen
and the production of antigenic peptides, which are presented by
phagocytic cells to activate specific lymphocytes, also link
phagocytosis to adaptive immunity (Jutras et al., 2005, Annual
Review in Cell Development Biology. 21:511-27).
[0016] The proteins present on engulfed particles encounter an
array of degrading proteases in phagosomes. Yet, this destructive
environment generates peptides that are capable of binding to MHC
class II molecules. Newly formed antigen-MHC class II complexes are
delivered to the cell surface for presentation to CD4+ T cells
(Boes et al. 2002 Nature 418:983-988). The activation of these
cells induces the Th2 subset of cytokines such as IL-4 and IL-5
that help B cells to proliferate and differentiate, and is
associated with humoral-type immune response.
[0017] A large body of evidence indicates that, in addition to the
clear involvement of the MHC class II pathway in the immune
response against phagocytosed pathogens, antigens from pathogens,
including mycobacteria, Salmonella, Brucella, and Leishmania, can
elicit an antigen cross-presentation. That is to say, the
presentation of engulfed antigen by phagocytosis by the MHC class
I-dependent response promotes the proliferation of CD8+ cytotoxic T
cells (Ackerman et al., 2004 Nature Immunology 5(7):678-684;
Kaufmann et al., 2005 Current Opinions in Immunology
17(1):79-87).
[0018] Dendritic cells play a central antigen presentation role to
induce the immune system (Blander et al., Nature Immunology 2006
10:1029-1035). Although rare, dendritic cells are the most highly
specialized APC, with the ability both to instigate and regulate
immune reactivity (Lau et al. 2003 Gut 52:307-314). Although
dendritic cells are important in presenting antigens, particularly
to initiate primary immune responses, macrophages are the APC type
most prominent in inflammatory sites and are specialized for
clearing necrotic and apoptotic material. Macrophages can act not
only as APCs, but can also perform-either pro- or anti-inflammatory
roles, dependent on the means by which they are activated.
[0019] Considering that APCs play a central role in the induction
and regulation of the adaptive immunity (humoral and cellular), the
recognition and phagocytosis of an antigen by those cells can be
considered a key step in the immunization process. A wide variety
of techniques based on the uptake of fluorescent particles have
been developed to study phagocytosis by the macrophages (Vergne et
al., 1998 Analytical Biochemistry 255:127-132).
[0020] An important aspect in veterinary vaccines is the genetic
diversity of the species being considered and the requirement for
generic systems that work across different species. To a large
degree, this diversity limits the use of molecular targeting
techniques to cell surface markers and immune modulators such as
cytokines, because for many species including wildlife, only
minimal knowledge of these molecules is available. Thus, adjuvants
that rely on universal activation signals of the innate immune
response (i.e. that are identical in different species) are to be
preferred. Taking these requirements into consideration,
particulate vaccine delivery systems are well suited for veterinary
and wildlife vaccine strategies (Scheerlinck et al., 2004 Methods
40:118-124).
[0021] As is discussed in greater detail hereinafter, the present
invention discloses that the expression of a fusion protein
comprised of (i) a protein sequence that mediates induction of
recombinant protein body-like assemblies (RPBLAs) linked to (ii) a
biologically active polypeptide (protein of interest or target)
induces the accumulation of those RPBLAs in cells of eukaryotic
organisms such as plants, fungi, algae and animals, producing a
biologically active target (protein).
BRIEF SUMMARY OF THE INVENTION
[0022] The present invention provides a system and method for
producing a fusion protein containing a protein body-inducing
sequence (PBIS) and a biologically active peptide or protein of
interest (often collectively referred to herein as a polypeptide or
target) in eukaryotic cells. The fusion proteins containing the
polypeptide of interest stably accumulate as recombinant protein
body-like assemblies (RPBLAs) in the eukaryotic cells, which can be
plant, animal, fungal or algal cells.
[0023] Cells of higher plants are preferred eukaryotic host cells
in some embodiments, whereas cells of lower plants such as algae
are preferred in other embodiments, cells of animals such as
mammals and insects are preferred eukaryotic host cells in further
embodiments and fungi are preferred eukaryotic host cells in still
other embodiments. The fusion protein can be expressed
constitutively or preferentially in particular cells in
multi-cellular eukaryotes. The PBISs are able to mediate the
induction of RPBLA formation and fusion protein entry and/or
accumulation in these organelles, with appropriate folding and/or
post-translational modifications such as basal glycosylation and
disulfide bond formation to provide biological activity to the
expressed peptide or protein of interest (targets).
[0024] Thus, a eukaryotic host cell that contains a biologically
active recombinant fusion protein within recombinant protein
body-like assemblies (RPBLAs) is contemplated as one aspect of the
present invention. The fusion protein contains two sequences linked
together in which one sequence is a protein body-inducing sequence
(PBIS) and the other is the sequence of at least 20 amino acid
residues of a biologically active polypeptide of interest. The
biologically active polypeptide, as found in nature, can be
heterologous to the recited eukaryotic host cells and is thus
expressed in a second cell type that is different from the
first-mentioned eukaryotic host cell, or it is produced
synthetically. In addition, the eukaryotic host cell does not
produce PBs in the absence of the fusion protein. Thus, it is the
expression of the fusion protein and the PBIS portion of that
fusion protein that causes the host cell to form protein body-like
assemblies or RPBLAs.
[0025] In a particular embodiment, the nucleic acid sequence used
for transformation comprises (i) a nucleic acid sequence coding for
a PBIS, and (ii) a nucleic acid sequence comprising the nucleotide
sequence coding for a product of interest. In one embodiment, the
3' end of nucleic acid sequence (i) is linked to the 5' end of said
nucleic acid sequence (ii). In another embodiment, the 5' end of
nucleic acid sequence (i) is linked to the 3' end of nucleic acid
sequence (ii). Thus, the PBIS sequence can be at the N-terminus or
the C-terminus of the fusion protein. It is to be understood that
all of the DNA linkages discussed herein for the expression of a
fusion protein are such that the two components of the fusion
protein are expressed in frame.
[0026] The biologically active polypeptide of the fusion protein
exhibits at least 25 percent, preferably at least 50 percent, more
preferably 75 percent, and most preferably at least 90 percent of
the biological activity of the same polypeptide isolated from the
above second cell type in an assay of the activity of that
polypeptide.
[0027] In another particular embodiment, the nucleic acid sequence
used for transformation comprises, in addition to the
before-mentioned nucleic acid sequences (i) and (ii), a nucleic
acid sequence comprising the nucleotide sequence coding for a
linker or spacer amino acid sequence. The spacer amino acid
sequence can be an amino acid sequence cleavable, or not cleavable,
by enzymatic or autoproteolytic or chemical means. In a particular
embodiment, the nucleic acid sequence (iii) is placed between the
nucleic acid sequences (i) and (ii), e.g., the 3' end of nucleic
acid sequence (iii) is linked to the 5' end of said nucleic acid
sequence (ii). In another embodiment, the 5' end of said nucleic
acid sequence (iii) is linked to the 3' end of nucleic acid
sequence (ii).
[0028] Also, in a particular embodiment, the nucleic acid sequence
used for transformation purposes encodes a sequence in accord with
patent application WO 2004003207, wherein the nucleic acid sequence
coding for the amino acid sequence that is specifically cleavable
by enzymatic or chemical means is present or absent. In a further
embodiment, the fusion proteins can be a direct fusion between the
PBIS and the peptide or protein of interest.
[0029] In a further embodiment, the method of the invention further
comprises the isolation and purification of the biologically active
fusion protein.
[0030] In another embodiment, the method of the invention further
comprises the isolation and purification of the fusion protein, and
obtaining a biologically active fusion protein. Thus, where the
fusion protein is tightly assembled and enclosed within a membrane,
it can be difficult to illustrate that the polypeptide is
biologically active. As a consequence, the biological activity can
be assayed after removal of the membrane, and if it is required,
the solubilization of the fusion protein. A method of preparing a
biologically active polypeptide is therefore contemplated.
[0031] In this method, recombinant protein body-like assemblies
(RPBLAs) are provided that comprise a membrane-enclosed fusion
protein. The RPBLAs are usually present in a generally spherical
form having a diameter of about 0.5 to about 3 microns (.mu.), but
in some instances are amorphous in shape and can vary widely in
dimensions, but are still derived from the ER. The fusion protein
contains two sequences linked together in which one sequence is a
protein body-inducing sequence (PBIS) and the other is a
biologically active polypeptide. The RPBLAs are contacted with an
aqueous buffer containing a membrane-disassembling amount of a
detergent (surfactant). That contact is maintained for a time
period sufficient to disassemble the membrane and at a temperature
that does not denature the biologically active polypeptide to
separate the membrane and fusion protein. The separated fusion
protein is thereafter collected in a usual manner, or can be acted
upon further without collection.
[0032] In some embodiments, the separated fusion protein exhibits
the biological activity of the biologically active polypeptide. In
other embodiments, biological activity of the polypeptide is
exhibited after the fusion protein is dissolved or dispersed in an
appropriate buffer. In yet other embodiments, the fusion protein
has to be cleaved into its constituent parts before biological
activity of the polypeptide is exhibited. Thus, the biologically
active polypeptide can be linked to the PBIS by a spacer amino acid
sequence that is cleavable by enzymatic or chemical means. Then,
upon cleavage, the biologically active polypeptide exhibits
biological activity when cleaved from the PBIS of the fusion
protein. In some embodiments, the fusion protein retains its
activity even when still incorporated into the intact RPBLA.
[0033] In another embodiment, the biologically active polypeptide
contains at least two N-linked glycosylation sequences.
[0034] In yet another preferred embodiment, the polypeptide of
interest is fused to a natural or modified storage protein, as for
instance, natural or modified prolamins or prolamin domains.
[0035] In another embodiment, the RPBLAs containing the
biologically active polypeptide are used as a delivery system for
the biologically active polypeptide. The benefits of this invention
could be applied in drug delivery, vaccines and nutrition.
[0036] In yet another embodiment, the RPBLAs containing a
polypeptide antigen can be used as a delivery system to provide
adjuvanticity (increase the immune response). The administration of
these RPBLAs can represent an improvement in the immunization
parameters such as the speed, quantity, quality and duration of the
immunization. The beneficial effect of administrating antigens in
RPBLAs can be achieved because (i) the antigen is encapsulated and
remains longer in the blood or in the gastrointestinal tract (slow
release effect) and/or (ii) the antigen is better exposed to the
immune system (RPBLAs as an antigen presentation vehicle) and/or
(iii) the presence of adjuvant molecules in the RPBLAs
preparations, and/or (iv) the RPBLAs are carriers able to cross
membranes that themselves provide adjuvanticity, and/or others.
[0037] Thus, another aspect of the invention is a vaccine or
inoculum (immunogenic composition) that comprises an immunogenic
effective amount of RPBLAs that contain biologically active
recombinant fusion protein dissolved or dispersed in a
pharmaceutically acceptable diluent. The recombinant fusion protein
contains two sequences linked together in which one sequence is a
PBIS and the other is a biologically active polypeptide to which an
immunological response is to be induced by said vaccine or
inoculum. The pharmaceutically acceptable diluent composition
typically also contains water. In another embodiment an RPBLA not
incorporating an antigen but possessing active adjuvant properties
is co-delivered with an isolated antigen to induce an immunological
response.
[0038] In another embodiment, the PBIS can be used as a carrier to
cross membranes. In a specific embodiment the PBIS is ZERA (RX3) or
a fragment of it.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] In the drawings forming a portion of this disclosure,
[0040] FIG. 1, Panel A is the schematic representation of the
constructs used for the CHO cells transfection studies. The
construct pECFP-N1 corresponds to the control expressing the ECFP
in the cytosol. The pRX3-ECFP and pRX3-Gx5-ECFP are the constructs
expressing the fusion protein RX3-ECFP, in the absence or presence
of a spacer formed by five glycine amino acids (Gx5), respectively.
The p22aZ-ECFP is the constructs coding for the maize alpha zein
(22 KDa) fused to ECFP. On the bottom, the pcDNA3.1(-) (Invitrogen)
based vectors are represented along with several constructs
discussed hereinafter. Panel B shows the schematic representation
of binary vectors for plant transformation (upper) and the
baculovirus vectors for insect infection (bottom). "RX3"=N-terminal
proline-rich gamma-zein sequence; "(Gly)x5"=spacer formed by five
glycines; "ECFP"=enhanced cyan fluorescent protein gene;
"P.sub.CMV"=human cytomegalovirus promoter; "P.sub.PH"=Polyhedrin
promoter; "P.sub.SV40"=SV40 early promoter; "CaMV35S x2"=Double
cauliflower mosaic virus promoter; "P.sub.cbhl"=major cellulase
promoter; "t35S"=Cauliflower mosaic virus terminator;
"TEV"=Translational enhancer of the tobacco etch virus ; "SV40
ter"=SV40 terminator; "HSV ter"=herpes simplex virus thymidine
kinase polyadenylation signal; "cbhl ter"=major cellulase
polyadenylation signal; "Kana/Neo" kanamycin/neomycin resistance
gene; "Amp R"=Ampicilin resistance gene; "Gentamicine"=Gentamicin
resistance gene; "SP.sub.cbhl"=major cellulase signal peptide; "Ori
f1"=f1 single strand DNA origin; "Ori pUC"=plasmid replication
origin; "BGH ter"=Bovine growth hormone terminator; "P BLA"=beta
lactamase gene promoter; "GFP"=Green fluorescent protein;
"DsRED"=Dicosoma red fluorescent protein; "hGH"=human growth
hormone; "EGF"=human epidermal growth factor; "EK"=bovine
enterokinase; "GUS"=glucuronidase; "RTB"=lectin subunit of ricin
(Ricinus comunis); "Casp2"=Human Caspase 2; "Casp3"=Human Caspase
3; "Int"=Ssp DNAb intein (New England Biolabs); "mInt"=mutated
version of Ssp DNAb intein (Asp154.fwdarw.Ala substitution).
[0041] FIG. 2 shows immunoblots from subcellular fractionation
studies of CHO cells transfected with pRX3-ECFP, pRX3-G-ECFP and
pECFP-N1 as a control (Panel A); p3.1-RX3-hGH, p3.1-RX3,
p3.1-RX3-EK, p3.1-RX3-C3, p3.1-RX3-C2, p3.1-RX3-GUS and
p3.1-RX3-I-hGH plasmids (Panel B). In panel B the immunoblot from
subcellular fractionation studies of tobacco plants agroinfiltrated
with pB-RX3-RTB are also shown. Panel C corresponds to subcellular
fractionation studies of insect larvae infected with pF-RX3-DsRED
and pF-DsRED as a control. Transfected cell homogenates were loaded
on step sucrose gradients, and after centrifugation, the
accumulation of the corresponding fusion proteins in the
supernatant, interphase and pellet fractions was analyzed by
immunoblot. The molecular weights and the antibody used in the
immunoblot are indicated on the right. H, homogenate loaded in the
density gradient; S, supernatant; F.sub.x, upper interphase of the
X % w/w sucrose cushion; P, pellet under 56% sucrose cushion.
[0042] FIG. 3 is a confocal microscopy photographic montage in six
panels showing the localization of the fusion proteins RX3-ECFP
(panel A), RX3-Gx5-ECFP (panel B), 22aZ-ECFP (panel D), RX3-GFP
(panel E) and RX3-DsRED (panel F) in RPBLAs within transfected CHO
cells. Some of the RPBLA structures containing the active
(fluorescent) fusion proteins are indicated by arrows. The
localization of the ECFP in the cytosol and the nucleus (panel C)
in CHO cells transfected by pECFP-N1 are shown as a control.
"N"=nucleus.
[0043] FIG. 4 is a confocal microscopy photograph in four panels
showing the localization of fluorescent RX3 fusion proteins in
different hosts. In panel A is shown the confocal optical sections
of epidermal leaf tissue from tobacco plants co-agroinfiltrated
with pB-RX3-GFP and a binary vector coding for HcPRO, a suppressor
of gene silencing. It can be observed a lot fluorescent RPBLAs
containing the active RX3-GFP fusion protein. On the right, in
Panel B, the merging of the RX3-GFP fluorescence and the contrast
phase shows the high percentage of transiently transfected cells.
The projection of optical sections of SF9 insect cells infected
with pF-RX3-DsRED is shown in Panel C. One micrometer optical
sections of fat tissue from insect larvae infected with
pF-RX3-DsRED are shown in Panel D. Some of the RPBLA structures
containing the active (fluorescent) fusion proteins are indicated
by arrows.
[0044] FIG. 5 shows the localization of RX3 fusion proteins inside
RPBLAs in CHO cells, four days after transfection. Optical
microscopy was used to show CHO cells expressing RX3-hGH (Panels A
and B) immunolocalized by using anti-RX3 and anti-hGH serum,
respectively. Panel C shows RX3 protein immunolocalized with RX3
antiserum. Anti-hGH serum was used in Panel D to immunolocalize the
RX3-I-hGH fusion protein. The incubation of CHO cells expressing
RX3-GUS fusion protein with RX3 antiserum is shown in Panel E.
Smaller RPBLAs were observed in CHO cells expressing RX3-EK,
incubated with anti-RX3 serum (Panel F). The endoplasmic reticulum
(ER) and the RPBLAs are indicated.
[0045] FIG. 6 shows western blots that illustrate the induction of
Ssp DNAb intein self-cleavage after RX3-I-hGH fusion protein
solubilization from a RPBLA preparation by low speed
centrifugation. Panels A and B illustrate the self-cleavage of the
RX3-I-hGH (wild type Ssp DNAb intein) fusion protein, after
solubilization. The RX3-Im-hGH (mutated Ssp DNAb intein) fusion
protein was included as a negative control. Equivalent volumes of
the samples were loaded per lane, and the western blot was
performed with anti-RX3 serum (Panel A) or anti-hGH serum (Panel
B). The full length fusion proteins are indicated with white
arrowheads and the products of the Ssp DNAb intein self-cleavage
(RX3-I in Panel A, and hGH in Panel B) are indicated with black
arrowheads. Panel C illustrates the comparison of RX3-I-hGH fusion
protein self-cleavage efficiency after 0.1% SDS (S1) and biphasic
solubilization (S2). Equivalent volumes of the samples were loaded
per lane, except T0 that was overloaded 4-fold. The incubation with
anti-hGH serum shows the full length fusion protein RX3-I-hGH
(white arrowhead) and the liberated hGH (black arrowhead).
"S"=Soluble fraction; "U"=insoluble fraction; "T0"=Sample before
induction of intein self-cleavage.
[0046] FIG. 7 shows the uptake and processing by macrophages of
RX3-DsRED RPBLAs produced in insect larvae. In panel A, confocal
microscopy analysis of macrophages 1 hour after incubation with
insect RX3-DsRED RPBLAs is shown. On the left, 2 macrophages can be
observed by phase contrast microscopy. On the right is a merged
image of DsRED fluorescence (black arrowheads) and the
self-fluorescence of the macrophages (white arrowheads) from 1
micrometer optical section of the same cells. The position of the
nucleus (N) in this optical section indicates that the RPBLAs have
been taken up and are now intracellular. Panel B shows a time
course study (1 and 10 hours) of DsRED fluorescence emitted by the
macrophages, after incubation for 1 hour with RPBLAs containing
RX3-DsRED. On the left, phase contrast microscopy shows the
presence of macrophages. On the right, the DsRED fluorescence of 1
micrometer optical sections shows the presence of undigested RPBLAs
at 1 hour (white arrowhead) and a more homogeneous DsRED
fluorescence pattern at 10 hours indicative of digested and
dispersed RPBLAs. The inset image corresponds to a higher
magnification of the undigested RPBLAs observed at 1 hour.
[0047] FIG. 8 shows the uptake of RX3-DsRED RPBLAs from insect
larvae by dendritic cells. The micrographs correspond to dendritic
cells incubated with RPBLAs (Panel A) and membrane-less RPBLAs
(Panel B) over time (2, 5 and 10 hours). In the upper portion of
each panel are phase contrast images showing the presence of
dendritic cells. At the bottom, the DsRED fluorescence from the
same dendritic cells shows the presence of RPBLAs absorbed to the
plasma membrane (2 hours) or phagocytosed inside the cell (5 and 10
hours). "N"=nucleus.
[0048] The present invention has several benefits and
advantages.
[0049] One benefit is that use of the invention enables relatively
simple and rapid expression of a desired recombinant biologically
active protein in a eukaryotic cell of choice.
[0050] An advantage of the invention is that it provides a source
of readily obtainable and purifiable recombinant biologically
active protein due to the unique properties of expression in
RPBLAs.
[0051] Another benefit of the invention is that the fusion
protein-containing RPBLAs can be used for delivery of vaccines,
including oral delivery.
[0052] Another advantage of the present invention is that the
fusion protein-containing RPBLAs can be used as is in an immunogen
in an injectable vaccine.
[0053] Another advantage of the present invention is that RPBLAs
can be used as insulators, membrane-bound structures that isolate
the expressed polypeptide from the rest of the cell components.
These insulators protect the cell from the polypeptide activity,
and the polypeptide from the cell, increasing the accumulation
rate. Thus, difficult biologically-active polypeptides that are
toxic and/or labile can be successfully expressed.
[0054] Still further benefits and advantages will be apparent to
the skilled worker from the discussion that follows.
DETAILED DESCRIPTION OF THE INVENTION
[0055] A contemplated recombinant biologically active polypeptide
is a portion of a fusion protein that forms recombinant protein
body-like assemblies (RPBLAS), frequently membrane-enclosed, in the
host cells in which they are expressed. RPBLA formation is induced
by a protein body-inducing sequence (PBIS) comprised of a signal
peptide and storage protein domain that forms high density deposits
inside the cells. These dense deposits can accumulate in the
cytosol, an endomembrane system organelle, mitochondria, plastid or
can be secreted. With the exception of certain cereal plant seeds,
the eukaryotic host cell does not itself produce protein bodies
(PBs) in the absence of the fusion protein. Thus, it is the
expression of the fusion protein and its PBIS portion that causes
the host cell to form protein body-like assemblies or RPBLAs.
[0056] A contemplated fusion protein comprises two polypeptide
sequences linked together directly or indirectly by a peptide bond,
in which one sequence is that of a protein body-inducing sequence
(PBIS) linked to the second sequence that is a biologically active
polypeptide product (e.g., peptide or protein) of interest
(target). The biologically active polypeptide, as found in nature,
is heterologous to the recited eukaryotic host cells and is thus
expressed in a second cell type that is different from the
first-mentioned eukaryotic host cell, or it is produced
synthetically. That is, the biologically active polypeptide is
heterologous to the recited eukaryotic host cells. PBIS are protein
or polypeptide amino acid sequences that mediate the induction of
RPBLA formation and the protein entry and/or accumulation in
organelles such as the ER. The fusion protein when free and
separated from the PBIS exhibits a biological activity similar to
that of the polypeptide.
[0057] The biologically active polypeptide of the fusion protein
exhibits at least 25 percent, preferably at least 50 percent, more
preferably at least 75 percent and most preferably at least 90
percent of the biological activity of the same polypeptide isolated
from the above second cell type, or synthesized in vitro. A
material is considered "biologically active" or "bioactive" if it
has interaction with or effect on any metabolite, protein,
receptor, organelle, cell or tissue in an organism.
[0058] These biological activities can be readily determined and
quantified using standard techniques for measuring the activity of
that polypeptide. For example, assay results for biological
activity between the polypeptide isolated from the second cell
type, or synthesized in vitro, and the expressed polypeptide can be
compared. When comparing the activity of a fusion protein, the
proportion of that material provided by the PBIS and any linker
sequence are taken into account in the assay comparison. Biological
activity can be exhibited by the expressed RPBLAs, the fusion
protein as a protein free of a surrounding membrane or as a target
polypeptide that is free of its PBIS.
[0059] In a particular embodiment, the nucleic acid sequence used
for transformation comprises (i) a nucleic acid sequence coding for
a PBIS, and (ii) a nucleic acid sequence comprising the nucleotide
sequence coding for a product of interest. In one embodiment, the
3' end of nucleic acid sequence (i) is linked to the 5' end of said
nucleic acid sequence (ii). In another embodiment, the 5' end of
nucleic acid sequence (i) is linked to the 3' end of nucleic acid
sequence (ii). Thus, the PBIS sequence can be at the N-terminus or
the C-terminus of the fusion protein. It is to be understood that
all of the DNA linkages discussed herein for the expression of a
fusion protein are such that the two components of the fusion
protein are expressed in-frame.
[0060] Most protein bodies are round-shaped (generally spherical)
structures, with diameters of about 0.5 to about 3.0.mu.. When
expressed in animal cells, the RPBLAs are generally spherical in
shape, have diameters of about 0.5 to about 3 microns (.mu.) and
have a surrounding membrane, RPBLAs expressed in plants are also
usually generally spherical, have diameters of about 0.5 to about
2.mu., and are surrounded by a membrane. RPBLAs expressed in either
plants, animals or fungi are derived from the ER if targeted there
by an ER-specific secretion signal and accumulate externally to the
ER envelope of the host cell following assembly. It is noted that
EGF-containing RPBLAs expressed in the ER of plant cells were not
generally spherical, and were amorphous in shape and of non-uniform
size.
[0061] The recombinant protein body-like assemblies have a
predetermined density that can differ among different fusion
proteins, but is predictable across hosts for a particular fusion
protein being prepared. That predetermined density of the RPBLAs is
typically greater than that of substantially all of the endogenous
host cell proteins present in the homogenate, and is typically
about 1.1 to about 1.35 g/ml. The high density of novel RPBLAs is
due to the general ability of the recombinant fusion proteins to
self-assemble and accumulate into ordered aggregates associated
with membranes. The contemplated RPBLAs are expressed in eukaryotes
and can be characterized by their densities as noted above, and
their size and shape.
[0062] The polypeptide portion of the fusion protein is believed to
obtain its biological activity from folding and assembly within the
ER, including disulfide bond formation, and in some instances from
basal glycosylation in the ER. Interestingly, most plants and
animals as well as lower eukaryotes such as fungi, N-glycosylate
proteins in the same pattern based upon the tripeptide
glycosylation recognition sequence Asn-X-Ser or Asn-X-Thr, where
"X" is any amino acid residue but proline. Thus, a
Glc.sub.3Man.sub.9(GlcNAc).sub.2 N-linked polypeptide is formed
initially, and is trimmed back after formation to a
Man.sub.7-9(GlcNAc).sub.2 N-linked polypeptide that can be excreted
to the Golgi or retained within the ER. This basal glycosylation is
remarkably similar across eukaryotic genera. Further
post-translational modification that are host-specific such as
terminal glycosylation can occur in the Golgi for proteins not
maintained in RPBLAs as are the fusion proteins contemplated
here
[0063] In this method, recombinant protein body-like assemblies
(RPBLAs) are provided that typically comprise a membrane-enclosed
fusion protein ordered assembly, and are preferably present in a
generally spherical form having a diameter of about 0.5 to about 3
microns. The fusion protein contains two sequences linked together
in which one sequence is a protein body-inducing sequence (PBIS)
and the other is a biologically active polypeptide. The RPBLAs are
contacted with an aqueous buffer containing a
membrane-disassembling amount of a detergent (surfactant). That
contact is maintained for a time period sufficient to disassemble
the membrane and at a temperature that does not denature the
biologically active polypeptide (e.g., above freezing to about
40.degree. C.) to separate the membrane and fusion protein. The
separated fusion protein is thereafter collected in a usual manner,
or can be acted upon further without collection. Illustrative
useful surfactants include Triton-X 100, CHAPS and the like are
well-known in biochemistry for solubilizing lipids under mild
conditions.
[0064] The separated fusion protein is typically in an insoluble
form due to the interactions among the PBIS portions of the fusion
protein mediated at least in part by the presence of cysteine
residues. However, the polypeptide of interest is complexed with
eukaryotic chaperones and foldases derived from the ER and hence is
held in a correctly folded conformation despite being tethered to
the assembled (and hence insoluble) PBIS domain. The PBIS-PBIS
interactions can be disrupted and the fusion protein solubilized by
contacting the fusion protein with an aqueous buffer that contains
a reducing agent such as dithiothreitol or 2-mercaptoethanol or
.beta.-mercaptoethanol (.beta.-ME). Conditions are chosen so as to
not disrupt and unfold the attached biologically active protein of
interest. The separated, solubilized fusion protein that contains
the biologically active polypeptide is then collected or otherwise
used. In addition, the two portions of the fusion can be cleaved
from each other upon solubilization. It is to be understood that
that cleavage need not be at the exact borders between the two
portions.
[0065] In some embodiments, the separated fusion protein exhibits
the biological activity of the biologically active polypeptide. In
other embodiments, the fusion protein is dissolved or dispersed in
a suitable buffer to exhibit the biological activity of the
polypeptide. For example, as discussed in detail hereinafter, human
growth hormone (hGH) expressed in RPBLAs in mammalian cells
exhibited activities substantially similar to that of the native
polypeptide both when solubilized as a fusion protein directly from
RPBLAs and also as a cleaved, isolated polypeptide produced from
the fusion protein.
[0066] In yet other embodiments, for steric interaction or size
reasons the fusion protein has to be cleaved into its constituent
parts before biological activity of the polypeptide is revealed.
Thus, the biologically active polypeptide can be linked to the PBIS
by a by a spacer amino acid sequence that is cleavable by enzymatic
or chemical means. Then, upon cleavage from the BPIS of the fusion
protein and assay, the target (biologically active) polypeptide
exhibits biological activity. Studies discussed hereinafter
illustrate biological activity of the T-20 polypeptide cleaved from
its fusion partner and produced in plants.
Protein Body-Inducing Sequences
[0067] A contemplated protein body-inducing sequences (PBIS) and
the host cell are preferably of different biological phyla. Thus,
the PBIS is preferably from a higher plant, a spermatophyte,
whereas the host cell is a eukaryote that is other than a
spermatophyte and can be an animal cell, as for instance mammalian
or insect cells, a fungus, or an algal cell, all of which are of
different phyla from spermatophytes. A PBIS and the host cell can
also be from the same phylum so that both can be from a higher
plant, for example. Illustrative, non-limiting examples of PBIS
include storage proteins or modified storage proteins, as for
instance, prolamins or modified prolamins, prolamin domains or
modified prolamin domains, Prolamins are reviewed in Shewry et al.,
2002 J. Exp. Bot. 53(370):947-958. Preferred PBIS are those of
prolamin compounds such as gamma-zein, alpha-zein, delta-zein,
beta-zein, rice prolamin and the gamma-gliadin that are discussed
hereinafter.
[0068] A PBIS also includes a sequence that directs a protein
towards the endoplasmic reticulum (ER) of a plant cell. That
sequence often referred to as a leader sequence or signal peptide
can be from the same plant as the remainder of the PBIS or from a
different plant or an animal or fungus. Illustrative signal
peptides are the 19 residue gamma-zein signal peptide sequence
shown in WO 2004003207 (US 20040005660), the 19 residue signal
peptide sequence of alpha-gliadin or 21 residue gamma-gliadin
signal peptide sequence (see, Altschuler et al., 1993 Plant Cell
5:443-450; Sugiyama et al., 1986 Plant Sci. 44:205-209; and
Rafalski et al., 1984 EMBO J. 3(6):1409-11415 and the citations
therein.) The pathogenesis-related protein of PR10 class includes a
25 residue signal peptide sequence that is also useful herein.
Similarly functioning signal peptides from other plants and animals
are also reported in the literature.
[0069] The characteristics of the signal peptides responsible for
directing the protein to the ER have been extensively studied (von
Heijne et al., 2001 Biochim. Biophys. Acta December 12
1541(1-2):114-119). The signal peptides do not share homology at a
primary structure, but have a common tripartite structure: a
central hydrophobic h-region and hydrophilic N- and C-terminal
flanking regions. These similarities, and the fact that proteins
are translocated through the ER membrane using apparently common
pathways, permits interchange of the signal peptides between
different proteins or even from different organisms belonging to
different phyla (See, Examples 1 and 2 hereinafter, and Martoglio
et al., 1998 Trends Cell Biol. October; 8(10):410-415). Thus, a
PBIS can include a signal peptide of a protein from a phylum
different from higher plants.
[0070] Gamma-Zein, a maize storage protein whose DNA and amino acid
residue sequences are shown hereinafter, is one of the four maize
prolamins and represents 10-15 percent of the total protein in the
maize endosperm. As other cereal prolamins, alpha- and gamma-zeins
are biosynthesized in membrane-bound polysomes at the cytoplasmic
side of the rough ER, assembled within the lumen and then
sequestered into ER-derived protein bodies (Herman et al., 1999
Plant Cell 11:601-613; Ludevid et al., 1984 Plant Mol. Biol.
3:277-234; Torrent et al., 1986 Plant Mol. Biol. 7:93-403).
[0071] Gamma-Zein is composed of four characteristic domains i) a
peptide signal of 19 amino acids, ii) the repeat domain containing
eight units of the hexapeptide PPPVHL (SEQ ID NO:1) [(53 amino acid
residues (aa)], iii) the ProX domain where proline residues
alternate with other amino acids (29 aa) and iv) the hydrophobic
cysteine rich C-terminal domain (lll aa).
[0072] The ability of gamma-zein to assemble in ER-derived RPBLAs
is not restricted to seeds. In fact, when gamma-zein-gene was
constitutively expressed in transgenic Arabidopsis plants, the
storage protein accumulated within ER-derived PBLS in leaf mesophyl
cells (Geli et al., 1994 Plant Cell 6:1911-1922). Looking for a
signal responsible for the gamma-zein deposition into the
ER-derived protein bodies (prolamins do not have KDEL signal for
ER-retention), it has been demonstrated that the proline-rich
N-terminal domain including the tandem repeat domain was necessary
for ER retention. In this work, it was also suggested that the
C-terminal domain could be involved in protein body formation,
however, recent data (WO2004003207A1) demonstrate that the
proline-rich N-terminal domain is necessary and sufficient to
retain in the ER and to induce the protein body formation. However,
the mechanisms by which these domains promote the protein body
assembly are still unknown, but evidence from in vitro studies
suggests that the N-terminal portion of gamma-zein is able to
self-assemble into ordered structures.
[0073] It is preferred that a gamma-zein-based PBIS include at
least one repeat and the amino-terminal nine residues of the ProX
domain, and more preferably the entire Pro-X domain. The C-terminal
portion of gamma-zein is not needed, but can be present. Those
sequences are shown in US 20040005660 and designated as RX3 and P4,
respectively, and are noted hereinafter.
[0074] Inasmuch as PBs are appropriately so-named only in seeds,
similar structures produced in other plant organs and in non-higher
plants are referred to generally as synthetic PBs or recombinant
protein body-like assemblies (RPBLAs).
[0075] Zeins are of four distinct types: alpha, beta, delta, and
gamma. They accumulate in a sequential manner in the ER-derived
protein bodies during endosperm development. Beta-zein and
delta-zein do not accumulate in large amount in maize PBs, but they
were stable in the vegetative tissues and were deposited in
ER-derived protein body-like structures when expressed in tobacco
plants (Bagga et al., 1997 Plant Cell September 9(9):1683-1696).
This result indicates that beta-zein, as well as delta-zein, can
induce ER retention and PB formation.
[0076] The wheat prolamin storage proteins, gliadins, are a group
of K/HDEL-less proteins whose transport via the ER appears to be
complex. These proteins sequester in to the ER where they are
either retained and packaged into dense protein bodies, or are
transported from the ER via the Golgi into vacuoles. (Altschuler et
al., 1993 Plant Cell 5:443-450.)
[0077] The gliadins appear to be natural chimeras, containing two
separately folded autonomous regions. The N-terminus is composed of
about 7 to about 16 tandem repeats rich in glutamine and proline.
The sequence of the tandem repeats varies among the different
gliadins, but are based on one or the other consensus sequences
PQQPFPQ (SEQ ID NO:47), PQQQPPFS (SEQ ID NO:48) and PQQPQ (SEQ ID
NO:49). The C-terminal region of the protein contains six to eight
cysteines that form intramolecular disulfide bonds. The work of the
Altschuler et al. indicates that the N-terminal region and
consensus sequences are responsible for PB formation in the ER from
gamma-gliadin. (Altschuler et al., 1993 Plant Cell 5:443-450.)
[0078] Illustrative useful prolamin-type sequences are shown in the
Table below along with their GenBank identifiers.
TABLE-US-00001 PROTEIN NAME GENBANK ID .alpha.-Zein (22 kD) M86591
Albumin (32 kD) X70153 .gamma.-Zein (27 kD) X53514 .gamma.-Zein (50
kD) AF371263 .delta.-Zein (18 kD) AF371265 7S Globulin or Vicilin
type NM_113163 11S Globulin or Legumin type DQ256294 Prolamin 13 kD
AB016504 Prolamin 16 kD AY427574 Prolamin 10 kD AF294580
.gamma.-Gliadin M36999 .gamma.-Gliadin precursor AAA34272
[0079] Further useful sequences are obtained by carrying out a
BLAST search in the all non-redundant GenBank CDS
translations+PDB+SwissProt+PIR+PRF (excluding environmental
samples) data base as described in Altschul et al., 1997 Nucleic
Acids Res. 25:3389-3402 using a query such as those shown
below:
TABLE-US-00002 RX3 query SEQ ID NO: 2
PPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHVPPPVHLP PPP Alpha-zein
SEQ ID NO: 3 QQQQQFLPALSQLDVVNPVAYLQQQLLASNPLALANVAAYQQQQQLQQFL
PALSQLAMVNPAAYL Rice prolamin query SEQ ID NO: 4
QQVLSPYNEFVRQQYGIAASPFLQSATFQLRNNQVWQQLALVAQQSHCQD
INIVQAIAQQLQLQQFGDLY
[0080] An illustrative modified prolamin includes (a) a signal
peptide sequence, (b) a sequence of one or more copies of the
repeat domain hexapeptide PPPVHL (SEQ ID NO: 1) of the protein
gamma-zein, the entire domain containing eight hexapeptide units;
and (c) a sequence of all or part of the ProX domain of gamma-zein.
Illustrative specific modified prolamins include the polypeptides
identified below as R3, RX3 and P4 whose DNA and amino acid residue
sequences are also shown below.
[0081] Particularly preferred prolamins include gamma-zein and its
component portions as disclosed in published application
WO2004003207, the rice rP13 protein and the 22 kDa maize alpha-zein
and its N-terminal fragment. The DNA and amino acid residue
sequences of the gamma-zein, rice and alpha-zein proteins are shown
below.
Gamma-Zein of 27 kD
DNA Sequence %
TABLE-US-00003 [0082] SEQ ID NO: 5 atgagggtgt tgctcgttgc cctcgctctc
ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg gcggctgcgg 80
ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120 catctgccac
ctccggttca cctgccacct ccggtgcatc 160 tcccaccgcc ggtccacctg
ccgccgccgg tccacctgcc 200 accgccggtc catgtgccgc cgccggttca
tctgccgccg 240 ccaccatgcc actaccctac tcaaccgccc cggcctcagc 280
ctcatcccca gccacaccca tgcccgtgcc aacagccgca 320 tccaagcccg
tgccagctgc agggaacctg cggcgttggc 360 agcaccccga tcctgggcca
gtgcgtcgag tttctgaggc 400 atcagtgcag cccgacggcg acgccctact
gctcgcctca 440 gtgccagtcg ttgcggcagc agtgttgcca gcagctcagg 480
caggtggagc cgcagcaccg gtaccaggcg atcttcggct 520 tggtcctcca
gtccatcctg cagcagcagc cgcaaagcgg 560 ccaggtcgcg gggctgttgg
cggcgcagat agcgcagcaa 600 ctgacggcga tgtgcggcct gcagcagccg
actccatgcc 640 cctacgctgc tgccggcggt gtcccccacg cc 672
Protein Sequence:
TABLE-US-00004 [0083] SEQ ID NO: 6 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45 Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55 60
His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro 65
70 75 80 Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln Pro
His Pro 85 90 95 Gln Pro His Pro Cys Pro Cys Gln Gln Pro His Pro
Ser Pro Cys Gln 100 105 110 Leu Gln Gly Thr Cys Gly Val Gly Ser Thr
Pro Ile Leu Gly Gln Cys 115 120 125 Val Glu Phe Leu Arg His Gln Cys
Ser Pro Thr Ala Thr Pro Tyr Cys 130 135 140 Ser Pro Gln Cys Gln Ser
Leu Arg Gln Gln Cys Cys Gln Gln Leu Arg 145 150 155 160 Gln Val Glu
Pro Gln His Arg Tyr Gln Ala Ile Phe Gly Leu Val Leu 165 170 175 Gln
Ser Ile Leu Gln Gln Gln Pro Gln Ser Gly Gln Val Ala Gly Leu 180 185
190 Leu Ala Ala Gln Ile Ala Gln Gln Leu Thr Ala Met Cys Gly Leu Gln
195 200 205 Gln Pro Thr Pro Cys Pro Tyr Ala Ala Ala Gly Gly Val Pro
His Ala 210 215 220
RX3
DNA Sequence:
TABLE-US-00005 [0084] SEQ ID NO: 7 atgagggtgt tgctcgttgc cctcgctctc
ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg gcggctgcgg 80
ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120 catctgccac
ctccggttca cctgccacct ccggtgcatc 160 tcccaccgcc ggtccacctg
ccgccgccgg tccacctgcc 200 accgccggtc catgtgccgc cgccggttca
tctgccgccg 240 ccaccatgcc actaccctac tcaaccgccc cggcctcagc 280
ctcatcccca gccacaccca tgcccgtgcc aacagccgca 320 tccaagcccg
tgccagacc 339
Protein Sequence:
TABLE-US-00006 [0085] SEQ ID NO: 8 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45 Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55 60
His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro 65
70 75 80 Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln Pro
His Pro 85 90 95 Gln Pro His Pro Cys Pro Cys Gln Gln Pro His Pro
Ser Pro Cys Gln 100 105 110 Tyr
R3
DNA Sequence:
TABLE-US-00007 [0086] SEQ ID NO: 9 atgagggtgt tgctcgttgc cctcgctctc
ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg gcggctgcgg 80
ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120 catctgccac
ctccggttca cctgccacct ccggtgcatc 160 tcccaccgcc ggtccacctg
ccgccgccgg tccacctgcc 200 accgccggtc catgtgccgc cgccggttca
tctgccgccg 240
Protein Sequence:
TABLE-US-00008 [0087] SEQ ID NO: 10 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45 Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55 60
His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro 65
70 75 80 Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Tyr 85 90
P4
DNA Sequence:
TABLE-US-00009 [0088] SEQ ID NO: 11 atgagggtgt tgctcgttgc
cctcgctctc ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg
gcggctgcgg 80 ctgccagcca ccgccgccgg ttcatctgcc gccgccacca 120
tgccactacc ctacacaacc gccccggcct cagcctcatc 160 cccagccaca
cccatgcccg tgccaacagc cgcatccaag 200 cccgtgccag acc 213
Protein Sequence:
TABLE-US-00010 [0089] SEQ ID NO: 12 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Pro Cys His Tyr Pro Thr Gln Pro Pro 35 40 45 Arg Pro
Gln Pro His Pro Gln Pro His Pro Cys Pro Cys Gln Gln Pro 50 55 60
His Pro Ser Pro Cys Gln Tyr 65 70
X10
DNA Sequence:
TABLE-US-00011 [0090] SEQ ID NO: 13 atgagggtgt tgctcgttgc
cctcgctctc ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg
gcggctgcgg 80 ctgccaatgc cactacccta ctcaaccgcc ccggcctcag 120
cctcatcccc agccacaccc atgcccgtgc caacagccgc 160 atccaagccc
gtgccagacc 180
Protein Sequence:
TABLE-US-00012 [0091] SEQ ID NO: 14 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala 1 5 10 Leu Ala Ala Ser Ala Thr Ser Thr His Thr Ser
Gly 15 20 Gly Cys Gly Cys Gln Cys His Tyr Pro Thr Gln Pro 25 30 35
Pro Arg Pro Gln Pro His Pro Gln Pro His Pro Cys 40 45 Pro Cys Gln
Gln Pro His Pro Ser Pro Cys Gln Tyr 50 55 60
[0092] rP13-rice prolamin of 13 kD homologous to the clone-AB016504
Sha et al., 1996 Biosci, Biotechnol. Biochem. 60(2):335-337; Wen et
al., 1993 Plant Physiol. 101(3):1115-1116; Kawagoe et al., 2005
Plant Cell 17(4):1141-1153; Mullins et al., 2004 J. Agric. Food
Chem. 52(8):2242-2246; Mitsukawa et al., 1999 Biosci. Biotechnol,
Biochem. 63(11):1851-1858
Protein Sequence:
TABLE-US-00013 [0093] SEQ ID NO: 15
MKIIFVFALLAIAACSASAQFDVLGQSYRQYQLQSPVLLQQQVLSPYN
EFVRQQYGIAASPFLQSATFQLRNNQVWQQLALVAQQSHCQDINIVQA
IAQQLQLQQFGDLYFDRNLAQAQALLAFNVPSRYGIYPRYYGAPSTIT TLGGVL
DNA Sequence
TABLE-US-00014 [0094] SEQ ID NO: 16
atgaagatcattttcgtctttgctctccttgctattgctgcatgcagc
gcctctgcgcagtttgatgttttaggtcaaagttataggcaatatcag
ctgcagtcgcctgtcctgctacagcaacaggtgcttagcccatataat
gagttcgtaaggcagcagtatggcatagcggcaagccccttcttgcaa
tcagctacgtttcaactgagaaacaaccaagtctggcaacagctcgcg
ctggtggcgcaacaatctcactgtcaggacattaacattgttcaggcc
atagcgcagcagctacaactccagcagtttggtgatctctactttgat
cggaatctggctcaagctcaagctctgttggcttttaacgtgccatct
agatatggtatctaccctaggtactatggtgcacccagtaccattacc
acccttggcggtgtcttg
[0095] 22aZt N-terminal fragment of the maize alpha-zein of 22
kD-V01475 Kim et al., 2002 Plant Cell 14(3):655-672; Woo et al.,
2001 Plant Cell 13(10):2297-2317; Matsushima et al., 1997 Biochim.
Biophys. Acta 1339(1):14-22; Thompson et al., 1992 Plant Mol. Biol,
18(4):827-833.
Protein Sequence (Full Length)
TABLE-US-00015 [0096] SEQ ID NO: 17
MATKILALLALLALFVSATNAFIIPQCSLAPSAIIPQFLPPVTSMGFE
HLAVQAYRLQQALAASVLQQPINQLQQQSLAHLTIQTIATQQQQQFLP
ALSQLDVVNPVAYLQQQLLASNPLALANVAAYQQQQQLQQFLPALSQL
DNA Sequence (Full Length):
TABLE-US-00016 [0097] SEQ ID NO: 18
atggctaccaagatattagccctccttgcgcttcttgccctttttgtg
agcgcaacaaatgcgttcattattccacaatgctcacttgctcctagt
gccattataccacagttcctcccaccagttacttcaatgggcttcgaa
cacctagctgtgcaagcctacaggctacaacaagcgcttgcggcaagc
gtcttacaacaaccaattaaccaattgcaacaacaatccttggcacat
ctaaccatacaaaccatcgcaacgcaacagcaacaacagttcctacca
gcactgagccaactagatgtggtgaaccctgtcgcctacttgcaacag
cagctgcttgcatccaacccacttgctctggcaaacgtagctgcatac
caacaacaacaacaattgcagcagtttctgccagcgctcagtcaacta
Gamma-Gliadin precursor--AAA34272--Scheets et al., 1988 Plant Sci.
57:141-150.
Protein Sequence:
TABLE-US-00017 [0098] SEQ ID NO: 19 NMQVDPSGQV QWPQQQPFPQ
PQQPFCQQPQ RTIPQPHQTF HHQPQQTFPQ PQQTYPHQPQ QQFPQTQQPQ QPFPQPQQTF
PQQPQLPFPQ QPQQPFPQPQ QPQQPFPQSQ QPQQPFPQPQ QQFPQPQQPQ QSFPQQQQPA
IQSFLQQQMN PCKNFLLQQC NHVSLVSSLV SIILPRSDCQ VMQQQCCQQL AQIPQQLQCA
AIHSVAHSII MQQEQQQGVP ILRPLFQLAQ GLGIIQPQQP AQLEGIRSLV LKTLPTMCNV
YVPPDCSTIN VPYANIDAGI GGQ
TABLE-US-00018 DNA Sequence (M36999) SEQ ID NO: 20 gcatgcattg
tcaaagtttg tgaagtagaa ttaataacct tttggttatt gatcactgta tgtatcttag
atgtcccgta gcaacggtaa gggcattcac ctagtactag tccaatatta attaataact
tgcacagaat tacaaccatt gacataaaaa ggaaatatga tgagtcatgt attgattcat
gttcaacatt actacccttg acataaaaga agaatttgac gagtcgtatt agcttgttca
tcttaccatc atactatact gcaagctagt ttaaaaaaga atyaaagtcc agaatgaaca
gtagaatagc ctgatctatc tttaaeaaca tgcacaagaa tacaaattta gtcccttgca
agctatgaag atttggttta tgcctaacaa catgataaac ttagatccaa aaggaatgca
atctagataa ttgtttqact tgtaaagtcg ataagatgag tcagtgccaa ttataaagtt
ttcgccactc ttagatcata tgtacaataa aaaggcaact ttgctgacca ctccaaaagt
acgtttgtat gtagtgccac caaacacaac acaccaaata atcagtttga taagcatcga
atcactttaa aaagtgaaag aaataatgaa aagaaaccta accatggtag ctataaaaag
cctgtaatat gtacactcca taccatcatc catccttcac acaactagag cacaagcatc
aaatccaagt aagtattagt t aacgcaaat ccaccatgaa gaccttactc atcctaacaa
tccttgcgat ggcaacaacc atcgccaccg ccaatatgca agtcgacccc agcggccaag
tacaatggcc acaacaacaa ccattccccc agccccaaca accattctgc cagcaaccac
aacgaactat tccccaaccc catcaaacat tccaccatca accacaacaa acatttcccc
aaccccaaca aacatacccc catcaaccac aacaacaatt tccccagacc caacaaccac
aacaaccatt tccccagccc caacaaacat tcccccaaca accccaacta ccatttcccc
aacaacccca acaaccattc ccccagcctc agcaacccca acaaccattt ccccagtcac
aacaaccaca acaacctttt ccccagcccc aacaacaatt tccgcagccc caacaaccac
aacaatcatt cccccaacaa caacaaccgg cgattcagtc atttctacaa caacagatga
acccctgcaa gaatttcctc ttgcagcaat gcaaccatgt gtcattggtg tcatctctcg
tgtcaataat tttgccacga agtgattgcc aggtgatgca gcaacaatgt tgccaacaac
tageacaaat tcctcaacag ctccagtacg cagccatcca cagcgtcgcg cattccatca
tcatgcaaca agaacaacaa caaggcgtgc cgatcctgcg gccactattt cagctcgccc
agggtctggg tatcatecaa cctcaacaac cagctcaatt ggaggggatc aggtcattgg
tattgaaaac tcttccaacc atgtgcaacg tgtatgtgcc acctgactgc tccaccatca
acgtaccata tgccaacata gacgctggca ttggtggcca atgaaaaatg caagatcatc
attgcttagc tgatqcacca atcgttgtag cgatgacaaa taaagtggtg tgcaccatca
tgtgtgaccc cgaccagtgc tagttcaagc ttgggaataa aagacaaaca aagttcttgt
ttgctagcat tgcttgtcac tgttacattc atttttcgat tcgatgttca tccctaaccg
caatcctagc cttacacgtc aatagctagc tgcttgtgct ggcaggttac tatataatct
atcaattaat ggtcgaccta ttaatccaag taataggcta ttgatagact gctcccaagc
cgaccgagca cctatcagtt acggatttct tgaacattgc acactataat aattcaacgt
atttcaacct ctagaagtaa aaggcatttt agtagc
Beta zein--AF371264--Woo et al., (2001) Plant Cell 13 (10),
2297-2317.
TABLE-US-00019 DNA SEQ ID NO: 21
atgaagatggtcatcgttctcgtcgtgtgcctggctctgtcagctgcc
agcgcctctgcaatgcagatgccctgcccctgcgcggggctgcagggc
ttgtacggcgctggcgccggcctgacgacgatgatgggcgccggcggg
ctgtacccctacgcggagtacctgaggcagccgcagtgcagcccgctg
gcggcggcgccctactacgccgggtgtgggcagccgagcgccatgttc
cagccgctccggcaacagtgctgccagcagcagatgaggatgatggac
gtgcagtccgtcgcgcagcagctgcagatgatgatgcagcttgagcgt
gccgctgccgccagcagcagcctgtacgagccagctctgatgcagcag
cagcagcagctgctggcagcccagggtctcaaccccatggccatgatg
atggcgcagaacatgccggccatgggtggactctaccagtaccagctg
cccagctaccgcaccaacccctgtggcgtctccgctgccattccgccc tactactga Protein
SEQ ID NO: 22 MKMVIVLVVCLALSAASASAMQMPCPCAGLQGLYGAGAGLTTMMGAGG
LYPYAEYLRQPQCSPLAAAPYYAGCGQPSAMFQPLRQQCCQQQMRMMD
VQSVAQQLQMMMQLERAAAASSSLYEPALMQQQQQLLAAQGLNPMAMM
MAQNMPAMGGLYQYQLPSYRTNPCGVSAAIPPYY
Delta zein 10 kD--AF371266--Woo et al., (2001) Plant Cell 13 (10),
2297-2317. and Kirihara et al., (1988) Gene. November 30;
71(2):359-70.
TABLE-US-00020 DNA SEQ ID NO: 23
atggcagccaagatgcttgcattgttcgctctcctagctctttgtgca
agcgccactagtgcgacgcatattccagggcacttgccaccagtcatg
ccattgggtaccatgaacccatgcatgcagtactgcatgatgcaacag
gggcttgccagcttgatggcgtgtccgtccctgatgctgcagcaactg
ttggccttaccgcttcagacgatgccagtgatgatgccacagatgatg
acgcctaacatgatgtcaccattgatgatgccgagcatgatgtcacca
atggtcttgccgagcatgatgtcgcaaatgatgatgccacaatgtcac
tgcgacgccgtctcgcagattatgctgcaacagcagttaccattcatg
ttcaacccaatggccatgacgattccacccatgttcttacagcaaccc
tttgttggtgctgcattctag Protein SEQ ID NO: 24
MAAKMLALFALLALCASATSATHIPGHLPPVMPLGTMNPCMQYCMMQQ
GLASLMACPSLMLQQLLALPLQTMPVMMPQMMTPNMMSPLMMPSMMSP
MVLPSMMSQMMMPQCHCDAVSQIMLQQQLPFMFNPMAMTIPPMFLQQP FVGAAF Signal
Peptides Gamma-Zein SEQ ID NO: 25 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser Ala Thr Ser Alpha-Gliadin SEQ ID
NO: 26 Met Lys Thr Phe Leu Ile Leu Val Leu Leu Ala Ile Val Ala Thr
Thr Ala Thr Thr Ala Gamma-Gliadin SEQ ID NO: 27 Met Lys Thr Leu Leu
Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile Gly Thr Ala Asn Met
PR10 SEQ ID NO: 28 Met Asn Phe Leu Lys Ser Phe Pro Phe Tyr Ala Phe
Leu Cys Phe Gly Gln Tyr Phe Val Ala Val Thr His Ala
Proteins of Interest
[0099] Examples of polypeptides or proteins of interest (targets)
include any protein having therapeutic, nutraceutical,
agricultural, or industrial uses. Illustrative activities of such
proteins include (a) light capture and emission as are provided by
green fluorescent protein (GFP), enhanced cyan fluorescent protein
(ECFP), red fluorescent protein (DsRed) and the like; (b) enzymatic
activity that can be associated with primary and secondary
intracellular signaling and metabolic pathways, is exemplified by
enterokinase, beta-glucuronidase (GUS), phytase, carbonic
anhydrase, and industrial enzymes (hydrolases, glycosidases,
cellulases, oxido-reductases, and the like); (c) protein-protein,
protein-receptor, and protein-ligand interaction such as, for
example antibodies (mAbs such as IgG, IgM, IgA, etc.) and fragments
thereof, hormones [calcitonin, human growth hormone (hGH),
epidermal growth factor (EGF) and the like], protease inhibitors,
antibiotics, antimicrobials, HIV entry inhibitors [Ryser et al.,
2005 Drug Discov Today. Aug. 15; 10(10:1085-1094], collagen, human
lactoferrin, and cytokines; (d) protein and peptides antigens for
vaccines (human immunodeficiency virus, HIV; hepatitis B
pre-surface, surface and core antigens, Foot and Mouth Disease
Virus (FMDV) structural polyprotein gene P1 [Dus Santos et al.,
2005 Vaccine. March 7; 23(15):1838-1843] T cell stimulating
peptides of U.S. Pat. No. 4,882,145, gastroenteritis corona virus,
human papilloma virus, and the like); (e) protein-non protein
interactions such as, phytohaemagglutinin (PHA), the Ricin Toxin
subunit B (RTB) and other lectins.
[0100] Assays for the bioactivity of such expressed polypeptides
are well known in the art and are available in one or more
publications. For example, ECFP activity can be measured by
quantifying the fluorescence emitted at a 470-530 nm wavelength
when the protein has been exited at 458 nm. See, Richards et al.,
2003 Plant Cell Rep. 22:117-121. The enzymatic activity of
enterokinase (EK), for example, can be measured with two different
approaches. The activity can be determined by analyzing the
cleavage of a fusion protein containing the enterokinase specific
cleavage site by western blot, as discussed in the Invitrogen Life
Technologies catalog (E180-01 and E180-2), and also by quantifying
the EK activity using fluorogenic peptide substrate for EK (Sigma
G-5261, CAS.RTM. RN 70023-02-8); enzyme activity is measured by an
increase of fluorescence (excitation at 337 nm, emission at 420 nm)
caused by the release of .beta.-naphthylamine from the peptide over
time. See, LaVallie et al., 1993 J. Biol. Chem.
268(31):23311-23317. The activity of the enzyme beta-glucuronidase
(GUS) can be measured by the conversion of the substrate MUG
(4-methyl umbelliferyl glucuronide) to the product MU. This product
can be quantified by measuring the fluorescence with excitation at
365 nm, emission at 455 nm on a spectrofluorimeter. See, Pai-Hsiang
et al., 2001 J. Plant Physiol. 158(2):247-254; and Jefferson et
al., 1987 EMBO J. 6:3901-3907. Phytase assays are carried out by
the quantification of inorganic ortho phosphates liberated from the
AAM reagent consisting of acetone, 5.0 N sulfuric acid, and 10 mM
ammonium molybdate. See, Ullah et al., 1999 Biochem. Biophys. Res.
Commun. 264(1):201-206. Similar assays are available for other
biological proteins. The RTB activity assays can be performed by
measuring the binding of RTB to asialofetuin, lactose and
galactose, as described in Reed et al., 2005 Plant Cell Rep. April;
24(1):15-24.
[0101] EGF is a growth factor involved in fibroblasts
proliferation. EGF activity can be assayed by the quantification of
the induction of DNA synthesis measured by incorporation of the
pyrimidine analog 5-bromo-2'-deoxyuridine (BrdU), instead of
thymidine, into the DNA of proliferating cells using the cell
proliferation ELISA kit [Oliver, et al., 2004 Am. J. Physiol. Cell
Physiol. 286:1118-1129; Catalog no. 1647229, Roche Diagnostics,
Mannheim, Germany]
[0102] It is noted that light capture and emission constitutes a
separate and special type of "biological activity" in that such
activity does not provide therapeutic, nutraceutical, agricultural,
or industrial use as do the other types of activity noted above.
The polypeptides of this class of targets are included herein as
biologically active because they share some of the required
secondary, tertiary and quaternary structural features that are
possessed by the target molecules that provide therapeutic,
nutraceutical, agricultural, or industrial uses, These proteins are
useful, however, as reporter molecules in many types of assays or
screens used in the analysis or discovery of biologically important
molecules, and their luminescent activity requires the presence of
correct secondary and tertiary protein structure. It is possibly
more accurate to refer to the group of targets as those
polypeptides that are biologically active and/or luminescently
active.
[0103] Illustrative DNA and amino acid residue sequences for
illustrative proteins of interest are provided below.
TABLE-US-00021 ECFP SEQ ID NO: 29
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctg
gtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggc
gagggcgagggcgatgccacctacggcaagctgaccctgaagttcatc
tgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccacc
ctgacctggggcgtgcagtgcttcagccgctaccccgaccacatgaag
cagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggag
cgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgag
gtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggc
atcgacttcaaggaggacggcaacatcctggggcacaagctggagtac
aactacatcagccacaacgtctatatcaccgccgacaagcagaagaac
ggcatcaaggccaacttcaagatccgccacaacatcgaggacggcagc
gtgcagctcgccgaccactaccagcagaacacccccatcggcgacggc
cccgtgctgctgcccgacaaccactacctgagcacccagtccgccctg
agcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa ECFP SEQ ID NO: 30
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
CTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQE
RTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY
NYISHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDG
PVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK GUS1381 SEQ ID NO:
31 atggtagatctgactagtttacgtcctgtagaaaccccaacccgtgaa
atcaaaaaactcgacggcctgtgggcattcagtctggatcgcgaaaac
tgtggaattgatcagcgttggtgggaaagcgcgttacaagaaagccgg
gcaattgctgtgccaggcagttttaacgatcagttcgccgatgcagat
attcgtaattatgcgggcaacgtctggtatcagcgcgaagtctttata
ccgaaaggttgggcaggccagcgtatcgtgctgcgtttcgatgcggtc
actcattacggcaaagtgtgggtcaataatcaggaagtgatggagcat
cagggcggctatacgccatttgaagccgatgtcacgccgtatgttatt
gccgggaaaagtgtacgtatcaccgtttgtgtgaacaacgaactgaac
tggcagactatcccgccgggaatggtgattaccgacgaaaacggcaag
aaaaagcagtcttacttccatgatttctttaactatgccggaatccat
cgcagcgtaatgctctacaccacgccgaacacctgggtggacgatatc
accgtggtgacgcatgtcgcgcaagactgtaaccacgcgtctgttgac
tggcaggtggtggccaatggtgatgtcagcgttgaactgcgtgatgcg
gatcaacaggtggttgcaactggacaaggcactagcgggactttgcaa
gtggtgaatccgcacctctggcaaccgggtgaaggttatctctatgaa
ctgtgcgtcacagccaaaagccagacagagtgtgatatctacccgctt
cgcgtcggcatccggtcagtggcagtgaagggccaacagttcctgatt
aaccacaaaccgttctactttactggctttggtcgtcatgaagatgcg
gacttacgtggcaaaggattcgataacgtgctgatggtgcacgaccac
gcattaatggactggattggggccaactcctaccgtacctcgcattac
ccttacgctgaagagatgctcgactgggcagatgaacatggcatcgtg
gtgattgatgaaactgctgctgtcggctttcagctgtctttaggcatt
ggtttcgaagcgggcaacaagccgaaagaactgtacagcgaagaggca
gtcaacggggaaactcagcaagcgcacttacaggcgattaaagagctg
atagcgcgtgacaaaaaccacccaagcgtggtgatgtggagtattgcc
aacgaaccggatacccgtccgcaaggtgcacgggaatatttcgcgcca
ctggcggaagcaacgcgtaaactcgacccgacgcgtccgatcacctgc
gtcaatgtaatgttctgcgacgctcacaccgataccatcagcgatctc
tttgatgtgctgtgcctgaaccgttattacggatggtatgtccaaagc
ggcgatttggaaacggcagagaaggtactggaaaaagaacttctggcc
tggcaggagaaactgcatcagccgattatcatcaccgaatacggcgtg
gatacgttagccgggctgcactcaatgtacaccgacatgtggagtgaa
gagtatcagtgtgcatggctggatatgtatcaccgcgtctttgatcgc
gtcagcgccgtcgtcggtgaacaggtatggaatttcgccgattttgcg
acctcgcaaggcatattgcgcgttggcggtaacaagaaagggatcttc
actcgcgaccgcaaaccgaagtcggcggcttttctgctgcaaaaacgc
tggactggcatgaacttcggtgaaaaaccgcagcagggaggcaaacaa
gctagccaccaccaccaccaccacgtgtga GUS1381 SEQ ID NO: 32
MVDLTSLRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESR
AIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAV
THYGKVWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELN
WQTIPPGMVITDENGKKKQSYFHDFFNYAGIHRSVMLYTTPNTWVDDI
TVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQVVATGQGTSGTLQ
VVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGQQFLI
NHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHY
PYAEEMLDWADEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEA
VNGETQQAHLQAIKELIARDKNHPSVVMWSIANEPDTRPQGAREYFAP
LAEATRKLDPTRPITCVNVMFCDAHTDTISDLFDVLCLNRYYGWYVQS
GDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYTDMWSE
EYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIF
TRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQASHHHHHHV GUS1391Z SEQ ID NO: 33
atggtagatctgagggtaaatttctagtttttctccttcattttcttg
gttaggacccttttctctttttatttttttgagctttgatctttcttt
aaactgatctattttttaattgattggttatggtgtaaatattacata
gctttaactgataatctgattactttatttcgtgtgtctatgatgatg
atgatagttacagaaccgacgactcgtccgtcctgtagaacgtgaaat
caaaaaactcgacggcctgtgggcattcagtctggatcgcgaaaactg
tggaattgatcagcgttggtgggaaagcgcgttacaagaaagccgggc
aattgctgtgccaggcagttttaacgatcagttcgccgatgcagatat
tcgtaattatgcgggcaacgtctggtatcagcgcgaagtctttatacc
gaaaggttgggcaggccagcgtatcgtgctgcgtttcgatgcggtcac
tcattacggcaaagtgtgggtcaataatcaggaagtgatggagcatca
gggcggctatacgccatttgaagccgatgtcacgccgtatgttattgc
cgggaaaagtgtacgtatcaccgtttgtgtgaacaacgaactgaactg
gcagactatcccgccgggaatggtgattaccgacgaaaacggcaagaa
aaagcagtcttacttccatgatttctttaactatgccggaatccatcg
cagcgtaatgctctacaccacgccgaacacctgggtggacgatatcac
cgtggtgacgcatgtcgcgcaagactgtaaccacgcgtctgttgactg
gcaggtggtggccaatggtgatgtcagcgttgaactgcgtgatgcgga
tcaacaggtggttgcaactggacaaggcactagcgggactttgcaagt
ggtgaatccgcacctctggcaaccgggtgaaggttatctctatgaact
gtgcgtcacagccaaaagccagacagagtgtgatatctacccgcttcg
cgtcggcatccggtcagtggcagtgaagggcgaacagttcctgattaa
ccacaaaccgttctactttactggctttggtcgtcatgaagatgcgga
cttacgtggcaaaggattcgataacgtgctgatggtgcacgaccacgc
attaatggactggattggggccaactcctaccgtacctcgcattaccc
ttacgctgaagagatgctcgactgggcagatgaacatggcatcgtggt
gattgatgaaactgctgctgtcggctttaacctctctttaggcattgg
tttcgaagcgggcaacaagccgaaagaactgtacagcgaagaggcagt
caacggggaaactcagcaagcgcacttacaggcgattaaagagctgat
agcgcgtgacaaaaaccacccaagcgtggtgatgtggagtattgccaa
cgaaccggatacccgtccgcaagtgcacgggaatatttcgccactggc
ggaagcaacgcgtaaactcgacccgacgcgtccgatcacctgcgtcaa
tgtaatgttctgcgacgctcacaccgataccatcagcgatctctttga
tgtgctgtgcctgaaccgttattacggatggtatgtccaaagcggcga
tttggaaacggcagagaaggtactggaaaaagaacttctggcctggca
ggagaaactgcatcagccgattatcatcaccgaatacggcgtggatac
gttagccgggctgcactcaatgtacaccgacatgtggagtgaagagta
tcagtgtgcatggctggatatgtatcaccgcgtctttgatcgcgtcag
cgccgtcgtcggtgaacaggtatggaatttcgccgattttgcgacctc
gcaaggcatattgcgcgttggcggtaacaagaaagggatcttcactcg
cgaccgcaaaccgaagtcggcggcttttctgctgcaaaaacgctggac
tggcatgaacttcggtgaaaaaccgcagcagggaggcaaacaagctag
ccaccaccaccaccaccacgtgtga GUS1391Z SEQ ID NO: 34
MVDLRVNRRLVRPVEREIKKLDGLWAFSLDRENCGIDQRWWESALQES
RAIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDA
VTHYGKVWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNEL
NWQTIPPGMVITDENGKKKQSYFHDFFNYAGIHRSVMLYTTPNTWVDD
ITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQVVATGQGTSGTL
QVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGEQFL
INHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSH
YPYAEEMLDWADEHGIVVIDETAAVGFNLSLGIGFEAGNKPKELYSEE
AVNGETQQAHLQAIKELIARDKNHPSVVMWSIANEPDTRPQVHGNISP
LAEATRKLDPTRPITCVNVMFCDAHTDTISDLFDVLCLNRYYGWYVQS
GDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYTDMWSE
EYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIF
TRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQASHHHHHHV
Salmon Calcitonin BAC57417
Protein Sequence:
TABLE-US-00022 [0104] SEQ ID NO: 35
KCSNLSTCVLGKLSQELHKLQTYPRTNTGSGTPG
DNA Sequence:
TABLE-US-00023 [0105] SEQ ID NO: 36
aagtgctccaacctctctacctgcgttcttggtaagctctctcaggag
cttcacaagctccagacttaccctagaaccaacactggttccggtacc cctggt
hEGF--Construction Based in the AAF85790 Without the Signal
Peptide
Protein Sequence:
TABLE-US-00024 [0106] SEQ ID NO: 37 NSDSECPLSH DGYCLHDGVC
MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR
DNA Sequence:
TABLE-US-00025 [0107] SEQ ID NO: 38
aactctgattcagaatgcccactcagtcacgacggatattgtcttcac
gatggggtatgcatgtacatcgaggccttggacaagtacgcatgtaat
tgtgtagtgggatacattggtgaacgctgtcagtatcgagacttgaaa
tggtgggagcttaggtga
hGH--Construction Based in the P01241 Without the Signal
Peptide
Protein Sequence:
TABLE-US-00026 [0108] SEQ ID NO: 39
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNP
QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSV
FANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYS
KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF
DNA Sequence:
TABLE-US-00027 [0109] SEQ ID NO: 40
ttcccaaccattcccttatccaggctttttgacaacgctatgctccgcgc
ccatcgtctgcaccagctggcctttgacacctaccaggagtttgaagaag
cctatatcccaaaggaacagaagtattcattcctgcagaacccccagacc
tccctctgtttctcagagtctattccgacaccctccaacagggaggaaac
acaacagaaatccaacctagagctgctccgcatctccctgctgctcatcc
agtcgtggctggagcccgtgcagttcctcaggagtgtcttcgccaacagc
ctggtgtacggcgcctctgacagcaacgtctatgacctcctaaaggacct
agaggaaggcatccaaacgctgatggggaggctggaagatggcagccccc
ggactgggcagatcttcaagcagacctacagcaagttcgacacaaactca
cacaacgatgacgcactactcaagaactacgggctgctctactgcttcag
gaaggacatggacaaggtcgagacattcctgcgcatcgtgcagtgccgct
ctgtggagggcagctgtggcttctga
[0110] In another embodiment, the recombinant fusion protein
further comprises in addition to the sequences of the PBIS and
product of interest, a spacer amino acid sequence. The spacer amino
acid sequence can be an amino acid sequence cleavable by enzymatic
or chemical means or not cleavable. By "not cleavable" it is meant
that cleavage of the spacer does not occur without destruction of
some or all of the biologically active polypeptide.
[0111] In a particular embodiment, the spacer amino acid sequence
is placed between the PBIS and biologically active polypeptide. An
illustrative amino acid sequence is cleavable by a protease such as
an enterokinase, Arg-C endoprotease, Glu-C endoprotease, Lys-C
endoprotease, Factor Xa, SUMO proteases [Tauseef et al., 2005
Protein Expr. Purif. 2005 September 43(1):1-9] and the like.
Alternatively, the spacer amino acid sequence corresponds to an
auto-cleavable sequence such as the FMDV viral auto-processing 2A
sequence, protein introns (inteins) such as the Ssp DNAb intein and
the like as are commercially available from New England Biolabs and
others. The use of an intein linker sequence is preferred as such
sequences can be selectively induced to cause protein splicing and
thereby eliminate themselves from an expressed, recovered, protein.
Inteins are particularly interesting since they do not require
large protein enzymes to reach their target site in order to cleave
the PBIS from the protein of interest. This property may be
particularly useful for direct isolation of proteins of interest
from intact RPBLAs. Alternatively, an amino acid sequence is
encoded that is specifically cleavable by a chemical reagent, such
as, for example, cyanogen bromide that cleaves at methionine
residues.
[0112] In a further embodiment, the nucleic acid sequence used for
transformation or transduction purposes is as disclosed according
to co-assigned patent application WO 2004003207, with or without
the nucleic acid sequence coding for the cleavable amino acid
sequence.
Methods of Preparation
[0113] In a preferred embodiment, the fusion proteins are prepared
according to a method that comprises transforming a eukaryotic host
cell system such as an animal, animal cell culture, plant or plant
cell culture, fungus culture, insect cell culture or algae culture
with a nucleic acid (DNA or RNA) sequence comprising (i) a first
nucleic acid coding for a PBIS that is operatively linked in frame
to (ii) a second nucleic acid sequence comprising the nucleotide
sequence coding for a polypeptide product of interest that is
biologically active; that is, the nucleic acid sequence that
encodes the PBIS is chemically bonded (peptide bonded) to the
sequence that encodes the polypeptide of interest such that both
polypeptides are expressed from their proper reading frames and the
protein of interest is biologically active. It is also contemplated
that appropriate regulatory sequences be present on either side of
the nucleic acid sequences that encode the PBIS and protein of
interest as is discussed hereinafter. Such control sequences are
well known and are present in commercially available vectors. The
use of indirect means of introducing DNA, such as via viral
transduction or infection, is also contemplated, and shall be used
interchangeably with direct DNA delivery methods such as
transfection.
[0114] The transformed host cell or entity is maintained for a time
period and under culture conditions suitable for expression of the
fusion protein and assembly of the expressed fusion protein into
RPBLAs. Upon expression, the resulting fusion protein accumulates
in the transformed host-system as high density RPBLAs. The fusion
protein can then be recovered from the host cells or the host cells
containing the fusion protein can be used as desired, as for an
animal food containing an added nutrient or supplement. The fusion
protein can be isolated as part of the RPBLAs or free from the
RPBLAs.
[0115] Culture conditions suitable for expression of the fusion
protein are typically different for each type of host entity or
host cell. However, those conditions are known by skilled workers
and are readily determined. Similarly, the duration of maintenance
can differ with the host cells and with the amount of fusion
protein desired to be prepared. Again, those conditions are well
known and can readily be determined in specific situations.
Additionally, specific culture conditions can be obtained from the
citations herein.
[0116] In one embodiment, the 3' end of the first nucleic acid
sequence (i) is linked (bonded) to the 5' end of the second nucleic
acid sequence (ii). In other embodiment, the 5' end of the first
nucleic acid sequence (i) is linked (bonded) to the 3' end of the
second nucleic acid sequence (ii). In another embodiment, the PBIS
comprises a storage protein or a modified storage protein, a
fragment or a modified fragment thereof.
[0117] In another particular embodiment, a fusion protein is
prepared according to a method that comprises transforming the host
cell system such as an animal, animal cell culture, plant, plant
cell culture, fungus or algae with a nucleic acid sequence
comprising, in addition to the nucleic acid sequences (i) and (ii)
previously mentioned, an in frame nucleic acid sequence (iii) that
codes for a spacer amino acid sequence. The spacer amino acid
sequence can be an amino acid sequence cleavable by enzymatic or
chemical means or not cleavable, as noted before. In one particular
embodiment, the nucleic acid sequence (iii) is placed between said
nucleic acid sequences (i) and (ii), e.g., the 3' end of the third
nucleic acid sequence (iii) is linked to the 5' end of the second
nucleic acid sequence (ii). In another embodiment, the 5' end of
the third nucleic acid sequence (iii) is linked to the 3' end of
the second nucleic acid sequence (ii).
[0118] A nucleic acid sequence (segment) that encodes a previously
described fusion protein molecule or a complement of that coding
sequence is also contemplated herein. Such a nucleic acid segment
is present in isolated and purified form in some preferred
embodiments.
[0119] In living organisms, the amino acid residue sequence of a
protein or polypeptide is directly related via the genetic code to
the deoxyribonucleic acid (DNA) sequence of the gene that codes for
the protein. Thus, through the well-known degeneracy of the genetic
code additional DNAs and corresponding RNA sequences (nucleic
acids) can be prepared as desired that encode the same fusion
protein amino acid residue sequences, but are sufficiently
different from a before-discussed gene sequence that the two
sequences do not hybridize at high stringency, but do hybridize at
moderate stringency.
[0120] High stringency conditions can be defined as comprising
hybridization at a temperature of about 50.degree.-55.degree. C. in
6.times.SSC and a final wash at a temperature of 68.degree. C. in
1-3.times.SSC. Moderate stringency conditions comprise
hybridization at a temperature of about 50.degree. C. to about
65.degree. C. in 0.2 to 0.3 M NaCl, followed by washing at about
50.degree. C. to about 55.degree. C. in 0.2.times.SSC, 0.1% SDS
(sodium dodecyl sulfate).
[0121] A nucleic sequence (DNA sequence or an RNA sequence) that
(1) itself encodes, or its complement encodes, a fusion protein
containing a protein body-inducing sequence (PBIS) and a
polypeptide of interest is also contemplated herein. As is
well-known, a nucleic acid sequence such as a contemplated nucleic
acid sequence is expressed when operatively linked to an
appropriate promoter in an appropriate expression system as
discussed elsewhere herein. This nucleic acid sequence can be
delivered directly or indirectly (via an appropriate vector
organism such as a virus or bacterium) to the host eukaryotic cell,
and can be integrated stably into the host nuclear or organellar
genome, or transiently expressed without genome integration.
[0122] Different hosts often have preferences for a particular
codon to be used for encoding a particular amino acid residue. Such
codon preferences are well known and a DNA sequence encoding a
desired fusion protein sequence can be altered, using in vitro
mutagenesis for example, so that host-preferred codons are utilized
for a particular host in which the fusion protein is to be
expressed.
[0123] A recombinant nucleic acid molecule such as a DNA molecule,
comprising a gene vector or construct containing one or more
regulatory sequences (control elements) such as a promoter suitable
for driving the expression of the gene in a compatible eukaryotic
host cell organism operatively linked to an exogenous nucleic acid
segment (e.g., a DNA segment or sequence) that defines a gene that
encodes a contemplated fusion protein, as discussed above, is also
contemplated in this invention. More particularly, also
contemplated is a recombinant DNA molecule that comprises a gene
vector comprising a promoter for driving the expression of the
fusion protein in host organism cells operatively linked to a DNA
segment that defines a gene encodes a protein body-inducing
sequence (PBIS) linked to a polypeptide of interest. That
recombinant DNA molecule, upon suitable transfection and expression
in a host eukaryotic cell, provides a contemplated fusion protein
as RPBLAs.
[0124] As is well known in the art, so long as the required nucleic
acid, illustratively DNA sequence, is present, (including start and
stop signals), additional base pairs can usually be present at
either end of the DNA segment and that segment can still be
utilized to express the protein. This, of course, presumes the
absence in the segment of an operatively linked DNA sequence that
represses expression, expresses a further product that consumes the
fusion protein desired to be expressed, expresses a product that
consumes a wanted reaction product produced by that desired fusion
protein, or otherwise interferes with expression of the gene of the
DNA segment.
[0125] Thus, so long as the DNA segment is free of such interfering
DNA sequences, a DNA segment of the invention can be about 500 to
about 15,000 base pairs in length. The maximum size of a
recombinant DNA molecule, particularly an expression vector, is
governed mostly by convenience and the vector size that can be
accommodated by a host cell, once all of the minimal DNA sequences
required for replication and expression, when desired, are present.
Minimal vector sizes are well known. Such long DNA segments are not
preferred, but can be used.
[0126] A DNA segment that encodes a before-described fusion protein
can be synthesized by chemical techniques, for example, the
phosphotriester method of Matteucci et al., 1981 J. Am. Chem. Soc.,
103:3185. Of course, by chemically synthesizing the coding
sequence, any desired modifications can be made simply by
substituting the appropriate bases for those encoding the native
amino acid residue sequence. However, DNA segments including
sequences specifically discussed herein are preferred.
[0127] DNA segments containing a gene encoding the fusion protein
are preferably obtained from recombinant DNA molecules (plasmid
vectors) containing that gene. A vector that directs the expression
of a fusion protein gene in a host cell is referred to herein as an
"expression vector".
[0128] An expression vector contains expression control elements
including the promoter. The fusion protein-coding gene is
operatively linked to the expression vector to permit the promoter
sequence to direct RNA polymerase binding and expression of the
fusion protein-encoding gene. Useful in expressing the polypeptide
coding gene are promoters that are inducible, viral, synthetic,
constitutive as described by Paszkowski et al., 1989 EMBO J.,
3:2719 and Odell et al., 1985 Nature, 313:810, as well as
temporally regulated, spatially regulated, and spatiotemporally
regulated as given in Chua et al., 1989 Science, 244:174-181.
[0129] Expression vectors compatible with eukaryotic cells, such as
those compatible with cells of mammals, algae or insects and the
like, are contemplated herein. Such expression vectors can also be
used to form the recombinant DNA molecules of the present
invention. Eukaryotic cell expression vectors are well known in the
art and are available from several commercial sources. Normally,
such vectors contain one or more convenient restriction sites for
insertion of the desired DNA segment and promoter sequences.
Optionally, such vectors contain a selectable marker specific for
use in eukaryotic cells.
[0130] Production of a fusion protein by recombinant DNA expression
in mammalian cells is illustrated hereinafter using a recombinant
DNA vector that expresses the fusion protein gene in Chinese
hamster ovary (CHO) host cells, Cos1 monkey host and human 293T
host cells. This is accomplished using procedures that are well
known in the art and are described in more detail in Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold
Spring Harbor Laboratories (1989).
[0131] An insect cell system can also be used to express a
contemplated fusion protein. For example, in one such system
Autographa californica nuclear polyhedrosis virus (AcNPV) or
baculovirus is used as a vector to express foreign genes in
Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding a fusion protein can be cloned into a
non-essential region of the virus, such as the polyhedrin gene, and
placed under control of the polyhedrin promoter. Successful
insertion of a fusion protein sequence renders the polyhedrin gene
inactive and produces recombinant virus lacking coat protein. The
recombinant viruses can then be used to infect, for example, S.
Frugiperda cells or Trichoplusia larvae in which the fusion protein
can be expressed, for example as described in Engelhard et al.
(1994) Proc. Natl. Acad. Sci., USA, 91:3224-3227; and V. Luckow,
"Insect Cell Expression Technology", pages 183-218, in Protein
Engineering: Principles and Practice, J. L. Cleland et al. eds.,
Wiley-Liss, Inc, 1996). Heterologous genes placed under the control
of the polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcNPV) are often expressed at high levels
during the late stages of infection.
[0132] Recombinant baculoviruses containing the fusion protein gene
are constructed using the baculovirus shuttle vector system (Luckow
et al., 1993 J. Virol., 67:4566-4579], sold commercially as the
Bac-To-Bac.TM. baculovirus expression system (Life Technologies).
Stocks of recombinant viruses are prepared and expression of the
recombinant protein is monitored by standard protocols (O'Reilly et
al., Baculovirus Expression Vectors: ALaboratory Manual, W.H.
Freeman and Company, New York, 1992; and King et al., The
Baculovirus Expression System: A Laboratory Guide, Chapman &
Hall, London, 1992). Use of baculovirus or other delivery vectors
in mammalian cells, such as the `BacMam` system described by T.
Kost and coworkers (see, for example Merrihew et al., 2004 Methods
Mol. Biol. 246:355-365), or other such systems as are known to
those skilled in the art are also contemplated in the instant
invention.
[0133] The choice of which expression vector and ultimately to
which promoter a fusion protein-encoding gene is operatively linked
depends directly on the functional properties desired, e.g., the
location and timing of protein expression, and the host cell to be
transformed. These are well known limitations inherent in the art
of constructing recombinant DNA molecules. However, a vector useful
in practicing the present invention can direct the replication, and
preferably also the expression (for an expression vector) of the
fusion protein gene included in the DNA segment to which it is
operatively linked.
[0134] Typical vectors useful for expression of genes in cells from
higher plants and mammals are well known in the art and include
plant vectors derived from the tumor-inducing (Ti) plasmid of
Agrobacterium tumefaciens described by Rogers et al, (1987) Meth.
in Enzymol., 153:253-277 and mammalian expression vectors pKSV-10,
above, and pCI-neo (Promega Corp., #E1841, Madison, Wis.). However,
several other expression vector systems are known to function in
plants including pCaMVCN transfer control vector described by Fromm
et al. (1985) Proc. Natl. Acad. Sci. USA, 82:58-24. Plasmid pCaMVCN
(available from Pharmacia, Piscataway, N.J.) includes the
cauliflower mosaic virus CaMV 35S promoter.
[0135] The above plant expression systems typically provide
systemic or constitutive expression of an inserted transgene.
Systemic expression can be useful where most or all of a plant is
used as the source of RPBLAs and their fusion proteins. However, it
can be more efficacious to express RPBLAs and their fusion protein
contents in a plant storage organ such as a root, seed or fruit
from which the particles can be more readily isolated or
ingested.
[0136] One manner of achieving storage organ expression is to use a
promoter that expresses its controlled gene in one or more
preselected or predetermined non-photosynthetic plant organs.
Expression in one or more preselected storage organs with little or
no expression in other organs such as roots, seed or fruit versus
leaves or stems is referred to herein as enhanced or preferential
expression. An exemplary promoter that directs expression in one or
more preselected organs as compared to another organ at a ratio of
at least 5:1 is defined herein as an organ-enhanced promoter.
Expression in substantially only one storage organ and
substantially no expression in other storage organs is referred to
as organ-specific expression; i.e., a ratio of expression products
in a storage organ relative to another of about 100:1 or greater
indicates organ specificity. Storage organ-specific promoters are
thus members of the class of storage organ-enhanced promoters.
[0137] Exemplary plant storage organs include the roots of carrots,
taro or manioc, potato tubers, and the meat of fruit such as red
guava, passion fruit, mango, papaya, tomato, avocado, cherry,
tangerine, mandarin, palm, melons such cantaloupe and watermelons
and other fleshy fruits such as squash, cucumbers, mangos,
apricots, peaches, as well as the seeds of maize (corn), soybeans,
rice, oil seed rape and the like.
[0138] The CaMV 35S promoter is normally deemed to be a
constitutive promoter. However, research has shown that a 21-bp
region of the CaMV 35S promoter, when operatively linked into
another, heterologous usual green tissue promoter, the rbcS-3A
promoter, can cause the resulting chimeric promoter to become a
root-enhanced promoter. That 21-bp sequence is disclosed in U.S.
Pat. No. 5,023,179. The chimeric rbcS-3A promoter containing the
21-bp insert of U.S. Pat. No. 5,023,179 is a useful root-enhanced
promoter herein.
[0139] A similar root-enhanced promoter, which includes the above
21-bp segment, is the -90 to +8 region of the CAMV 35S promoter
itself. U.S. Pat. No. 5,110,732 discloses that that truncated CaMV
35S promoter provides enhanced expression in roots and the radicle
of seed, a tissue destined to become a root. That promoter is also
useful herein.
[0140] Another useful root-enhanced promoter is the -1616 to -1
promoter of the oil seed rape (Brassica napus L.) gene disclosed in
PCT/GB92/00416 (WO 91/13922 published Sep. 19, 1991), E. coli
DH5-alpha harboring plasmid pRlambdaS4 and bacteriophage
lambda.beta.l that contain this promoter were deposited at the
National Collection of Industrial and Marine Bacteria, Aberdeen, G
B on Mar. 8, 1990 and have accession numbers NCIMB40265 and
NCIMB40266. A useful portion of this promoter can be obtained as a
1.0 kb fragment by cleavage of the plasmid with HaeIII.
[0141] A preferred root-enhanced promoter is the mannopine synthase
(mas) promoter present in plasmid pKan2 described by DiRita and
Gelvin (1987) Mol. Gen. Genet, 207:233-241. This promoter is
removable from its plasmid pKan2 as a XbaI-XbaII fragment.
[0142] The preferred mannopine synthase root-enhanced promoter is
comprised of the core mannopine synthase (mas) promoter region up
to position -138 and the mannopine synthase activator from -318 to
-213, and is collectively referred to as AmasPmas. This promoter
has been found to increase production in tobacco roots about 10- to
about 100-fold compared to leaf expression levels.
[0143] Another root specific promoter is the about 500 bp 5'
flanking sequence accompanying the hydroxyproline-rich
glycopeprotein gene, HRGPnt3, expressed during lateral root
initiation and reported by Keller et al. (1989) Genes Dev.,
3:1639-1646. Another preferred root-specific promoter is present in
the about -636 to -1 5' flanking region of the tobacco
root-specific gene ToRBF reported by Yamamoto et al, (1991) Plant
Cell, 3:371-381. The cis-acting elements regulating expression are
more specifically located by those authors in the region from about
-636 to about -299 5' from the transcription initiation site.
Yamamoto et al, reported steady state mRNA production from the
ToRBF gene in roots, but not in leaves, shoot meristems or
stems.
[0144] Still another useful storage organ-specific promoter are the
5' and 3' flanking regions of the fruit-ripening gene E8 of the
tomato, Lycopersicon esculentum. These regions and their cDNA
sequences are illustrated and discussed in Deikman et al. (1988)
EMBO J., 7(11):3315-3320 and (1992) Plant Physiol.,
100:2013-2017.
[0145] Three regions are located in the 2181 bp of the 5' flanking
sequence of the gene and a 522 bp sequence 3' to the poly (A)
addition site appeared to control expression of the E8 gene. One
region from -2181 to -1088 is required for activation of E8 gene
transcription in unripe fruit by ethylene and also contributes to
transcription during ripening. Two further regions, -1088 to -863
and -409 to -263, are unable to confer ethylene responsiveness in
unripe fruit but are sufficient for E8 gene expression during
ripening.
[0146] The maize sucrose synthase-1 (Sh) promoter that in corn
expresses its controlled enzyme at high levels in endosperm, at
much reduced levels in roots and not in green tissues or pollen has
been reported to express a chimeric reporter gene,
.beta.-glucuronidase (GUS), specifically in tobacco phloem cells
that are abundant in stems and roots. Yang et al. (1990) Proc.
Natl. Acad. Sci., U.S.A., 87:4144-4148. This promoter is thus
useful for plant organs such as fleshy fruits like melons, e.g.
cantaloupe, or seeds that contain endosperm and for roots that have
high levels of phloem cells.
[0147] Another exemplary tissue-specific promoter is the lectin
promoter, which is specific for seed tissue. The lectin protein in
soybean seeds is encoded by a single gene (Le1) that is only
expressed during seed maturation and accounts for about 2 to about
5 percent of total seed mRNA. The lectin gene and seed-specific
promoter have been fully characterized and used to direct seed
specific expression in transgenic tobacco plants. See, e.g., Vodkin
et al. (1983) Cell, 34:1023 and Lindstrom et al. (1990)
Developmental Genetics, 11:160.
[0148] A particularly preferred tuber-specific expression promoter
is the 5' flanking region of the potato patatin gene. Use of this
promoter is described in Twell et al. (1987) Plant Mol. Biol.,
9:365-375. This promoter is present in an about 406 bp fragment of
bacteriophage LPOTI. The LPOTI promoter has regions of over 90
percent homology with four other patatin promoters and about 95
percent homology over all 400 bases with patatin promoter PGT5.
Each of these promoters is useful herein. See, also, Wenzler et al.
(1989) Plant Mol. Biol., 12:41-50.
[0149] Still further higher plant organ-enhanced and organ-specific
promoter are disclosed in Benfey et al. (1988) Science,
244:174-181.
[0150] Each of the promoter sequences utilized is substantially
unaffected by the amount of RPBLAs in the cell. As used herein, the
term "substantially unaffected" means that the promoter is not
responsive to direct feedback control (inhibition) by the RPBLAs
accumulated in transformed cells or transgenic plant.
[0151] Transfection of plant cells using Agrobacterium tumefaciens
is typically best carried out on dicotyledonous plants. Monocots
are usually most readily transformed by so-called direct gene
transfer of protoplasts. Direct gene transfer is usually carried
out by electroportation, by polyethyleneglycol-mediated transfer or
bombardment of cells by microprojectiles carrying the needed DNA.
These methods of transfection are well-known in the art and need
not be further discussed herein. Methods of regenerating whole
plants from transfected cells and protoplasts are also well-known,
as are techniques for obtaining a desired protein from plant
tissues, See, also, U.S. Pat. Nos. 5,618,988 and 5,679,880 and the
citations therein.
[0152] A transgenic plant formed using Agrobacterium
transformation, electroportation or other methods typically
contains a single gene on one chromosome. Such transgenic plants
can be referred to as being heterozygous for the added gene.
However, inasmuch as use of the word "heterozygous" usually implies
the presence of a complementary gene at the same locus of the
second chromosome of a pair of chromosomes, and there is no such
gene in a plant containing one added gene as here, it is believed
that a more accurate name for such a plant is an independent
segregant, because the added, exogenous chimer molecule-encoding
gene segregates independently during mitosis and meiosis. A
transgenic plant containing an organ-enhanced promoter driving a
single structural gene that encodes a contemplated HBc chimeric
molecule; i.e., an independent segregant, is a preferred transgenic
plant.
[0153] More preferred is a transgenic plant that is homozygous for
the added structural gene; i.e., a transgenic plant that contains
two added genes, one gene at the same locus on each chromosome of a
chromosome pair. A homozygous transgenic plant can be obtained by
sexually mating (selfing) an independent segregant transgenic plant
that contains a single added gene, germinating some of the seed
produced and analyzing the resulting plants produced for enhanced
chimer particle accumulation relative to a control (native,
non-transgenic) or an independent segregant transgenic plant. A
homozygous transgenic plant exhibits enhanced chimer particle
accumulation as compared to both a native, non-transgenic plant and
an independent segregant transgenic plant.
[0154] It is to be understood that two different transgenic plants
can also be mated to produce offspring that contain two
independently segregating added, exogenous (heterologous) genes.
Selfing of appropriate progeny can produce plants that are
homozygous for both added, exogenous genes that encode a chimeric
HBc molecule. Back-crossing to a parental plant and out-crossing
with a non-transgenic plant are also contemplated.
[0155] A transgenic plant of this invention thus has a heterologous
structural gene that encodes a contemplated chimeric HBc molecule.
A preferred transgenic plant is an independent segregant for the
added heterologous chimeric HBc structural gene and can transmit
that gene to its progeny. A more preferred transgenic plant is
homozygous for the heterologous gene, and transmits that gene to
all of its offspring on sexual mating.
[0156] The expressed RPBLAs and their fusion proteins can be
obtained from the expressing host cells by usual means utilized in
biochemical or biological recovery. Because the RPBLAs are dense
relative to the other proteins present in the host cells, the
RPBLAs are particularly amenable to being collected by
centrifugation of a cellular homogenate.
[0157] Thus, regions of different density are formed in the
homogenate to provide a region that contains a relatively enhanced
concentration of the RPBLAs and a region that contains a relatively
depleted concentration of the RPBLAs. The RPBLAs-depleted region is
separated from the region of relatively enhanced concentration of
RPBLAs, thereby purifying said fusion protein. The region of
relatively enhanced concentration of RPBLAs can thereafter be
collected or can be treated with one or more reagents or subjected
to one or more procedures prior to isolation of the RPBLAs or the
fusion protein therein. In some embodiments, the collected RPBLAs
are used as is, without the need to isolate the fusion protein, as
where the RPBLAs are used as an oral vaccine. The fusion protein
containing the biologically active polypeptide can be obtained from
the collected RPBLAs by dissolution of the surrounding membrane in
an aqueous buffer containing a detergent and a reducing agent as
discussed previously. Illustrative reducing agents include
2-mercaptoethanol, thioglycolic acid and thioglycolate salts,
dithiothreitol (DTT), sulfite or bisulfite ions, followed by usual
protein isolation methods, Sodium dodecyl sulfate (SDS) is the
preferred detergent, although other ionic (deoxycholate,
'N-Lauroylsarcosine, and the like), non-ionic (Tween.RTM. 20,
Nonidet.RTM. P-40, octyl glucoside and the like) and zwitterionic
(CHAPS, Zwittergent.TM. 3-X serie and the like) surfactants can be
used. A minimal amount of surfactant that dissolves or disperses
the fusion protein is utilized.
Vaccines and Inocula
[0158] In yet another embodiment of the invention, RPBLAs are used
as the immunogen of an inoculum or vaccine in a human patient or
other suitable animal host such as a chimpanzee, mouse, rat, horse,
sheep, bovine, dog, cat or the like. An inoculum can induce a B
cell or T cell response (stimulation) such as production of
antibodies that immunoreact with the immunogenic epitope or
antigenic determinant, or T cell activation to such an epitope,
whereas a vaccine provides protection against the entity from which
the immunogen has been derived via one or both of a B cell or T
cell response.
[0159] The RPBLAs of a contemplated vaccine or inoculum appear to
act upon antigen presenting cells (APCs) such as dendritic cells
and monocytes/macrophages that engulf the RPBLAs and process their
contents. In acting upon those cell types, the RPBLAs improve the
antigen delivery to antigen-presenting cells. Those RPBLAs also
improve the antigen processing and presentation to
antigen-presenting cells.
[0160] Thus, the invention also contemplates a vaccine or inoculum
that comprises an immunogenic effective amount of recombinant
protein body-like assemblies (RPBLAs) that are dissolved or
dispersed in a pharmaceutically acceptable diluent. The RPBLAs
contain a recombinant fusion protein recombinant fusion protein
that itself contains two sequences linked together in which one
sequence is a protein body-inducing sequence (PBIS) and the other
is a biologically active polypeptide to which an immunological
response is to be induced by said vaccine or inoculum.
[0161] T cell activation can be measured by a variety of
techniques. In usual practice, a host animal is inoculated with a
contemplated RPBLA vaccine or inoculum, and peripheral mononuclear
blood cells (PMBC) are thereafter collected. Those PMBC are then
cultured in vitro in the presence of the biologically active
polypeptide (T cell immunogen) for a period of about three to five
days. The cultured PMBC are then assayed for proliferation or
secretion of a cytokine such as IL-2, GM-CSF of IFN-.gamma.. Assays
for T cell activation are well known in the art. See, for example,
U.S. Pat. No. 5,478,726 and the art cited therein.
[0162] Using antibody formation as exemplary, a contemplated
inoculum or vaccine comprises an immunogenically effective amount
of RPBLAs that are dissolved or dispersed in a pharmaceutically
acceptable diluent composition that typically also contains water.
When administered to a host animal in which an immunological
response to the biologically active polypeptide is to be induced by
the vaccine or inoculum such as a host animal in need of
immunization or in which antibodies are desired to be induced such
as a mammal (e.g., a mouse, dog, goat, sheep, horse, bovine,
monkey, ape, or human) or bird (e.g., a chicken, turkey, duck or
goose), an inoculum induces antibodies that immunoreact with one or
more antigenic determinants of the target biologically active
polypeptide.
[0163] The amount of RPBLA immunogen utilized in each immunization
is referred to as an immunogenically effective amount and can vary
widely, depending inter glia, upon the RPBLA immunogen, patient
immunized, and the presence of an adjuvant in the vaccine, as
discussed below. Immunogenically effective amounts for a (i)
vaccine and an (ii) inoculum provide the (i) protection or (ii)
antibody or T cell activity, respectively, discussed
hereinbefore.
[0164] Vaccines or inocula typically contain a RPBLA immunogen
concentration of about 1 microgram to about 1 milligram per
inoculation (unit dose), and preferably about 10 micrograms to
about 50 micrograms per unit dose. The term "unit dose" as it
pertains to a vaccine or inoculum of the present invention refers
to physically discrete units suitable as unitary dosages for
animals, each unit containing a predetermined quantity of active
material calculated to individually or collectively produce the
desired immunogenic effect in association with the required
diluent; i.e., carrier, or vehicle.
[0165] Vaccines or inocula are typically prepared from a recovered
RPBLA immunogen by dispersing the immunogen, in particulate form,
in a physiologically tolerable (acceptable) diluent vehicle such as
water, saline, phosphate-buffered saline (PBS), acetate-buffered
saline (ABS), Ringer's solution, or the like to form an aqueous
composition. The diluent vehicle can also include oleaginous
materials such as peanut oil, squalane, or squalene as is discussed
hereinafter.
[0166] The preparation of inocula and vaccines that contain
proteinaceous materials as active ingredients is also well
understood in the art. Typically, such inocula or vaccines are
prepared as parenterals, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection can also be prepared. The preparation can also
be emulsified, which is particularly preferred.
[0167] The immunogenically active RPBLAs are often mixed with
excipients that are pharmaceutically acceptable and compatible with
the active ingredient. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol, or the like and combinations
thereof. In addition, if desired, an inoculum or vaccine can
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents that enhance the
immunogenic effectiveness of the composition.
[0168] The word "antigen" has been used historically to designate
an entity that is bound by an antibody or receptor, and also to
designate the entity that induces the production of the antibody.
More current usage limits the meaning of antigen to that entity
bound by an antibody or receptor, whereas the word "immunogen" is
used for the entity that induces antibody production or binds to
the receptor. Where an entity discussed herein is both immunogenic
and antigenic, reference to it as either an immunogen or antigen is
typically made according to its intended utility.
[0169] "Antigenic determinant" refers to the actual structural
portion of the antigen that is immunologically bound by an antibody
combining site or T-cell receptor. The term is also used
interchangeably with "epitope".
[0170] As used herein, the term "fusion protein" designates a
polypeptide that contains at least two amino acid residue sequences
not normally found linked together in nature that are operatively
linked together end-to-end (head-to-tail) by a peptide bond between
their respective carboxy- and amino-terminal amino acid residues.
The fusion proteins of the present invention are chimers of a
protein body-inducing sequence (PBIS) linked to a second sequence
that is a biologically active polypeptide product (e.g., peptide or
protein) of interest (target).
[0171] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description and the detailed
examples below utilize the present invention to its fullest extent.
The following preferred specific embodiments are, therefore, to be
construed as merely illustrative, and not limiting of the remainder
of the disclosure in any way whatsoever.
Example 1
Accumulation of RX3-ECFP Derived Fusion Proteins in Dense Fractions
of Transfected Mammal Cells
[0172] The polynucleotide sequence coding for the N-terminal
gamma-zein coding sequence RX3 (WO2004003207) was fused directly,
or through a linker consisting of five glycines, to the 5' end of
the sequence encoding ECFP, a cyan fluorescent variant of GFP.
Those constructs (FIG. 1A) that code for the fusion proteins
RX3-ECFP or RX3-Gx5-ECFP were introduced in cultured mammalian CHO
cells by the Lipofectamine-based transfection method (Invitrogen).
CHO cells transfected with plasmid pECFP-N1 (Clontech) containing
the gene sequence of a non-targeted (cytosolic) ECFP were used as
controls.
[0173] Transfected mammalian cell extracts were loaded on density
step gradients and centrifuged. The accumulation of recombinant
proteins in the different fractions was analyzed by immunoblot. The
results shown in FIG. 2A indicate that RX3-ECFP and RX3-Gx5-ECFP
sedimented in fractions F42, F56 and P corresponding to dense
RPBLAs, (FIG. 2A, lanes 3-5). This result demonstrates that the
fusion proteins are able to assemble and induce RPBLA formation.
Some fusion protein was also detected in the supernatant fraction
(FIG. 2A, lane 1), probably representing fusion proteins from the
RPBLAs solubilized partially during the extraction process, or
newly-synthesized fusion proteins that had not yet assembled.
[0174] In contrast, when mammalian cell extract transfected with
the control plasmid pECFP-N1 was loaded on the same density step
gradients, the ECFP protein was observed exclusively in the
supernatant. No traces of ECFP were detected in the dense fractions
indicating that the ECFP by itself is not able to aggregate and
form PB-like structures.
Example 2
Accumulation of Active ECFP Fused to PBIS Domains in RPBLAs of
Transfected Mammal Cells
[0175] To determine if the fusion proteins RX3-ECFP, RX3-Gx5-ECFP
and 22aZ-ECFP are active inside the RPBLAs, confocal microscopic
analysis was performed to visualize target protein fluorescence in
transfected CHO cells (FIG. 1A). Cyan fluorescence was imaged by
excitation at 458 nm with an argon ion laser with an emission
window set at 470-530 nm. As shown in FIG. 3, the corresponding
fusion proteins, RX3-ECFP (FIG. 3A) and RX3-Gx5-ECFP (FIG. 3B) and
22aZ-ECFP (FIG. 3D), were detected in proximity to the ER,
indicating that the gamma-zein and the alpha-zein signal peptides
are functional in mammalian cells where they mediates the
translocation of the fusion protein into the ER.
[0176] In addition, the fusion proteins surprisingly also
accumulated preferentially into large and dense spherical
structures that strongly resembled both authentic PBs of cereal
seed and RPBLAs in heterologous systems visualized by
immunodetection. The intense fluorescence emission observed in
these structures indicates that the fusion proteins remain properly
folded, and therefore active, in spite of being tightly packaged
inside the RPBLAs. It is also important to note that RX3 domains,
as well as other protein body inducing sequences (PBIS) responsible
for the formation of PBs and PB-like structures contain multiple
cysteine residues. Although it might be predicted that such
cysteines could form disulfide bonds with target protein cysteines
and hence interfere with the proper folding of the target proteins
this was not observed to be the case. Both active target protein
(ECFP fluorescence) and functional PBIS (formation of RPBLAs) were
observed.
[0177] As a control, the construct pECFP-N1 was used to transfect
CHO cells. The expression of a cytosolic ECFP showed a homogeneous
fluorescence pattern all along the cell, including the nucleus
(FIG. 3C).
Example 3
Subcellular Localization of Other Fluorescent Proteins Fused to RX3
in CHO Cells
[0178] The sub-cellular localization of RX3-DsRED and RX3-GFP
fusion proteins in transiently transfected CHO cells was analyzed
by confocal microscopy to analyze whether other fluorescent
proteins than ECFP fused to RX3 are properly folded and bioactive
inside RPBLAs. It is important to note that DsRED shares no
homology to ECFP, which implies a completely different folding
mechanism. Fluorescence images from the transfected cells were
obtained by using a confocal laser scanning microscope (Leica TCS
SP, Heidelberg, Germany) fitted with spectrophotometers for
emission band wavelength selection. Green fluorescent images were
collected by excitation at 488 nm with an Argon ion laser using an
emission window set at 495-535 nm. Red fluorescent images were
collected using 543 nm excitation with a HeNe laser and an emission
window of 550-600 nm. Optical sections were 0.5 .mu.m thick.
[0179] The expression of RX3-GFP (FIG. 3E) and RX3-DsRED (FIG. 3F)
fusion proteins in CHO cells produced a large amount of highly
fluorescent round-shaped RPBLAs. These results confirm that both
fusion proteins are properly folded and active inside the intact
RPBLAs.
Example 4
Subcellular Localization of Fluorescent RX3 Fusion Proteins in
Plants and Insects
[0180] In order to analyze whether host cells other than CHO cells
can produce RPBLAs containing active fluorescent proteins fused to
RX3 domains, tobacco plants were transiently transformed with
RX3-GFP by syringe agroinfiltration. The analysis by confocal
microscopy of the epidermal cells (FIGS. 4A and 4B) revealed the
presence of a large amount of fluorescent RPBLAs. Similar results
were obtained when transformed tobacco mesophyll cells were
analyzed.
[0181] Expression of fully functional DsRED fluorescent protein in
RPBLAs was also obtained when Spodoptera Sf9 insect cells or insect
larvae (Trichoplusia ni) were infected with baculovirus carrying
expression plasmids coding for the fusion protein RX3-DsRED under
control of the insect polyhedrin promoter. As shown in FIG. 4C,
infected insect cells accumulated a large number of fluorescent
RPBLAs containing active RX3-DsRED fusion protein. These structures
were approximately 0.5 micrometers in diameter. Confocal analysis
of infected larvae also showed an impressive amount of fluorescent
RPBLAs in all tissues analyzed. In FIG. 4D, epidermal cells from
infected larvae show RPBLAs containing active RX3-DsRED.
Interestingly, DsRED fluorescence was not observed in insect
haemolymph, suggesting that the expressed protein remained
sequestered entirely within RPBLAs.
Example 5
Activity of RX3-hGH Assembled in RPBLAs in CHO Cells
[0182] Studies were undertaken to determine if human growth hormone
(hGH) produced in RPBLAs still retained biological activity. Growth
hormone was chosen because this molecule contains 2 disulphide
bonds that are required for the proper folding of the protein into
an active conformation. The RX3 domain also contains cysteine
residues involved in disulphide bonds that are essential for the
assembly and stabilization of the RPBLAs, which could interfere in
the proper folding of the hGH.
[0183] The p3.1-RX3-hGH construct was introduced into CHO cells by
transient transfection with the lipofectamine protocol
(Invitrogen). Four days after transfection the cells were fixed and
permeabilized. In order to visualize RPBLAs, the cells were
incubated with primary anti-RX3 or anti-hGH antisera followed by a
secondary antibody conjugated to Alexa Fluor 488 (Invitrogen) Both
RX3 and hGH immunodetection (FIGS. 5A and 5B, respectively)
revealed the presence of identical large and densely immunostaining
structures of ca, 1-3 um containing the RX3-hGH fusion protein.
[0184] In order to verify that the PB-like organelles observed by
immunodetection were in fact RBPLAs, CHO cells expressing RX3-hGH
were homogenized, and the homogenates loaded on a density step
gradient and centrifuged as previously described. The accumulation
of RX3-hGH in the different fractions was analyzed by immunoblot.
As can be seen, part of the fusion protein was present in the
supernatant, representing non-assembled RX3-hGH, but most of the
fusion protein was detected in fraction F56 corresponding to dense
RPBLAs (FIG. 2B, lanes 2 and 5, respectively).
[0185] The isolated F56 fraction was diluted 3-fold in buffer PBP4
(100 mM Tris pH7.5, 50 mM KCl, 5 mM MgCl.sub.2, 5 mM EDTA) and
centrifuged at 8.0000.times.g in a swinging-bucket to recover the
RPBLAs in the pellet. The presence of hGH was quantified using an
ELISA assay (Active.RTM. Human Growth Hormone ELISA DSL-10-1900;
Diagnostic Systems Laboratories, Inc), which was able to detect the
hGH even in the presence of the intact RPBLA membrane.
[0186] The intact hGH RPBLA sample was analyzed for hormone
bioactivity using a commercial assay (Active.RTM. Bioactive Human
Growth Hormone ELISA DSL-10-11100; Diagnostic Systems Laboratories,
Inc). This bioactivity assay is based on the capacity of properly
folded hGH to interact with an hGH binding protein provided by the
kit, the interaction being dependent on correct functional
conformation of the hGH. The sample gave a positive result (24
ng/ml of bioactive protein), demonstrating that the hGH proteins
were correctly folded and presented on the outer surface of the
dense RPBLAs. Removal of the membrane surrounding the RPBLAs by
washing the preparation with 50 mM Tris pH 8 and 1% Triton X-100
and by sonicating at 50% amplitude and 50% cycle for 1 minute,
repeated times 5 (Ikasonic U200S--IKA Labortechnik) resulted in
greater specific activity (45 ng/ml) due to the exposure of
additional hGH molecules on the surface of the aggregates.
[0187] The activity of isolated RX3-hGH fusion proteins were also
determined following solubilized from RPBLAs isolated by density
gradient (F56, diluted 3-fold in buffer PBP4 and centrifuged at
80000.times.g in a swinging-bucket for 2 hours). The fusion protein
was solubilized in buffer S (Tris 50 mM, pH8 and 2% of .beta.-ME)
and sonicated (Clycle 5, Amplitude 50%, 1 minute, repeated five
times; Ikasonic U200S--IKA Labortechnik). After incubation at
37.degree. C. for 2 hours, the sample was centrifuged at
5000.times.g for 10 minutes, and the supernatant containing the
soluble RX3-hGH fusion protein was assayed to assess the bioactive
component of the fraction. The amount of fusion protein in the
supernatant was determined to be 250 ng/ml by ELISA (Active.RTM.
Human Growth Hormone ELISA--DSL-10-1900; Diagnostic Systems
Laboratories, Inc). The protein assayed in the bioactivity ELISA
assay (Active.RTM. Bioactive Human Growth Hormone
ELISA--DSL-10-11100; Diagnostic Systems Laboratories, Inc) gave a
result of 70 ng/ml, indicating that about 30% of the RX3-hGH fusion
protein was active. The loss of hGH activity could be a consequence
of the high concentration of reducing agent used in the
solubilization, or due to some impairing effect of the RX3 domain
over the hGH or the hGH binding protein.
[0188] Finally, the RX3-hGH fusion protein was cleaved by a site
specific protease to liberate the hGH from the fusion protein. The
solubilized RX3-hGH fusion protein was diluted 2-fold and the
digestion was performed using the EKmax kit as described by the
manufacturer (Invitrogen). Free hGH was isolated from the insoluble
uncleaved fusion protein fraction by centrifugation at
16000.times.g at 4.degree. C. for 1 hour. The soluble hGH was
recovered from the supernatant and assayed for bioactivity as
described above. The same value of 90 ng/ml was obtained for the
quantification and bioactivity ELISA assays (Active.RTM. Human
Growth Hormone ELISA--DSL-10-1900; Diagnostic Systems Laboratories,
Inc) and Active.RTM. Bioactive Human Growth Hormone
ELISA--DSL-10-11100; Diagnostic Systems Laboratories, Inc)
indicating that all the protein present as detected by the
quantification kit is also in the biologically active
conformation.
[0189] Summary table for the quantification and bioactivity of the
hGH protein in all the formulations is presented below:
TABLE-US-00028 Quantification Bioactivity Amount Amount Formulation
ng/ml ng/ml Intact RPBLAs 14 25 Membrane removed RPBLAs 35 45
Soluble RX3-hGH 250 70 Cleaved hGH 90 90
[0190] It is important to note that CHO cells stably transfected
with the vector p3.1-RX3 were used as a negative control. As shown
in FIG. 2B, RX3 without a fusion partner expressed in CHO cells
also accumulates in dense structures which can be isolated by
density step gradient in F56 (FIG. 2B, lane 5). Moreover, optical
analysis of CHO cells transfected with p3.1-RX3, showed that the
RX3 protein accumulate in RPBLAs (FIG. 5C) These control RX3 RPBLA
preparations and isolated RX3 protein showed no hGH activity in the
ELISA bioactivity assay.
Example 6
Activity of DNAb Intein After RX3-Int-hGH Solubilization from
RPBLAs from CHO Cells
[0191] The polynucleotide sequence coding for the Ssp DNAb intein
(New England Biolabs) was fused in frame to the 3' end of the RX3
sequence (WO2004003207), and to the 5' end of the hGH cDNA. The
resulting construct was cloned into vector pcDNA3.1(-) [FIG. 1A] to
form vector p3.1-RX3-I-hGH. As a negative control, an inactive
version of the same intein was produced by PCR where the amino acid
residue Asp154 was mutated to Ala [FIG. 1A] to form vector
p3.1-RX3-Im-hGH. The Asp154 amino acid residue has been reported to
be essential for the Ssp DNAb self-cleavage activity (Mathys et al,
GENE (1999) 231:1-13)
[0192] Immunochemical analysis of CHO cells transfected with
p3.1-RX3-I-hGH using anti-hGH antiserum revealed that the fusion
protein RX3-Int-hGH accumulated in large round-shaped RPBLAs,
similar to the ones observed in CHO cells expressing RX3-hGH
(compare FIGS. 5B and 5D). This result indicates that the fusion
protein containing the DNAb intein self-assembles and accumulates
into high density structures in a similar manner to fusions lacking
the intervening intein sequence.
[0193] CHO cells transfected with p3.1-RX3-I-hGH were homogenized,
the homogenates were loaded in density step gradients, and the
fractions corresponding to the different densities were analyzed by
immunoblot. Most of the RX3-I-hGH was detected in the fraction F56
corresponding to dense RPBLAs (FIG. 2B). As for other RX3 fusion
proteins, the presence of RX3-I-hGH fusion protein in the
supernatant probably represents the un-assembled fusion protein
contained in the ER and solubilized during the homogenization
process.
[0194] Once it was demonstrated that the RX3-I-hGH accumulated in
RPBLAs, these ER-derived organelles were isolated by low speed
centrifugation as described elsewhere herein. The centrifugation of
homogenates of CHO cells transfected with p3.1-RX3-I-hGH at
1500.times.g for 10 minutes permitted the separation of the
non-assembled RX3-Int-hGH fusion proteins in the supernatant from
the assembled in RPBLAs in the pellet. Equivalent studies were
performed with CHO cells expressing the inactive RX3-mInt-hGH
fusion protein.
[0195] The pellets containing the assembled RX3-Int-hGH and
RX3-mInt-hGH fusion proteins were solubilized in S1 buffer (20 mM
Tris pH7, 200 mM NaCl, 1 mM EDTA, 0.1% SDS and 0.1 mM TCEP) at
37.degree. C. for 2 hours, and the intein enzymatic activity was
induced by incubation at 25.degree. C. for 48 hours after dialysis
against the cleavage induction buffer: 20 mM Tris pH 7, 200 mM
NaCl, 1 mM EDTA. After induction of intein self-cleavage, the
composition was centrifuged at 16000.times.g for 10 minutes and the
supernatant and the pellet analyzed by immunoblot using anti-RX3
and anti-hGH antiserum.
[0196] Both fusion proteins were solubilized, but only the fusion
protein containing the active intein (RX3-Int-hGH) was able to
self-cleave (FIGS. 6A and 6B, black arrowheads). The absence of
self-cleavage of the mutated RX3-mInt-hGH fusion protein
demonstrates that the self-cleavage observed with the RX3-Int-hGH
is due to the specific activity of the intein, and not due to some
endogenous protease activity co-purified during the RPBLAs
isolation process.
[0197] To optimize the efficiency of intein self-cleavage,
alternative solubilization protocols were assayed. The intein
self-cleavage of the RX3-Int-hGH can be compared, after
solubilization with the S1 buffer and the biphasic extraction
protocol (S2) described elsewhere (FIG. 6C). From the ratio between
the remaining of the full-length fusion protein and the appearance
of the band corresponding to the liberated hGH, even though the
biphasic extraction protocol was the more efficient permitting more
than 50% of cleavage, it can be concluded that in both cases a
large proportion of DNAb intein was active and able to
self-cleave.
Example 7
Activity of RX3-EGF Assembled in RPBLAs in Tobacco Plants
[0198] RPBLAs from transgenic tobacco plants expressing the RX3-EGF
fusion protein were isolated by low speed centrifugation
essentially as described in U.S. Ser. No. 11/289,264. The fusion
protein was solubilized by sonication (Cycle 5, Amplitude 50%, 1
minute, repeated five times; Ikasonic U2005--TKA Labortechnik) in
50 mM Tris pH 8 and 2% of .beta.-ME and incubation at 37.degree. C.
for 2 hours. Afterwards, the solubilized material was centrifuged
at 16000.times.g at 4.degree. C. for 30 minutes to discard the
unsolubilized fusion protein in the pellet. The supernatant was
dialyzed against 50 mM Tris pH 8 to remove the .beta.-ME,
centrifuged once again at 16000.times.g at 4.degree. C. for 30
minutes, and the supernatant quantified by the hEGF kit from
Biosource International Inc, (KHG0062).
[0199] The bioactivity of EGF was analyzed by determining the
proliferation rate (radioactive thymidine incorporation to DNA) of
MDA-MB231 cells (breast cancer cells that overexpress EGF receptor)
incubated with 1.2 ng/mL of RX3-EGF fusion protein. As a positive
control, MDA-MB231 cells were incubated with 10 ng/mL of commercial
EGF (Promega) or fetal calf serum (FCS). The results, summarized in
the following Table, are represented as percentage (%) of
proliferation with regard to the basal proliferation rate of MB231
cells (100%), determined as the proliferation rate of these cells
cultivated in the absence of EGF (deprived).
TABLE-US-00029 Proliferation of MDA-MB231 cells % proliferation
with respect to Deprived cells Sample Concentration Mean STD
Deprived -- 100 -- FCS -- 145 1.27 EGF (Promega) 10 ng/mL 158 11.7
RX3-EGF 1.2 ng/mL 146 4
[0200] As expected, the supplementation of MB231 cell culture with
commercial EGF (Promega) or the FCS produced a significant increase
of the proliferation rate (158% and 1450, respectively).
Remarkably, the addition of 1.2 ng/mL of RX3-EGF also produced an
increase of 146% of the proliferation rate. It is important to note
that almost the same proliferation rate was observed with 10-fold
more concentration of commercial EGF than with RX3-EGF. This
surprising result could be explained by previous results showing
that saturation of the proliferation rate of MB231 cell was
observed at 5 ng/mL of the commercial EGF. Another possible
explanation could be a more active conformation of EGF when fused
to RX3. In any case, this result shows that RX3-EGF is at least as
active as the commercial hGH (Promega).
Example 8
Activity of RX3-GUS Assembled in RPBLAs in CHO Cells
[0201] The .beta.-glucuronidase enzyme (GUS) is a broadly used
reporter protein (Gilisen et al., Transgenic Res. (1998)
7(3):157-163). The expression of an active RX3-GUS fusion protein
in RPBLAs was predicted to be difficult due to the protein's large
size (ca. 70 kDa) and the presence of 9 cysteine residues.
[0202] The polynucleotide sequence coding for RX3 (WO2004003207)
was fused in frame to the 5' end of the sequence encoding GUS (FIG.
1A. RX3-GUS), and the resulting construct used to transfect CHO
cells as described in Example 7 above, Immunochemical analysis of
CHO cells transfected with p3.1-RX3-GUS incubated with anti-RX3
antiserum revealed the presence of large RPBLAs (FIG. 5E). To
verify the density of those RPBLAs, CHO cells transfected with the
same plasmid were homogenized and loaded onto step-density
gradients. The analysis of the different fractions by immunoblot
showed that the fusion proteins localized in the higher dense
fractions (FIG. 2B. F56), indicating that the RX3-GUS fusion
proteins are able to assemble and accumulate in dense RPBLAs. It is
important to note that no fusion protein was detected in the
supernatant, meaning that almost all RX3-GUS is assembled in dense
structures (RPBLAs).
[0203] Once it was demonstrated that the RX3-GUS accumulated in
RPBLAs, the fusion protein was recovered from the F56 fraction (as
described in Example 5 for RX3-hGH) and solubilized in 50 mM Tris,
pH 8, .beta.-ME 2% and SDS 0.1% at 37.degree. C. for 2 hours.
Afterwards, the solubilized material was centrifuged at
16000.times.g at room temperature for 10 minutes, and the
supernatant containing the soluble disassembled RX3-GUS fusion
protein was dialyzed at 4.degree. C. against a 50 mM Tris pH 8
solution over night (about 18 hours).
[0204] The GUS activity assay is based in the conversion of
metilumbeliferil-.beta.-glucuronide acid (MUG) to the
4-metilumbeliferone (4-MU) fluorescent product (Jefferson et al.
1987 EMBO J. 6(13):3901-3907). Fifty .mu.L of the solubilized
RX3-GUS fusion protein (about 0.25 ng of RX3-GUS/.mu.L) was
incubated in the presence of MUG at room temperature, and the
appearance of 4-MU was measured by fluorescence (excitation
wavelength 355 nm; emission wavelength 420 nm). To rule out the
possibility of an endogenous GUS-like activity in the RPBLA
preparation from CHO cells, RPBLAs from CHO cells transfected with
p3.1--RX3 were isolated, and the solubilized RX3 protein was
included in the activity assay as a control. The table below
summarizes the results obtained:
TABLE-US-00030 Absorbance at 420 nm Time RX3-GUS RX3 (minutes) Mean
STD Mean STD 0 337 24 227 6.4 30 534 4.2 236 15 60 690 12.7 265 9.2
90 909 30.4 299 21.2 120 1049 38.9 309 10.6 160 1141 21.9 311
82
[0205] From the results shown in this table, it is clear that the
RX3-GUS fusion protein remains active once solubilized from the
RPBLAs. The specific activity of the RX3-GUS calculated from these
experiments was 0.2 pmol of 4-MU/min-1*12.5 ng-l of RX3-GUS. No
significant endogenous GUS-like activity was observed using RX3
RPBLA preparations.
Example 9
Activity of RX3-EK Assembled in RPBLAs in CHO Cells
[0206] Bos taurus enterokinase (enteropeptidase) is a
membrane-bound serine protease of the duodenal mucosa, involved in
the processing of trypsinogen to trypsin (DDDK ) with a
chymotrypsin-like serine protease domain. The enteropeptidase is a
disulfide linked two-chain peptide formed by the heavy chain
(EK.sub.HC--120 kD) and the catalytic light chain (EK.sub.LC--47
kD). The catalytic subunit (here referred as EK) is almost as
active and specific by itself as the complete holoenzyme (LaVallie
et al. 1993 J. Biol. Chem. 268(31):23311-23317). It is important to
point out that bovine EK has 4 disulphide bonds. Moreover, the
N-terminal end of the protein is folded inside the protein, and it
is essential for the proper folding of a functional EK. These two
EK requirements make EK protein a challenging protein to be
expressed as an active protein in RPBLAs.
[0207] The polynucleotide sequence coding for RX3 (WO2004003207)
was fused through a linker comprising the FXa cleavage site (IEGR)
to the 5' end of the EK sequence, and cloned in pcDNA3.1(-) (FIG.
1A, p3.1--RX3-EK).
[0208] This construct was used to transfect CHO cells using
lipofectamine (Invitrogen). Immunocytochemistry analysis of those
transfected cells using anti-RX3 antiserum revealed the presence of
a large quantity of small RPBLAs. These organelles were visible all
along the cytoplasm of the transfected cells, but their size
usually did not exceed 0.5.mu. (FIG. 5F).
[0209] To verify the density of those small RPBLAs, CHO cells
transfected with the same plasmid were homogenized and loaded in
step-density gradients, The RX3-EK fusion protein was localized in
the F56 fraction (FIG. 2B). The high density of the RX3-GUS fusion
protein assemblies suggests that this fusion protein accumulates
into dense RPBLAs. It is important to note that no fusion protein
was detected in the supernatant, meaning that almost all RX3-EK is
assembled into aggregates. Interestingly, the molecular weight of
the RX3-EK fusion protein was estimated at 58 KDa, about 15 KDa
higher than the theoretical molecular weight expected. This result
suggests that the EK in the RPBLAs can be glycosylated, as has been
described for the natural protein (LaVallie et al., 1993 J. Biol,
Chem. 268(31):23311-23317).
[0210] The fusion protein was recovered from the F56 fraction (as
described in Example 5 for RX3-hGH) and solubilized in 50 mM Tris,
pH 8, .beta.-ME 2% and SDS 0.1% at 37.degree. C. for 2 hours. To
increase the solubilization, the sample was sonicated at 50%
amplitude and 50% cycle for 1 minute, repeated 5 times (Ikasonic
U200S--IKA Labortechnik), before the SDS addition. Afterwards, the
sample was centrifuged at 5000.times.g at room temperature for 10
minutes, and the supernatant containing the soluble disassembled
RX3-GUS fusion protein was dialyzed at 4.degree. C. against a 50 mM
Tris pH 8 solution for 18 hours. The fusion protein was digested by
FXa as described by the manufacturer (Quiagen), and the EK activity
was treasured by fluorimetric assay (Grant, et al., 1979 Biochim.
Biophys. Acta 567:207-215). The liberated EK from the RX3-EK had
enteropeptidase activity.
Example 10
Activity of RX3-Casp2 and RX3-Casp3 Assembled in RPBLAs in CHO
Cells
[0211] Studies were undertaken to determine the activity of
caspases produced in RPBLAs.
Caspases are a family of cysteine proteases involved in apoptosis
that cleave a unique consensus sequence with high specificity.
Caspases exist as inactive procaspases with a prodomain of variable
length followed by a large subunit (p20) and a small subunit (p10).
They are activated through proteolysis and mature active caspase
consists of the heterotetramer p20.sub.2-p10.sub.2 (Lavrik et al.,
2005 J. Clin. Invest. 115:266S-2671). Caspases are divided into
initiator caspases and executioner caspases that differ in their
mechanism of action. Caspase2 (initiator caspase) and caspase3
(executioner caspase) have been chosen as an example of proteins
which are active in the RPBLAs (Baliga et al., 2004 Cell Death and
Differentiation 11:1234-1241; Feeney et al., 2006 Protein
Expression and Purification 47(1):311-318) Those proteins are
especially challenging because they are synthesized as zymogens
that, to become active, need to be self-cleaved and to form a
heterotetramer.
[0212] The p3.1-RX3-C2 and p3.1-RX3-C3 constructs (FIG. 1) were
introduced into CHO cells by transient transfection with the
lipofectamine protocol (invitrogen). Four days after transfection,
to determine if caspases are accumulated in dense RPBLAs
organelles, CHO cells expressing RX3-Casp2 or RX3-Casp2 were
homogenized, loaded on a density step gradient and centrifuged as
described elsewhere.
[0213] The accumulation of both RX3-caspases fusion proteins in the
different fractions was analyzed by immunoblot (FIG. 2B). As can be
seen, most of the RX3-Casp2 or RX3-Casp2 fusion proteins sediment
to fraction F56 and F42 corresponding to dense RPBLAs. This result
indicates that these two fusion proteins are able to tightly
assemble in dense structures.
[0214] In the immunoblot presented in FIG. 2B, only the full length
fusion protein is shown, but bands of different molecular weight
are present in this fraction. These bands are cross-reactive to
either anti-RX3 antibody or anti-CASP (SA-320 and SA-325, Biomol
International) antibody correspond to the different caspase
subunits, indicating that autocatalytic activation has taken place
inside RPBLAs. These observations indicate that Caspase2 and
Caspase 3 are active in vivo.
[0215] The F56 and F42 fractions were diluted 4-fold in buffer PBP4
and centrifuged at 80000.times.g in a swinging-bucket to recover
the RPBLAs in the pellet. The ER membrane surrounding this
organelle was removed by washing the RPBLAs preparation with 50 mM
Tris pH 8 and 1% Triton X-100. Upon removal of the ER membrane,
activity of caspase is assayed using the BIOMOL QuantiZyme.TM.
Assay System, CASPASE-3 Cellular Activity Assay Kit PLUS-AK703
(caspase 3) and BIOMOL QuantiZyme.TM. Assay System, CASPASE-2
Cellular Activity Assay Kit PLUS-AK702 (Caspase 2). This kit
measures caspase activity colorimetrically with a specific
substrate, The RX3-Casp2 and the RX3-Casp3 RPBLAs show caspase
activity.
[0216] In determining the activity of caspases fused to RX3, the
fusion protein is solubilized from RPBLAs isolated by density
gradient (F56 and F42, diluted 4-fold in buffer PBP4 and
centrifuged at 80000.times.g in a swinging-bucket). The fusion
protein is solubilized in buffer CA (50 mM Hepes, pH 7.4, 100 mM
NaCl, 1 mM EDTA, 100 mM DTT, 1% CHAPS, 10% glycerol) after
sonication (50% amplitude and 50% cycle for 30 seconds, 5 times).
Solubilization is performed by a 2-hour incubation at 37.degree. C.
and insoluble material is discarded by centrifugation at
16000.times.g for 10 minutes. The supernatant containing the
soluble RX3-casp fusion protein is dialyzed against caspase kit
assay buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM
DTT, 0.1% CHAPS, 10% glycerol). Activity of the dialyzed sample
containing RX3-Casp2 and RX3-Casp3 are assessed with the BIOMOL
QuantiZyme.TM. Assay System, CASPASE-3 Cellular Activity Assay Kit
PLUS-AK703 (caspase 3) and BIOMOL QuantiZyme.TM. Assay System,
CASPASE-2 Cellular Activity Assay Kit PLUS-AK702 (caspase 2).
Caspase 2 and Caspase 3 are active.
Example 11
Activity of RX3-RTB Assembled in RPBLAs in Agroinfiltrated Tobacco
Plants
[0217] The polynucleotide sequence coding for RTB (Reed et al.,
2005 Plant Cell Report 24:15-24) was fused in frame to the 3' end
of RX3 domain and cloned in a binary vector (pB-RX3-RTB). This
construct was used in tobacco plants transformed by syringe
agroinfiltration, as described elsewhere. The agroinfiltrated
tobacco leaves were homogenized and loaded in step density
gradients. The RX3-RTB fusion protein was localized to fractions
F42 and F56 (FIG. 2B), consistent with the fusion protein
self-assembling and accumulating in dense RPBLAs. As described for
RX3-EK, the RX3-RTB fusion protein isolated from the RPBLAs has a
lower electrophoretic mobility compared to the theoretical
molecular weight. This results supports that RTB also can be
glycosylated in RPBLAs.
[0218] The fusion protein was recovered from those dense fractions
(as described in Example 5 for RX3-hGH) and solubilized in 50 mM
Tris, pH 8, .beta.-ME 0.8% at 37.degree. C. for 2 hours. To
increase the solubilization, the sample is sonicated at 50%
amplitude and 50% cycle for 1 minute, repeated 5 times (Ikasonic
U200S-IKA Labortechnik). Subsequently, the sample is centrifuged at
5000.times.g at room temperature for 10 minutes, and the
supernatant containing the soluble disassembled RX3-RTB is analyzed
by ELISA for binding to the glycoprotein fetuin treated with
sialydase to expose galactose-terminated glycans, and they
bind.
Example 12
Plasmid Construction for Plant Transformation
[0219] The coding sequences of human epidermal growth factor (hEGF)
were obtained synthetically and were modified in order to optimize
its codon usage for expression in plants.
TABLE-US-00031 hEGF protein SEQ ID NO: 41 NSDSECPLSH DGYCLHDGVC
MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR hEGF DNA SEQ ID NO: 42
aactctgatt cagaatgccc actcagtcac gacggatatt gtcttcacga tggggtatgc
atgtacatcg aggccttgga caagtacgca tgtaattgtg tagtgggata cattggtgaa
cgctgtcagt atcgagactt gaaatggtgg gagcttaggt ga
[0220] The synthetic gene encoding the 53 amino acids of active
hEGF was obtained by primer overlap extension PCR method, using 4
oligonucleotides of around 60 bases, with 20 overlapping bases. The
synthetic hEGF cDNA included a 5' linker sequence corresponding to
the Factor Xa specific cleavage site. The oligonucleotides were
purified by polyacrylamide denaturing gel.
[0221] Synthetic hEGF cDNA was purified from agarose (Amersham)
following gel electrophoresis and cloned into the pGEM vector
(Promega), The RX3 cDNA fragment (coding for an N-terminal domain
of gamma-zein) containing cohesive ends of BspHI and NcoI, was
inserted into the vector pCKGFPS65C (Reichel et al., 1996 Proc.
Natl. Acad. Sci, USA 93:5888-5893) previously digested with NcoI
(as described in patent application WO2004003207). The sequence
coding for EGF was fused in frame to the RX3 sequence. The
constructs RX3-EGF was prepared by substitution of the GFP coding
sequence for the EGF synthetic gene.
[0222] The resulting construct named pCRX3EGF contained a nucleic
acid sequence that directs transcription of a protein as the
enhanced 35S promoter, a translation enhancer as the tobacco etch
virus (TEV), the EGF coding sequence and the 3' polyadenylation
sequences from the cauliflower mosaic virus (CaMV). Effective plant
transformation vector p19RX3EGF was ultimately obtained by
inserting the HindIII/HindIII expression cassettes into the binary
vector pBin19 (Bevan, 1984 Nucleic Acids Research
12:8711-8721).
[0223] The cDNA encoding the alpha-zein of 22 kD (22aZ) and the
rice prolamin of 13 kD (rPl3) were amplified by RT-PCR from a cDNA
library from maize W64A and Senia rice cultivar, respectively. The
oligonucleotides used in the PCR reaction were:
TABLE-US-00032 22aZ-5' SEQ ID NO: 43
5'GAGGATCCGCATGGCTACCAAGATATTAGCCCT3' 22aZ-3' SEQ ID NO: 44
5'CATTCATGATTCCGCCACCTCCACCAAAGATGGCACCTCCAACGATGG 3' Rice13Pro1-5'
SEQ ID NO: 45 5'GAGTCGACGGATCCATGAAGATCATTTTCGTCTTTGCTCTCC3'
Rice13Pro1-3': SEQ ID NO: 46
5'CATCCATGGTTCCGCCACCTCCACCCAAGACACCGCCAA- GGGTGGTAATGG3'
[0224] The corresponding PCR fragments were cloned in the pCRII
vector (Invitrogen), sequenced and cloned in pUC18 vectors
containing the enhanced CaMV 35S promoter, the TEV sequence and 3'
ocs terminator. The pCRII-rP13 was digested by SalI and NcoI, and
cloned in the pUC18RX3Ct, pUC18RX3hGH and pUC18RX3EGF plasmids
digested by the same enzymes to obtain plasmid pUC18rP13EGF. The
pCRII-22aZ was digested by SalI/NcoI and cloned in the pUC18RX3EGF
plasmid digested by the same enzymes to obtain plasmid
pUC1822aZtEGF. Finally, the pUC18-derived vector was cloned in
pCambia 5300 by HindIII/EcoRI.
[0225] The construct pBIN m-gfp4-ER, contain an optimized GFP for
expression in plants (Haseloff et al., 1997 Proc. Natl. Acad. Sci.
USA 94:2122-2127). This construct was used as template for PCR
amplification of the GFP. The oligonucleotides were designed to
eliminate the signal peptide and HDEL motif present in the original
sequence as well as to introduce the restriction sites for further
cloning.
Primers:
TABLE-US-00033 [0226] GFP 5': SEQ ID NO: 50 5'
AATTCATGAGCAGTAAAGGAGAAGAACTTTTCAC 3' GFP 3': SEQ ID NO: 51 5'
ATTGGATCCTCATTATTTGTATAGTTCATCCATGC 3'
[0227] The PCR product was cloned in a PCR cloning vector
(PCR.RTM.II Vector, Invitrogen)) and the sequence verified. The GFP
fragment containing cohesive ends RcaI/BamHI was cloned into
pUC18RX3hGH (US2006123509 (A1)), giving the cassette RX3-GFP in a
pUC18 vector. This cassette was liberated by HindIII/BamHI
digestion and subsequently inserted in a pCAMBIA 2300 vector
(pB-RX3-GFP)
[0228] The RTB clone (GenBank accession no. X03179) was amplified
by PCR (RTB5 and RTB3) and digested by RcaI/SmaI. The digested PCR
fragment was cloned in pUC18RX3hGH (US2006123509 (A1)) digested by
NcoI/SmaI to obtain pUC18RX3RTB. Then, this vector was digested by
HindIII/EcoRI and the liberated fragment cloned in a pCAMBIA 2300
vector digested by the same restriction enzymes (pB-RX3-RTB)
Primers:
TABLE-US-00034 [0229] RTB5: SEQ ID NO: 52 5'
AATTCATGAGCAGTAAAGGAGAAGAACTTTTCAC 3' RTB3: SEQ ID NO: 53 5'
TTACCATTATTTTGATACCCGGGAAG 3'
Plant material
[0230] Tobacco (Nicotiana tabacum var. Wisconsin) plants were grown
in an in vitro growth chamber at 24-26.degree. C. with a 16 hour
photoperiod. Adult plants were grown in greenhouse between at
18-28.degree. C., humidity was maintained between 55 and 65% with
average photoperiod of 16 hours.
[0231] Plantlets for agroinfiltration (Vaquero et al., 1999 Proc.
Natl. Acad. Sci., USA 96(20):11128-11133; Kapila et al., 1997 Plant
Sci. 122:101-108) method were grown from seeds for 4-6 weeks in the
in vitro conditions described above.
Tobacco Stable Transformation
[0232] The binary vectors were transferred into LBA4404 strain of
A. tumefaciens. Tobacco (Nicotiana tobaccum, W38) leaf discs were
transformed as described by Draper and Hamil 1988, In: Plant
Genetic Transformation and Gene Expression. A Laboratory Manual
(Eds. Draper, J., Scott, R., Armitage, P. and Walden, R.),
Blackwell Scientific Publications. Regenerated plants were selected
on medium containing 200 mg/L kanamycin and transferred to a
greenhouse. Transgenic tobacco plants having the highest transgene
product levels were cultivated in order to obtain T1 and T2
generations.
[0233] Recombinant protein levels were detected by immunoblot.
Total protein extracts from tobacco leaves were quantified by
Bradford assay, separated onto 15% SDS-PAGE and transferred to
nitrocellulose membranes using a Mini Trans-Blot Electrophoretic
Transfer Cell (Bio Rad). Membranes were incubated with gamma-zein
antiserum (dilution 1/7000) (Ludevid et al. 1985, Plant Science
41:41-48) and were then incubated with horseradish
peroxidase-conjugated antibodies (dilution 1/10000, Amersham
Pharmacia) Immunoreactive bands were detected by enhanced
chemiluminescence (ECL western blotting system, Amersham
Pharmacia).
Tobacco Agroinfiltration
[0234] Vacuum Agroinfiltration
[0235] Plantlets for agroinfiltration method were grown from seeds
for 4-6 weeks in an in vitro growth chamber at 24-26.degree. C.
with a 16 hour photoperiod.
[0236] A. tumefaciens strain LB4404 containing a desired construct
was grown on LB medium (Triptone 10 g/l, yeast extract 5 g/l, NaCl
10 g/l) supplemented with kanamycin (50 mg/l) and rifampin (100
mg/l) at 28.degree. C. with shaking (250 rpm) overnight (about 18
hours). Agrobacterium cells were inoculated into 30 ml of LB also
supplemented with kanamycin (50 mg/l) and rifampin (100 mg/l).
After overnight culture at 28.degree. C. cells were collected by
centrifugation for 10 minutes at 3000.times.g and resuspended in 10
ml of liquid MS medium with MES (Sigma Chemical) 4.9 g/l and
sucrose 30 g/l at pH 5.8. Bacterial cultures were adjusted to a
final OD.sub.600 of 0.1 and supplemented with acetosyringone to a
final concentration of 0.2 mM and incubated for 90 min at
28.degree. C.
[0237] For agroinfiltration, plantlets were totally covered with
the suspension and vacuum was applied (100 KPa) for 5-6 seconds.
The suspension was removed and plantlets maintained in a growth
chamber at 24-26.degree. C. under a photoperiod of 16 hours for
four days. Plant material was recovered and total protein extracts
were analyzed by immunoblot using anti-gamma zein antibody.
[0238] Agroinfiltration by Syringe
[0239] Agrobacterium tumefaciens strain EHA 105 was grown to
stationary phase at 28.degree. C. in L-broth supplemented with 50
.mu.g mL.sup.-1 kanamycin and 50 .mu.g mL.sup.-1 rifampin. Bacteria
were sedimented by centrifugation at 5000 g for 15 minutes at room
temperature and resuspended in 10 mM MES buffer pH 5, 6, 10 mM
MgCl.sub.2 and 200 .mu.M acetosyringone to a final OD.sub.600 of
0.2. Cells were left in this medium for 3 hr at room temperature.
Individual Agrobacterium cultures carrying the RX3 constructs and
the HC-Pro silencing suppressor constructs (Goytia et al., 2006)
were mixed together and infiltrated into the abaxial face of leaves
of 2-4-week-old Nicotiana benthamiana plants (Voinnet et al.,
2003).
Example 13
Isolation (Purification) of RPBLAs by Density Gradient from
Transgenic Plant Vegetative Tissues
[0240] The gene coding for RX3-EGF gamma-zein derived fusion
proteins was introduced into tobacco plants via Agrobacterium
tumefaciens. Transformed plants were analyzed by immunoblot to
identify those with higher recombinant protein expression. The
fusion proteins usually accumulated as multimers and the amount of
monomers and oligomers detected in the immunoblots depended on the
disulfide bond reduction state.
[0241] Tobacco leaf extracts were loaded on density step gradients
and the accumulation of recombinant proteins in the different
fractions was analyzed by immunoblot. The results indicate that
RX3-EGF appeared in fractions corresponding to dense RPBLAs, Most
of these organelles exhibited densities higher than 1.2632
g/cm.sup.3 and a significant portion of them showed a density
higher than 1.3163 g/cm.sup.3. These densities were comparable to
or higher than that of natural maize PBs (Ludevid et al., 1984
Plant Mol. Biol. 3:227-234; Lending et al., 1989 Plant Cell
1:1011-1023).
[0242] It was estimated that more than 90 percent of the
recombinant protein was recovered in the dense RPBLAs fractions and
pellet. Thus, isolation of RPBLAs by density appears to be a useful
system to purify (concentrate) the fusion proteins.
[0243] To evaluate the purification of the recombinant protein
RX3-EGF by RPBLAs isolation, the different density fractions were
analyzed by silver stain. More than 90 percent of tobacco
endogenous proteins were located in the soluble and the interphase
fractions of the gradient where the RX3-EGF protein was absent or
barely detected. Thus, soluble proteins and the bulk of proteins
present in less dense organelles could be discarded by selecting
one or two fractions of the gradient.
[0244] In respect to the degree of fusion proteins purification in
the RPBLAs fractions, it was estimated that RX3-EGF protein
represents approximately 80 percent of the proteins detected in the
PBLS-containing fractions. This result indicates that, using an
RPBLA isolation procedure, one can achieve an important enrichment
of fusion proteins in only one step of purification.
Example 14
Recombinant Proteins Recovery in RPBLAs Isolated From Dryplant
Tissues
[0245] The ability of RPBLAs to stabilize and protect recombinant
proteins of interest was assessed by means of accelerated stability
testing using heat-dried plant leaf biomass. Transformed tobacco
leaves accumulating RX3-EGF fusion protein were dried at 37 C. for
one week and then stored at room temperature under low humidity
conditions. After 5 months of dry storage, the levels of
recombinant proteins were compared to those of identical leaves
harvested at the same time but frozen at -80 C. instead of being
heat-dried. Protein extracts from equivalent amounts of
fresh-frozen and dried leaf tissue were analyzed by immunoblot. The
RX3-EGF protein was found to be highly stable in desiccated
transformed plants, as the amount recovered in wet and dry plants
was virtually identical.
[0246] The distribution in step density gradients of RX3EGF fusion
protein from homogenates of dried leaves was analyzed by
immunoblot. The fusion protein was mainly recovered in dense
structures exhibiting densities higher than 1.1868 g/cm.sup.3 and
1.2632 g/cm.sup.3.
[0247] Given the abundant proteases and metabolites present in
tobacco leaves, the remarkable ability of RPBLA sequestration to
protect recombinant proteins was unexpected. However, similar
results were subsequently obtained using rice seed expressing RX3
which had been dried for prolonged periods.
Example 15
Recombinant Protein Recovery by Isolation of RPBLAs from
Transiently Transformed Tobacco Plantlets
[0248] The transient expression systems can be a convenient tool to
test the accumulation of recombinant proteins over a short period
of time. Thus, the recombinant protein RX3-EGF was also expressed
and accumulated in transiently transformed tobacco plantlets via
agroinfiltration. The protein extracts from transformed plantlets
analyzed by immunoblot show the characteristic complex
electrophoretic pattern observed from stably-transformed plants,
indicating that the fusion proteins assemble correctly using this
method of transformation.
Example 16
Recovery of Recombinant Proteins by Low and Medium Speed
Centrifugation
[0249] To simplify the procedure used to purify recombinant
proteins via dense recombinant protein body-like assemblies, two
additional alternative methods were performed: i) clarified
homogenates were centrifuged through only one dense sucrose cushion
and ii) clarified homogenates were centrifuged without gradients at
low speed (i.e. 1000-2500.times.g for 10 minutes).
[0250] In agreement with the previously described results, the
RX3-EGF protein was recoverable at high yields (greater than 90%)
in pellets obtained after centrifugation through 1.1868 g/cm3
sucrose cushions. In addition, the purification of RX3-EGF protein
was still effective in that contaminating endogenous tobacco
proteins were barely detected in the corresponding pellet.
[0251] The principal advantage of this method as compared to
step-density gradients lies in its easy scalability for industrial
production of recombinant proteins. It should be noted that the
cushion density as well other properties such as its viscosity and
osmolarity can be adjusted in each case in order to optimize
recovery and purification of the recombinant proteins.
[0252] In addition, low speed centrifugation (LSC) was also assayed
to concentrate and purify fusion protein-containing protein
body-like structures. The results indicated that, after
1000.times.g for 10 minutes, practically all the RX3-EGF fusion
protein was recovered in the pellet. But the staining of the
proteins contained in this pellet revealed that the fusion protein
was not highly purified as compared with that obtained after
centrifugation through 1.1868 g/cm3 sucrose cushion.
[0253] Thereafter, the first pellet obtained by low speed
centrifugation was washed by using a buffer containing 5%
Triton.RTM. X-100. After washing, the sample was centrifuged at
12,000.times.g for 5 minutes and, interestingly, the bulk of
contaminating proteins present in the P1 pellet were eliminated
after washing and centrifugation and the new pellet contained a
highly enriched RX3-EGF protein. The amount as well the pattern of
proteins noted in this study was similar to those obtained after
washing the pellet obtained by centrifugation through the sucrose
cushion in the Triton X-100-containing buffer. The low speed
centrifugation alternative is based on the high density of the
structures containing fusion proteins and centrifugation conditions
can be optimized for every target before scale-up.
[0254] Transgenic tobacco plants expressing fusion proteins
including EGF linked to rice prolamin or alpha-zein rather than
RX3, rP13-EGF and the 22aZ-EGF, were produced by Agrobacterium
tumefasciens transformation. Highly-expressing lines were
identified by immunoblot using an antibody against EGF, and those
cell lines were used in a comparative analysis with tobacco
plantlets agroinfiltrated with the same constructs. In all cases,
the RPBLAs where recovered in unique interface, suggesting that the
RPBLAs are very dense and homogeneous.
[0255] Taking all these results together, it is clear that a broad
range of prolamins are able to induce high-density RPBLAs, even
when they are fused to heterologous proteins. This was an
unexpected result, as almost no homology exists between the various
prolamins of diverse origin, and suggests that structural rather
than sequence-specific motifs may mediate RPBLA formation.
Moreover, some data suggests that the different prolamins interact
to stabilize the natural PBs of cereals. In contrast to gamma-zein,
alpha-zein for example might not be stable when expressed alone in
vegetative tissue (Coleman et al., 1996 Plant Cell
8:2335-2345).
Example 17
Extraction of Recombinant Proteins from Isolated RPBLAs
[0256] Like isolation of bacterial inclusion bodies, isolation of
dense recombinant PB-like assemblies is an advantageous method to
recover recombinant proteins with high yield and high
pre-purification levels from transgenic organisms. Here it is shown
that these recombinant proteins can be extracted from the storage
organelles.
[0257] RX3-EGF proteins were solubilized following overnight (ca.
18 hr) incubation of RPBLA fractions at 37.degree. C. in a buffer
containing a detergent and reducing agents (SB buffer with sodium
borate 12.5 mM pH 8, 0.1% SDS and 2% .beta.-mercaptoethanol). The
extracted fusion proteins were recovered in a soluble form for
further purification or use directly as partially-purified
extracts.
Example 18
Plasmid Construction for Animal Cell Transformation
[0258] The RX3 sequence was amplified by PCR to obtain the cDNA
fragments corresponding to RX3 and RX3-(Gly)x5. These fragments
were digested by SalI/BamHI cloned in plasmid pECFP-N1 (Clontech)
digested by the same enzymes to obtain pRX3-ECFP and pRX3-G-ECFP
plasmids, respectively.
Primers:
TABLE-US-00035 [0259] SPfor: SEQ ID NO: 54 5'
CAGTCGACACCATGAGGGTGTTGCTCGTTGCCCTCGCTC 3' RX3ECFP3': SEQ ID NO: 55
5' GGTGGATCCCTAGAATCCATGGTCTGGCAC 3' RX3G5ECFP3': SEQ ID NO: 56 5'
GGTGGATCCCTAGAGCCACCGCCACCTCCATCCATGGTCTGGCA 3'
[0260] The p22aZ-ECFP vector corresponds to the following
HindIII/XbaI DNA fragment in pEGFP-N1 plasmid (Clontech).
TABLE-US-00036 SEQ ID NO: 57
aagcttcgaattctgcagtcgacaacatggctaccaagatattagccctc
cttgcgcttcttgccctttttgtgagcgcaacaaatgcgttcattattcc
acaatgctcacttgctcctagtgccattataccacagttcctcccaccag
ttacttcaatgggcttcgaacacctagctgtgcaagcctacaggctacaa
caagcgcttgcggcaagcgtcttacaacaaccaattaaccaattgcaaca
acaatccttggcacatctaaccatacaaaccatcgcaacgcaacagcaac
aacagttcctaccagcactgagccaactagatgtggtgaaccctgtcgcc
tacttgcaacagcagctgcttgcatccaacccacttgctctggcaaacgt
agctgcataccaacaacaacaacaattgcagcagtttctgccagcgctca
gtcaactagccatggtgaaccctgccgcctacctacaacagcaacaactg
ctttcatctagccctctcgctgtgggtaatgcacctacatacctgcaaca
acaattgctgcaacagattgtaccagctctgactcagctagctgtggcaa
accctgctgcctacttgcaacagctgcttccattcaaccaactgactgtg
tcgaactctgctgcgtacctacaacagcgacaacagttacttaatccact
agaagtgccaaacccattggtcgctgccttcctacagcagcaacaattgc
taccatacagccagttctctttgatgaaccctgccttgtcgtggcagcaa
cccatcgttggaggtgccatctttggtggaggtggcggaatcatggtgag
caagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctgg
acggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggc
gatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaa
gctgcccgtgccctggcccaccctcgtgaccaccctgacctggggcgtgc
agtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaag
tccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaagga
cgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccc
tggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaac
atcctggggcacaagctggagtacaactacatcagccacaacgtctatat
caccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgcc
acaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaac
acccccatcggcgacggccccgtgctgctgcccgacaaccactacctgag
cacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatgg
tcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgag
ctgtacaagtaaagcggccgcgactctaga
[0261] The GFP was obtained by PCR amplification of the plasmid
pEGFP-N1 (Clontech) with specific oligonucleotides containing
enzyme restriction sites for further cloning:
TABLE-US-00037 ECFP NcoI 5': SEQ ID NO: 58 5'
GTACCATGGTGAGCAAGGGCGAGGAGCTG 3' ECFPN1 BamNotSac 3': SEQ ID NO: 59
5' GCAGAGCTCGCGGCCGCGGATCCTTACTTGTACAGCTCGTCCATGCC G3'
[0262] The PCR product (GFP) was cloned in a PCR cloning vector
(PCR.RTM.II Vector, Invitrogen) and the sequence verified. The GFP
fragment was excised by NcoI/BamHI digestion and cloned into
pUC18RX3hGH (US2006123509 (A1)), giving the cassette RX3-GFP in a
pUC18 vector. This cassette was liberated by SalI/BamHI digestion
and subsequently cloned into a pcDNA3.1(-) (Invitrogen) previously
digested by XhoI/BamHI (p3.1-RX3-GFP)
[0263] A construct containing the coding sequence of an improved
monomeric DS Red protein (mCherry; Shaner et al., 2004 Nat.
Biotechnol. 22:1567-1572) was used as a template in a PCR reaction
(mCherry RcaI 5'/ECFPN1 BamNotSac 3').
TABLE-US-00038 mCherry RcaI 5': SEQ ID NO: 60 5'
ATCATGATGGTGAGCAAGGGCGAG 3'
[0264] The PCR product (DsRed) was cloned in a PCR cloning vector
(PCR.RTM.II Vector, Invitrogen)) and the sequence verified. The
DsRed fragment was excised by RcaI/BamHI digestion and cloned into
pUC18RX3hGH (US2006123509 (A1)), giving the cassette RX3-DsRed in a
pUC18 vector. This cassette was liberated by SalI/BamHI digestion
and subsequently cloned into a pcDNA3.1(-) (Invitrogen) previously
digested by XhoI/BamHI (p3.1-RX3-DsRED)
[0265] To obtain a RX3 cDNA with a STOP codon at the 3' end, the
RX3 fragment was amplified by PCR (SPFOR/RX3STOP) and digested by
SalI/BamHI. The fragment was cloned in pcDNA3.1(-) digested by the
same restriction enzymes to obtain p3.1-RX3.
TABLE-US-00039 RX3STOP3': SEQ ID NO: 61 5'
TCGGATCCTTCTAGAATCATCAGGTCT 3'
[0266] The cDNA encoding the hGH were fused to the RX3 N-terminal
gamma-zein coding sequence (patent WO2004003207) and was introduced
into the vector pcDNA3.1(-) (Invitrogen) as described elsewhere. In
the resulting construct named p3.1RX3hGH, the fusion protein
sequences were under the CMV promoter and the terminator pA
BGH.
[0267] The Ssp DNAb intein from pTWIN1 plasmid (New England
Biolabs) and the hGH cDNA were amplified by PCR. Both PCR fragments
were fused in frame, also by PCR, digested by NcoI/BamHI and cloned
in pUC18RX3hGH (US2006121573 (A1)) vector also digested by
NcoI/BamHI. The RX3-Int-hGH insert was obtained by SalI/BamHI
digestion of this intermediate vector and cloned in pcDNA3.1(-)
(Invitrogen) digested by XhoI/BamHI. The resulting contruct was
named p3.1-RX3-I-hGH. The PCR product was digested by BsRGI/BamHI
and cloned in p3.1-RX3-I-hGH plasmid digested with the same
restriction enzymes.
Primers:
TABLE-US-00040 [0268] 5'DNAb: SEQ ID NO: 62 5'
AGCCATGGCGCGAGTCCGGAGCTATCTCTG 3' 3'DNAb: SEQ ID NO: 63 5'
GTTGTGTACAATGATGTCATTCG 3' DNAb-hGH: SEQ ID NO: 64 5'
GAATGACATCATTGTACACAACTTCCCAACCATTCCCTTATCC 3' 3'hGH: SEQ ID NO: 65
5' ATGGTACCACGCGTCTTATCAGAAGCCACAGCTGCCCTCC 3'
[0269] As a negative control for cleavage induction, a
non-cleavable Ssp DnaB was engineered. The mutated
(Asp154.fwdarw.Ala154) Ssp DnaB intein fused in frame to the hGH
was obtained by PCR from p3.1-RX3-I-hGH.
Primers:
TABLE-US-00041 [0270] IM-for: SEQ ID NO: 66 5'
ATCATTGTACACGCCTTCCCAACCATTCCCTTATCC 3' IM-rev: SEQ ID NO: 67 5'
TCAGGATCCTTATCAGAAGCCACAGCTGCCCTCCA 3'
[0271] Full length cDNAs of human caspase-2 (IRAUp969A0210D6) and
caspase-3 (IRATp970B0521D6) were acquired from RZPD GmbH (Berlin),
based on an original sequence reference from the Lawrence Livermore
National Laboratory.
[0272] The caspase-3 and the caspase-2 specific cleavage sites
(DEVD and DEHD, respectively) were added by PCR at the 5' termini
of the corresponding caspase sequences. It is important to note
that amplified fragment corresponding to caspase-2 did not contain
the pro-domain.
TABLE-US-00042 Casp3 forward SEQ ID NO: 68 5'
GACTCATGATCGATGAGGTGGACATGGAGAACACTGAAAACTCAG 3' Casp3 reverse SEQ
ID NO: 69 5' CTGGGTACCATGTCTAGATCATTAGTGATAAAAATAGAGTTCTTTTG TG3'
Casp2 for SEQ ID NO: 70 5'
GACTCATGATCGATGAGCACGACGGTCCTCTCTGCCTTCAGGT 3' Casp2 reverse SEQ ID
NO: 71 5' CTGGGTACCATGTCTAGATAATCATGTGGGAGGGTGTCCTGGG 3'
[0273] The amplified sequences were cloned into pUC18RX3hGH
(US2006123509 (A1)) by digesting with NcoI and KpnI. The resulting
construct was then digested by SalI/KpnI and cloned to a pCDNA3.1
(Invitrogen) vector digested by XhoI/KpnI. The corresponding
vectors were named (p3.1-RX3-C2 and p3.1-RX3-C3).
[0274] The pUC18RX3hGH (US2006123509 (A1)) vector was digested by
HindIII/EcoRI, and the liberated insert cloned into pCambia2300
also digested by these enzymes. The corresponding vector was
digested by HindIII/NcoI and the insert cloned in pCambia1381
opened by HindIII/NcoI (p4-17). The DNA comprising the
RX3-(gly)x5-GUS fragment was obtained by digesting p4-17 with
BstEII, filling in the overhang with Klenow and finally digesting
with SalI. The resulting fragment was cloned into pcDNA3.1(-)
digested by XhoI/EcoRV to obtain the p3.1-RX3-GUS clone.
[0275] The p3.1-RX3-EK corresponds to the following NheI/HindIII
DNA fragment in pcDNA3.1(-) (Invitrogen)
TABLE-US-00043 SEQ ID NO: 72
gctagcgtttaaacgggccctctagactcgacaccatgagggtgttgctc
gttgccctcgctctcctggctctcgctgcgagcgccacctccacgcatac
aagcggcggctgcggctgccagccaccgccgccggttcatctaccgccgc
cggtgcatctgccacctccggttcacctgccacctccggtgcatctccca
ccgccggtccacctgccgccgccggtccacctgccaccgccggtccatgt
gccgccgccggttcatctgccgccgccaccatgccactaccctactcaac
cgccccggcctcagcctcatccccagccacacccatgcccgtgccaacag
ccgcatccaagcccgtgccaaaggcgcgccggtggaggcggaggtaccat
gattgagggtaggattgttggtggaagtgattcccgtgaaggtgcttggc
cttgggttgtggctctttatttcgatgatcagcaagtttgtggagcctcc
cttgtttctagagattggcttgtgtctgctgcacattgcgtgtatggaag
aaatatggaaccaagtaagtggaaggcagttcttggattgcatatggctt
caaatcttacaagtccacagattgaaactcgtctcatcgatcaaattgtt
atcaacccacactataacaagaggagaaaaaacaatgatattgctatgat
gcatcttgagatgaaagtgaactacacagattacattcagccaatttgtc
ttccagaggaaaaccaagttttcccacctggaaggatttgttctattgcc
ggttggggagcacttatctatcaaggatcaactgcagatgttcttcaaga
agcagatgttccacttttgtcaaatgagaaatgccaacagcaaatgcctg
agtataacattactgagaatatggtgtgtgctggatacgaggcaggaggt
gtggattcttgtcagggagattctggaggtcctcttatgtgccaggagaa
taacagatggcttttagccggagttacttctttcggataccaatgcgcat
tgccaaatagacctggtgtgtatgctagagttccaaggtttacagagtgg
attcaatcatttctacattgataaggatccgagctcggtaccaagctt
Example 19
Plasmid Construction for Insect Infection
[0276] The RX3-DsRED fragment from p3.1-RX3-DsRED was digested by
XbaI/HindIII and cloned into pFastBac1 (Invitrogen) digested also
by these two enzymes in order to obtain pF-RX3-DsRED vector.
[0277] The DsRED cDNA was amplified by PCR from pF-RX3-DsRED by
using the following primers:
TABLE-US-00044 bGH rev: SEQ ID NO: 73 5' CCTCGACTGTGCCTTCTA 3' bGH
rev2: SEQ ID NO: 74 5' CCTCTAGACTCGACCCATGGTGAGCAAGGGCGAGGAG 3'
To obtain the pF-DsRED vector, the PCR-amplified DNA fragment was
digested by XbaI/HindIII and cloned into pFastBac1 (Invitrogen)
also digested by XbaI/HindIII.
Example 20
Insect Cell and Larvae Infection
[0278] Baculovirus and Larvae
[0279] The pFastBac baculoviral expression vector system
(Invitrogen) was used for all experiments. The recombinant virus
was produced and amplified according to the manufacturer's
instructions. Eggs of cabbage looper, Trichoplusia ni, were
obtained from Entopath, Inc. (Easton, Pa.). The eggs were hatched
and larvae reared according to the directions provided by the
manufacturer, and fourth instar larvae were used for baculovirus
infection.
[0280] Larvae Infection
[0281] Various amounts of baculovirus stock solution, consisting of
occluded recombinant virus were spread on the larval diet, which
was ordered premade in Styrofoam cups from Entopath, Inc. (Easton,
Pa.). The cups were covered and allowed to stand for an hour so
that the virus was completely absorbed by the media. The fourth
instar larvae were then placed into the cups (approximately 10 to
15 larvae per cup), and the cups were inverted. The larvae fed from
the top (bottom of cup) so that fecal matter dropped on to the lid
where it was discarded daily. The quantity of food was sufficient
for at least 5 days of growth. Three to five larvae were collected
daily for RX3-DsRED and DsRED analysis.
[0282] SF9 Infection
[0283] Spodoptera Sf9 cell cultures were obtained from Invitrogen
(San Diego, Calif., U.S.A.) and cultured as previously (O'Reilly et
al., 1992) using Grace's insect medium supplemented with
lactalbumin hydrolysate, yeastolate, L-glutamine, 10%
heat-inactivated fetal bovine serum and 1% penicillin/streptomycin
solution (Gibco). Cells were grown in either spinner flasks (Bellco
Glass, Vineland, N.J., U.S.A.) or 100 mm plastic tissue culture
dishes (Falcon). Recombinant virus was produced using the
BaculoGold Transfection Kit (PharMingen, San Diego, Calif.,
U.S.A.). Single plaques were isolated and amplified two to four
times to obtain a high-titer viral stock which was stored at
4.degree. C. until use. For routine infection, Sf9 cells in Grace's
medium were allowed to attach to the bottom of a 100 mm plastic
culture dish (10.sup.7 cells/dish). After incubation for 15 min to
1 h, a portion of viral stock was added and the cultures were
maintained at 27.degree. C. in a humidified air atmosphere. Cells
were typically used at 30-36 hours after infection.
Example 21
RPBLAs Preparation from Mammal Cells and Insect Larvae
[0284] Homogenization
[0285] Mammalian Cells
[0286] Transfected cells were recovered from culture plates by
scraping and were suspended in the homogenization B medium (10 mM
Tris-HCl pH 8.0, 0.9% NaCl, 5 mM EDTA with protease inhibitors).
The cell suspension was aspirated into a 5 ml syringe fitted with a
23 gauge needle and it was taken up and expelled approximately 30
times. Cell rupture was monitored by a phase contrast
microscope.
[0287] Insect Larvae
[0288] Frozen Trichoplusia ni larvae expressing RX3-DsRED and DsRED
proteins were homogenized in PBP5 buffer (20 mM Hepes pH 7.5, 5 mM
EDTA) by polytron for 2 minutes at 13500 rpm and/or by Potter for 5
minutes in ice at 2000 rpm. This homogenate was centrifuged at 200
g 10 minutes to remove cuticle and tissue debris and the
supernatant was loaded on a density step-gradient.
[0289] RPBLAs isolation by Density
[0290] RPBLAs from mammal cells and frozen insect larvae were
isolated essentially as described for plants (density step gradient
or low-speed centrifugation).
Example 22
Solubilization by Triton X-114-Based Biphasic Separation
[0291] Cell homogenates were diluted with PBS and centrifuged at
16,000.times.g for 15 minutes. The supernatant was removed and the
pellet dried. 2 ml of ice cold Solubilization Buffer (50 mM Tris pH
7, 5% Triton X-114, 20 mM TCEP, 20 mM NDSB195 and 100 mM
MgCl.sub.2) was added to the pellet followed by 1 ml of PBS
containing 1M Urea, 10% Glycerol and 100 mM MgCl.sub.2. This
composition was incubated on ice for 15 min with occasional
vortexing. The suspension was then sonicated for 20 seconds X 4 at
50% potential, keeping it on ice between bursts for 1 minute to
maintain cold temperature. The suspension was then incubated at
37.degree. C. for 15 minutes to form the 2 phases. Three ml of 10%
PEG were added to the lower hydrophobic layer (Triton X-114 rich)
and the composition was incubated on ice for 20 minutes. The
solution was then incubated at 37.degree. C. for 15 minutes to
re-form the 2 phases. The upper phase (4 ml) was recovered and
stored for analysis.
Example 23
Immunolocalization
[0292] Immunocytochemistry was performed two to four days after
transfection using a fluorescent microscope (Vertical Eclipse
Microscope Nikon E600A), Cells were fixed for 30 minutes in a 1%
paraformaldehyde solution, washed with phosphate saline buffer, and
incubated for 45 minutes with antibodies raised against either (i)
hGH (dilution 1/150), (ii) EK (dilution 1/500), or (iii) RX3
(dilution 1/700). In order to detect the antigen-antibody reaction,
cells were incubated for 45 minutes with anti-rabbit antisera
conjugated to Alexa Fluor 488(Invitrogen).
[0293] Confocal analysis was performed using a Confocal laser
scanning microscope (Leica TCS SP, Heidelberg, Germany) fitted with
spectrophotometers for emission band wavelength selection. Green
fluorescent images were collected following excitation at 488 nm
with an Argon ion laser using an emission window set at 495-535 nm.
Red fluorescent images were collected using 543 nm excitation with
a HeNe laser and emission window of 550-600 nm. Optical sections
were 0.5 to 1 .mu.m thick.
Example 24
Activity Assays
EGF Activity Assay
[0294] MDA-MB231 breast cancer cells over-expressing the human EGF
receptor were seeded in 96-well plates at 5,500 cells/well. Cells
were allowed to adhere for 8 hours in growth medium supplemented
with 10% FCS (Fetal calf serum) and then starved overnight in
medium with 0.1% of FCS. Afterwards, the medium was removed and the
positive-control EGF from Promega or the corresponding experimental
sample (solubilyzed RX3-EGF) was added at different concentrations.
Radioactive thymidine was added to a final concentration of 0.5
.mu.Ci. The cells were washed twice with cold PBS and kept on ice
to stop the cell metabolism, and proliferation was studied at 48
hours after stimulation at 37.degree. C. A 10% trichloroacetic acid
(TCA) solution was added, and the cells were incubated for 20
minutes at 4.degree. C. Once the TCA solution was removed, the
plates were washed twice with 70% Ethanol, and the cells were
incubated for 20 minutes at 37.degree. C. in 0.5 mL of lysis
solution (2% CO.sub.3Na.sub.2, 0.1N NaOH and 10% SDS) Plates were
mixed by vortex agitation and the sample was not measured before 12
hours to avoid undesired chemo-luminescent phenomena.
[0295] EK Activity Assay
[0296] The enzymatic activity was measured by fluorometric assay
(Grant et al, (1979) Biochim. Biophys. Acta 567:207-215). The
reaction was initiated by adding the enzyme to 0.3 to 1.0 mM of the
fluorogenic substrate Gly-(Asp)4-Lys-.beta.-naphtylamide (Sigma) In
25 mM Tris-HCl (pH 8.4), 10 mM CaCl.sub.2, 10% DMSO (dimethyl
sulfoxide) at 37.degree. C. Free .beta.-naphtylamine concentration
was determined from the increment of fluorescence (.lamda.ex=337 nm
and .lamda.em=420 nm) continuously monitored for 1 minute. The
activity was calculated as change in fluorescence over time.
[0297] GUS Activity Assay
[0298] GUS activity assay is based in the catalysis of
metilumbeliferil-.beta.-glucuronide acid (MUG) to the
4-metilumbeliferone (4-MU) fluorescent product, by the GUS enzyme
(Jefferson et al. 1987 EMBO J. 6(13):3901-3907). 50 .mu.L of
solubilyzed RX3-GUS (or solubilyzed RX3 as a control) was added to
200 .mu.L of Reaction buffer (50 mM of phosphate buffer pH 7, 10 mM
EDTA, 0.1% SDS and 0.1% Triton X100) plus 66 .mu.L of Methanol. The
substrate (MUG) was added to a final concentration of 10 mM. The
standard was prepared by adding 0, 50, 100, 200, 300 or 500 pmols
of 4-MU (the product of the reaction) to 200 .mu.L of Reaction
buffer of the reaction (4-MU).
[0299] The samples and the standard were mixed and they were
measured in a fluorimeter (Victor, Perkin-Elmer) at .lamda.ex=355
nm and .lamda.em=460 nm. The samples were measured each 30 minutes
for 3 hours. The specific activity was calculated by the formula:
GUS activity (pmols 4-MU/min-l*mg-l)=(.lamda.em(T1)-(80
em(T0))/(k*(T1-T0)). "K"=ratio (Units of fluorescence)/(pmol
4-MU).
[0300] RTB Activity Assay
[0301] (Asialofetuin-BINDINN ELISA)
[0302] The functionality of RX3-RTB in the protein extracts from
RPBLAs was determined via binding to asialofetuin, the glycoprotein
fetuin treated with sialydase to expose galactose-terminated
glycans. Two hundred microliters of asialofetuin (Sigma) at a
concentration of 300 mg/mL in modified PBS (mPBS) buffer (100 mM
Na-phosphate, 150 mM NaCl, pH 7.0) was bound to the wells of an
Immulon 4HBX (Fisher, Pittsburg, Pa.) microtiter plate for 1 hour
at RT. The coating solution was discarded and the wells blocked
with 200 ml 3% BSA, 0.1% Tween 20 in mPBS for 1 hour at RT. After
the blocking solution was discarded, 100 ml of RTB standards and
protein extracts (see below) were applied and incubated for 1 hr at
RT. The wells were then washed three times with 200 ml PBS, 0.1%
Tween 20. Rabbit anti-R. communis lectin (RCA60) polyclonal Ab
(Sigma) at 1:4000 in blocking buffer (as above) was applied and
incubated for 1 hour at RT. The wells were then washed as before.
AP-conjugated goat-anti-rabbit IgG (Bio-Rad) was applied at a
dilution of 1:3000 in blocking buffer and incubated for 1 hour at
RT. The wells were washed three times as described above and 100 ml
pNPP (p-nitrophenyl phosphate disodium salt) substrate (Pierce,
Rockford, Ill.) was applied. The reaction was stopped after 15
minutes by the addition of 50 .mu.l of 2N NaOH. Absorbance
(A.sub.405) was read in a Bio-Tek EL808 Ultra Microplate Reader.
Protein extracts were prepared at a ratio of 1 g FW leaf to 3 ml of
Tris-acorbate buffer (above), and the samples compared against a
standard curve consisting of serially-diluted castor bean-derived
RTB (Vector Labs, Burlingame, Calif.) in Tris-acorbate buffer, with
the concentrations ranging from 5 ng to 500 ng per well.
Example 25
Enhanced Uptake of RX3-DsRED Assembled in RPBLAs from Insect Larvae
by Macrophages
[0303] The cDNA coding for RX3-DsRED and DsRED were cloned in the
baculovirus FastBac vector (Invitrogen) to obtain pFB-RX3-DsRED and
pFB-DsRED. These constructs were used to infect Trichoplusia ni
larvae. Frozen larvae expressing RX3-DsRED and DsRED proteins were
homogenized and loaded on a density step gradient. After
centrifugation at 80000.times.g in a swinging-bucket for 2 hours,
the analysis of the RX3-DsRED fusion protein and the control
corresponding to DsRED expressed in the cytosol was performed by
immunoblot (FIG. 2C). As expected, when expressed in the larval
cells cytosol, the DsRED protein did not assemble in highly dense
structures and was localized in the supernatant and the F35
fraction (FIG. 2C, lane 2 and 3). On the other hand, RX3-DsRED
fusion protein was able to assemble and accumulate in dense
structures that can be isolated from F56 (FIG. 2C, lane 5). As
shown by confocal microscopy analysis in Example 4 (FIG. 4), the
RX3-DsRED accumulated in round-shaped RPBLAs.
[0304] The RPBLAS of RX3-DsRED from F56 were diluted 3-fold in PBP5
(10 mM HEPES pH 7.4, 2 mM EDTA) and collected in the pellet by
centrifugation at 80000.times.g at 4.degree. C. in a
swinging-bucket for 2 hours. The pellet was resuspended in PBS
buffer and the number of RPBLAs was quantified by FACS. From one
larva infected with the pFB-RX3-DsRED vector, approximately
1.times.10.sup.9 RPBLAs particles were obtained at a concentration
of 500,000 RPBLAs per microliter (.mu.l).
[0305] It has been reported that antigen presentation by the
antigen presentation cells (APC) such as macrophages and dendritic
cells is a key process necessary to induce an immune response
(Greenberg et al, Current Op. Immunology (2002), 14:136-145). In
this process, the APC takes up (phagocytoses) the antigen, which is
subsequently cleaved into small peptides in the phagolysosome.
These peptides interact with the major histocompatibility II
(MHCII) protein and are sorted to the plasma membrane to be
presented to the cell- and antibody-mediated immunity responses
(Villandagos et al., Immunological Reviews (2005) 207:101-205).
[0306] To determine the antigenicity of RX3 fusion proteins present
inside the RPBLAs, a macrophage cell culture was incubated with
these organelles at different RPBLA/cell ratios (100:1 and 1000:1).
The macrophage cell cultures were grown on starved conditions or in
the presence of GM-CSF. These cell cultures were incubated with
RPBLAs for 1 hour, and 1, 2, 5 and 10 hours after RPBLA removal the
macrophages were extensively washed with PBS and fixed with 2%
paraformaldehyde. Afterwards, these fixed macrophages were analyzed
by FACS to quantify the amount of fluorescent RPBLAs up taken by
the macrophages as well as the percentage of macrophages that had
phagocytosed the fluorescent RX3-DsRED RPBLAs
Percentage of Fluorescent Macrophages
TABLE-US-00045 [0307] Starved M-CSF M-CSF (RPBLA/cell (RPBLA/cell
(RPBLA/cell Time ratio 100:1) ratios 100:1) ratio 1000:1) (hours)
Mean STD Mean STD Mean STD zero 1.19 1.21 0.82 0.35 0.82 0.35 1
65.42 2.29 65.19 3.2 85.78 1.65 2 79.64 1.66 75.08 3.94 91.55 1.5 5
91.85 2.17 87.68 1.58 91.53 1.09 10 88.91 0.7 90.54 1.59 94.4
0.08
[0308] From these results, it is clear that the macrophages
phagocytosed the RX3-DsRED RPBLAs with an unexpected avidity. Even
at the lower RPBLAs/cells ratio (1:100) and in the presence of
M-CSF, at 1 hour after RPBLAs addition, 65% of macrophages are
fluorescent. Even the presence of a mitogenic cytokine, such as
M-CSF, which has a negative effect on macrophage phagocytosis could
not impair significantly the RPBLA uptake. At 5 hours, almost all
(more than 80%) of the macrophages were fluorescent, meaning that
the majority of the cells had up taken some RPBLAs from the
medium.
[0309] When the amount of fluorescence associated with the
macrophages was analyzed over the time of incubation, the results
were even more surprising, as no saturation of the capacity of the
macrophages to uptake the RPBLAs was observed. If the results of
the Tables above and below are compared at 5 and 10 hours of
incubation, it is seen that almost all the macrophages are
fluorescent, but there is a continuous increase in the total
fluorescence associated with the macrophages. This result is
consistent with the macrophages phagocytosing a large quantity of
fluorescent RPBLAs particles.
Time Dependent Macrophage Fluorescence
TABLE-US-00046 [0310] Starved M-CSF M-CSF (RPBLA/cell (RPBLA/cell
(RPBLA/cell Time ratio 100:1) ratios 100:1) ratio 1000:1) (hours)
Mean STD Mean STD Mean STD 0 0.975 0.31 0.725 0.1 0.725 0.1 1 8.9
0.42 10.3 1.13 24 1.7 2 16.35 0.07 16.25 0.5 41.5 0.3 5 64.65 2.05
42.35 4.45 93.3 2.2 10 120.7 1.84 79.9 5.66 125.65 13.08
[0311] To demonstrate that RPBLAs containing the RX3-DsRED fusion
protein were inside the macrophages and not simply adsorbed
externally to the plasma membrane, confocal microscopy analysis was
performed. FIG. 7A (left panel) shows macrophage cells incubated
with RX3-DsRED particles (at 100:1) for 1 hour. On the left panel
of the same figure, a section of 1 micrometer of the same cells
shows the typical green auto-fluorescence of macrophages observed
with a green filter (FIG. 7A, white arrowhead). The presence of the
nucleus and the red-fluorescent RPBLA particles (FIG. 7A, black
arrowhead) in the same optical section indicates that the RPBLAs
had been taken up inside the cells.
[0312] Another important factor to be analyzed is protein
degradation following macrophage phagocytosis, Antigen degradation
is needed to produce the antigenic peptides that are presented on
the MHCII receptor. Analysis of the DsRED fluorescent pattern of
the macrophages over time is consistent with the RPBLA particles
being actively digested.
[0313] Another set of micrographs shows that after 1 hr of
incubation, the RPBLA particles were still not fully degraded and
could still be observed inside the cells (FIG. 7B, upper panels).
After 10 hours, the red fluorescence pattern was more homogenous,
however, indicating that the macrophages had begun to degrade the
RPBLA particles (FIG. 7B, bottom panels).
Example 26
Enhanced Uptake of RX3-DsRED in RPBLAs from Insect Larvae by
Dendritic Cells
[0314] To assess the capacity of dendritic cells to phagocytose
RX3-DsRED fusion proteins assembled in RPBLAs from insect larvae, a
dendritic cell culture was incubated with these organelles at a 100
RPBLAs/cell ratio. Two kinds of RPBLAs were prepared: (i) RPBLAs
isolated as described previously and (ii) the same RPBLAs washed in
50 mM Tris pH 8, 1% Triton X-100, in order to remove the
surrounding membrane. The dendritic cell cultures were grown on
starved conditions in the presence of RPBLAs, and samples were
analyzed at 0, 1, 2, 5 and 10 hours.
Percentage of Fluorescent Dendritic Cells
TABLE-US-00047 [0315] % of fluorescent dendritic cells Time RPBLAs
Membrane-less RPBLAs (hours) Mean STD Mean STD 0 1.43 -- 1.41 -- 1
26.76 -- 36.46 0.28 2 33.79 0.6 50.785 0.21 5 45.845 0.07 67.275
3.4 10 61.885 5.73 74.97 4.17
Time Dependent Dendritic Cells Fluorescence
TABLE-US-00048 [0316] Fluorescence associated ot dendritic cells
Time RPBLAs Membrane-less RPBLAs (hours) Mean STD Mean STD 0 0.5 --
1.1 -- 1 3.1 -- 5.1 0.28 2 3.55 0.6 5.05 0.21 5 25.15 0.07 54 3.4
10 37.05 5.73 74.05 4.17
[0317] As can be concluded from Tables above, the dendritic cells
show a surprising avidity for RPBLAs. As expected, they have a
slower phagocytosis rate compared to the macrophages (compare the
previous tables), as is described elsewhere. The percentage of
fluorescent dendritic cells increases all along the time course
analyzed, and no saturation effect was observed even at 10 hours
after RPBLAs incubation. Similar conclusions can be drawn when the
amount of fluorescence associated with the macrophages over time
was analyzed.
[0318] The dendritic cells' capacity to take up the RPBLAs did not
exhibit a saturation effect. This lack of effect can be explained
by the fact that more and more dendritic cells are induced to
phagocytosis (and becoming fluorescent) over time. Nevertheless, it
is also possible that the phagocytosis capacity of individual cells
is not saturated, as has been observed with macrophages.
[0319] Unexpectedly, the FACS analysis of dendritic cells incubated
with membrane-less RPBLAs showed a significantly higher percentage
of fluorescent dendritic cells than the same cells incubated with
membrane-containing RPBLAs. Moreover, the fluorescence of these
dendritic cells was also higher as well. Similar results were
obtained using macrophages with membrane-less RPBLAs. This was
somewhat surprising as it was expected that the presence of
insect-derived membrane proteins in the membrane-containing RPBLAs
would be recognized as foreign proteins by the murine dendritic
cells, and hence enhance phagocytosis. It is thus apparent that
insect-derived RPBLAs in the presence or absence of the surrounding
membrane are very efficient antigen presentation vehicles.
[0320] To demonstrate that RPBLAs and membrane-less RPBLAs
containing the RX3-DsRED fusion protein were taken up by the
dendritic cells, optical microscopy analysis was done. FIG. 8A
(upper) shows dendritic Cells incubated for 2, 5 and 10 hours with
RX3-DsRED RPBLAs (100:1 ratio). On the bottom of FIG. 8B, the red
fluorescence of the DsRED protein illustrates the uptake of the
RPBLAs by those cells. At 2 hours of incubation, some phagocytosis
can be observed, but most of the RPBLAs are only adsorbed to the
plasmatic membrane. At 5 hours, and even more at 10 hours, many
phagocytosed red fluorescent RPBLAs were observed. Similar results
were obtained when dendritic cells were incubated with
membrane-less RPBLAs (FIG. 8B).
[0321] It is important to note that even at 10 hours of incubation
with RPBLAs or membrane-less RPBLAs, most of the phagocytosed
particles remained visible as particles, meaning that little
proteolysis had take place. This observation agrees with previous
observation showing that the kinetics of protease acquisition, and
hence, of proteolysis, is slower in dendritic cells than in
macrophages (Lennon-Dum enil et al. (2002) J. Exp. Med.
196:529-540). These conditions may limit the proteolysis of
proteins in dendritic cells and favor the generation of peptide
antigens of appropriate length for loading onto MHC class II
molecules.
Example 27
Phagocytosis of Macrophages and Dendritic Cells
[0322] Macrophages
[0323] Macrophages were obtained from the marrow of Balb/C mice.
Mice were sacrificed by a cervical dislocation and femur and tibia
were extracted. The bones were cut and the marrow was extracted
with DMEM medium using a syringe. The marrow was cultivated on a
150 mm Petri plate with complete DMEM medium (supplemented with 20%
FCS and 30% L-cell). A murine macrophage culture of 99% purity was
obtained after 7 days of incubation at 37.degree. C.
[0324] The differentiated macrophages were cultivated in complete
medium to give rise 350,000 cells per well. When the cells were
adhered, the medium was removed and cells were incubated with new
medium that contained RX3-DsRED RPBLAs from larvae. The experiment
was repeated with either 100 or 1000 particles per cell. The number
of particles (RPBLAs) was counted by Coulter Epics XL FACS using an
Argon laser at 488 nm for excitation and FL2 at 575 nm+/-30 for
emission. Flow-count from Beckman Coulter ref. 7547053 (lot
754896F) was used to verify the flow.
[0325] After 0, 1, 2, 5 and 10 hours the medium was removed and two
washings with PBS were performed. Cells were permitted to
recuperate and then were fixed with 2% paraformaldehyde in PBS. The
treated macrophages were stored at 4.degree. C. and the cell
fluorescence was analyzed by FACS using the same program as for
counting.
[0326] Immunohistochemistry was performed to verify that RX3-DsRED
particles were located inside the cells following phagocytosis. The
differentiated macrophages (50,000 cells/well) were incubated with
100:1 particles of RX3-DsRED for 1 hour. After incubation, cells
were washed twice with PBS and fixed with PBS/2% formaldehyde for
15 minutes. Treated cells were analyzed by confocal microscopy.
[0327] Dendritic Cells
[0328] The marrow from Balb/C mice was cultivated with complete
medium (DMEM, 10% FCS, 5 ng/ml GM-CSF) for one day. In order to
remove granulocytes, plates were agitated and medium was changed
twice. Medium was then changed twice without agitation and plates
were incubated 2 days to obtain immature dendritic cells. Dendritic
cells were incubated with 100:1 particles of RX3-DsRED for 1, 5 and
10 hours. After treatment, cells were fixed with 2%
paraformaldehyde, stored at 4.degree. C. and the fluorescence was
analyzed by FACS.
[0329] Each of the patent applications, patents and articles cited
herein is incorporated by reference. The use of the article "a" or
"an" is intended to include one or more.
[0330] The foregoing description and the examples are intended as
illustrative and are not to be taken as limiting. Still other
variations within the spirit and scope of this invention are
possible and will readily present themselves to those skilled in
the art.
Sequence CWU 1
1
7416PRTArtificial sequencepart of Promoter 2xCaMV 1Pro Pro Pro Val
His Leu1 5253PRTArtificial sequenceRX3 query aa sequence 2Pro Pro
Pro Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro1 5 10 15Val
His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu Pro 20 25
30Pro Pro Val His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His
35 40 45Leu Pro Pro Pro Pro 50365PRTArtificial sequenceAlpha zein
aa sequence 3Gln Gln Gln Gln Gln Phe Leu Pro Ala Leu Ser Gln Leu
Asp Val Val1 5 10 15Asn Pro Val Ala Tyr Leu Gln Gln Gln Leu Leu Ala
Ser Asn Pro Leu 20 25 30Ala Leu Ala Asn Val Ala Ala Tyr Gln Gln Gln
Gln Gln Leu Gln Gln 35 40 45Phe Leu Pro Ala Leu Ser Gln Leu Ala Met
Val Asn Pro Ala Ala Tyr 50 55 60Leu65470PRTArtificial sequencerice
prolamin query aa sequence 4Gln Gln Val Leu Ser Pro Tyr Asn Glu Phe
Val Arg Gln Gln Tyr Gly1 5 10 15Ile Ala Ala Ser Pro Phe Leu Gln Ser
Ala Thr Phe Gln Leu Arg Asn 20 25 30Asn Gln Val Trp Gln Gln Leu Ala
Leu Val Ala Gln Gln Ser His Cys 35 40 45Gln Asp Ile Asn Ile Val Gln
Ala Ile Ala Gln Gln Leu Gln Leu Gln 50 55 60Gln Phe Gly Asp Leu
Tyr65 705672DNAMaize 5atgagggtgt tgctcgttgc cctcgctctc ctggctctcg
ctgcgagcgc cacctccacg 60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg
ttcatctacc gccgccggtg 120catctgccac ctccggttca cctgccacct
ccggtgcatc tcccaccgcc ggtccacctg 180ccgccgccgg tccacctgcc
accgccggtc catgtgccgc cgccggttca tctgccgccg 240ccaccatgcc
actaccctac tcaaccgccc cggcctcagc ctcatcccca gccacaccca
300tgcccgtgcc aacagccgca tccaagcccg tgccagctgc agggaacctg
cggcgttggc 360agcaccccga tcctgggcca gtgcgtcgag tttctgaggc
atcagtgcag cccgacggcg 420acgccctact gctcgcctca gtgccagtcg
ttgcggcagc agtgttgcca gcagctcagg 480caggtggagc cgcagcaccg
gtaccaggcg atcttcggct tggtcctcca gtccatcctg 540cagcagcagc
cgcaaagcgg ccaggtcgcg gggctgttgg cggcgcagat agcgcagcaa
600ctgacggcga tgtgcggcct gcagcagccg actccatgcc cctacgctgc
tgccggcggt 660gtcccccacg cc 6726224PRTMaize 6Met Arg Val Leu Leu
Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala Thr Ser Thr
His Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30Pro Val His
Leu Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55 60His
Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro65 70 75
80Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln Pro His Pro
85 90 95Gln Pro His Pro Cys Pro Cys Gln Gln Pro His Pro Ser Pro Cys
Gln 100 105 110Leu Gln Gly Thr Cys Gly Val Gly Ser Thr Pro Ile Leu
Gly Gln Cys 115 120 125Val Glu Phe Leu Arg His Gln Cys Ser Pro Thr
Ala Thr Pro Tyr Cys 130 135 140Ser Pro Gln Cys Gln Ser Leu Arg Gln
Gln Cys Cys Gln Gln Leu Arg145 150 155 160Gln Val Glu Pro Gln His
Arg Tyr Gln Ala Ile Phe Gly Leu Val Leu 165 170 175Gln Ser Ile Leu
Gln Gln Gln Pro Gln Ser Gly Gln Val Ala Gly Leu 180 185 190Leu Ala
Ala Gln Ile Ala Gln Gln Leu Thr Ala Met Cys Gly Leu Gln 195 200
205Gln Pro Thr Pro Cys Pro Tyr Ala Ala Ala Gly Gly Val Pro His Ala
210 215 2207339DNAArtificial sequenceRX3 DNA sequence 7atgagggtgt
tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg 60catacaagcg
gcggctgcgg ctgccagcca ccgccgccgg ttcatctacc gccgccggtg
120catctgccac ctccggttca cctgccacct ccggtgcatc tcccaccgcc
ggtccacctg 180ccgccgccgg tccacctgcc accgccggtc catgtgccgc
cgccggttca tctgccgccg 240ccaccatgcc actaccctac tcaaccgccc
cggcctcagc ctcatcccca gccacaccca 300tgcccgtgcc aacagccgca
tccaagcccg tgccagacc 3398113PRTArtificial sequenceRX3 aa 8Met Arg
Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala
Thr Ser Thr His Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25
30Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu
35 40 45Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro
Val 50 55 60His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu
Pro Pro65 70 75 80Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro
Gln Pro His Pro 85 90 95Gln Pro His Pro Cys Pro Cys Gln Gln Pro His
Pro Ser Pro Cys Gln 100 105 110Tyr9240DNAArtificial sequenceR3 DNA
9atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg
60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctacc gccgccggtg
120catctgccac ctccggttca cctgccacct ccggtgcatc tcccaccgcc
ggtccacctg 180ccgccgccgg tccacctgcc accgccggtc catgtgccgc
cgccggttca tctgccgccg 2401092PRTArtificial sequencechemically
synthesized 10Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu
Ala Ala Ser1 5 10 15Ala Thr Ser Thr His Thr Ser Gly Gly Cys Gly Cys
Gln Pro Pro Pro 20 25 30Pro Val His Leu Pro Pro Pro Val His Leu Pro
Pro Pro Val His Leu 35 40 45Pro Pro Pro Val His Leu Pro Pro Pro Val
His Leu Pro Pro Pro Val 50 55 60His Leu Pro Pro Pro Val His Val Pro
Pro Pro Val His Leu Pro Pro65 70 75 80Pro Pro Cys His Tyr Pro Thr
Gln Pro Pro Arg Tyr 85 9011213DNAArtificial sequenceP4 DNA
11atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg
60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctgcc gccgccacca
120tgccactacc ctacacaacc gccccggcct cagcctcatc cccagccaca
cccatgcccg 180tgccaacagc cgcatccaag cccgtgccag acc
2131271PRTArtificial sequenceP4 protein 12Met Arg Val Leu Leu Val
Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala Thr Ser Thr His
Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30Pro Val His Leu
Pro Pro Pro Pro Cys His Tyr Pro Thr Gln Pro Pro 35 40 45Arg Pro Gln
Pro His Pro Gln Pro His Pro Cys Pro Cys Gln Gln Pro 50 55 60His Pro
Ser Pro Cys Gln Tyr65 7013180DNAArtificial sequenceX10 DNA
13atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg
60catacaagcg gcggctgcgg ctgccaatgc cactacccta ctcaaccgcc ccggcctcag
120cctcatcccc agccacaccc atgcccgtgc caacagccgc atccaagccc
gtgccagacc 1801460PRTArtificial sequenceX10 protein 14Met Arg Val
Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala Thr
Ser Thr His Thr Ser Gly Gly Cys Gly Cys Gln Cys His Tyr 20 25 30Pro
Thr Gln Pro Pro Arg Pro Gln Pro His Pro Gln Pro His Pro Cys 35 40
45Pro Cys Gln Gln Pro His Pro Ser Pro Cys Gln Tyr 50 55
6015150PRTArtificial sequencerP13 protein 15Met Lys Ile Ile Phe Val
Phe Ala Leu Leu Ala Ile Ala Ala Cys Ser1 5 10 15Ala Ser Ala Gln Phe
Asp Val Leu Gly Gln Ser Tyr Arg Gln Tyr Gln 20 25 30Leu Gln Ser Pro
Val Leu Leu Gln Gln Gln Val Leu Ser Pro Tyr Asn 35 40 45Glu Phe Val
Arg Gln Gln Tyr Gly Ile Ala Ala Ser Pro Phe Leu Gln 50 55 60Ser Ala
Thr Phe Gln Leu Arg Asn Asn Gln Val Trp Gln Gln Leu Ala65 70 75
80Leu Val Ala Gln Gln Ser His Cys Gln Asp Ile Asn Ile Val Gln Ala
85 90 95Ile Ala Gln Gln Leu Gln Leu Gln Gln Phe Gly Asp Leu Tyr Phe
Asp 100 105 110Arg Asn Leu Ala Gln Ala Gln Ala Leu Leu Ala Phe Asn
Val Pro Ser 115 120 125Arg Tyr Gly Ile Tyr Pro Arg Tyr Tyr Gly Ala
Pro Ser Thr Ile Thr 130 135 140Thr Leu Gly Gly Val Leu145
15016450DNAArtificial sequencerP13 DNA 16atgaagatca ttttcgtctt
tgctctcctt gctattgctg catgcagcgc ctctgcgcag 60tttgatgttt taggtcaaag
ttataggcaa tatcagctgc agtcgcctgt cctgctacag 120caacaggtgc
ttagcccata taatgagttc gtaaggcagc agtatggcat agcggcaagc
180cccttcttgc aatcagctac gtttcaactg agaaacaacc aagtctggca
acagctcgcg 240ctggtggcgc aacaatctca ctgtcaggac attaacattg
ttcaggccat agcgcagcag 300ctacaactcc agcagtttgg tgatctctac
tttgatcgga atctggctca agctcaagct 360ctgttggctt ttaacgtgcc
atctagatat ggtatctacc ctaggtacta tggtgcaccc 420agtaccatta
ccacccttgg cggtgtcttg 45017144PRTArtificial sequence22aZt protein
17Met Ala Thr Lys Ile Leu Ala Leu Leu Ala Leu Leu Ala Leu Phe Val1
5 10 15Ser Ala Thr Asn Ala Phe Ile Ile Pro Gln Cys Ser Leu Ala Pro
Ser 20 25 30Ala Ile Ile Pro Gln Phe Leu Pro Pro Val Thr Ser Met Gly
Phe Glu 35 40 45His Leu Ala Val Gln Ala Tyr Arg Leu Gln Gln Ala Leu
Ala Ala Ser 50 55 60Val Leu Gln Gln Pro Ile Asn Gln Leu Gln Gln Gln
Ser Leu Ala His65 70 75 80Leu Thr Ile Gln Thr Ile Ala Thr Gln Gln
Gln Gln Gln Phe Leu Pro 85 90 95Ala Leu Ser Gln Leu Asp Val Val Asn
Pro Val Ala Tyr Leu Gln Gln 100 105 110Gln Leu Leu Ala Ser Asn Pro
Leu Ala Leu Ala Asn Val Ala Ala Tyr 115 120 125Gln Gln Gln Gln Gln
Leu Gln Gln Phe Leu Pro Ala Leu Ser Gln Leu 130 135
14018432DNAArtificial sequence22aZt DNA 18atggctacca agatattagc
cctccttgcg cttcttgccc tttttgtgag cgcaacaaat 60gcgttcatta ttccacaatg
ctcacttgct cctagtgcca ttataccaca gttcctccca 120ccagttactt
caatgggctt cgaacaccta gctgtgcaag cctacaggct acaacaagcg
180cttgcggcaa gcgtcttaca acaaccaatt aaccaattgc aacaacaatc
cttggcacat 240ctaaccatac aaaccatcgc aacgcaacag caacaacagt
tcctaccagc actgagccaa 300ctagatgtgg tgaaccctgt cgcctacttg
caacagcagc tgcttgcatc caacccactt 360gctctggcaa acgtagctgc
ataccaacaa caacaacaat tgcagcagtt tctgccagcg 420ctcagtcaac ta
43219283PRTArtificial sequencegamm gliadin precursor 19Asn Met Gln
Val Asp Pro Ser Gly Gln Val Gln Trp Pro Gln Gln Gln1 5 10 15Pro Phe
Pro Gln Pro Gln Gln Pro Phe Cys Gln Gln Pro Gln Arg Thr 20 25 30Ile
Pro Gln Pro His Gln Thr Phe His His Gln Pro Gln Gln Thr Phe 35 40
45Pro Gln Pro Gln Gln Thr Tyr Pro His Gln Pro Gln Gln Gln Phe Pro
50 55 60Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Thr
Phe65 70 75 80Pro Gln Gln Pro Gln Leu Pro Phe Pro Gln Gln Pro Gln
Gln Pro Phe 85 90 95Pro Gln Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln
Ser Gln Gln Pro 100 105 110Gln Gln Pro Phe Pro Gln Pro Gln Gln Gln
Phe Pro Gln Pro Gln Gln 115 120 125Pro Gln Gln Ser Phe Pro Gln Gln
Gln Gln Pro Ala Ile Gln Ser Phe 130 135 140Leu Gln Gln Gln Met Asn
Pro Cys Lys Asn Phe Leu Leu Gln Gln Cys145 150 155 160Asn His Val
Ser Leu Val Ser Ser Leu Val Ser Ile Ile Leu Pro Arg 165 170 175Ser
Asp Cys Gln Val Met Gln Gln Gln Cys Cys Gln Gln Leu Ala Gln 180 185
190Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Val Ala His Ser
195 200 205Ile Ile Met Gln Gln Glu Gln Gln Gln Gly Val Pro Ile Leu
Arg Pro 210 215 220Leu Phe Gln Leu Ala Gln Gly Leu Gly Ile Ile Gln
Pro Gln Gln Pro225 230 235 240Ala Gln Leu Glu Gly Ile Arg Ser Leu
Val Leu Lys Thr Leu Pro Thr 245 250 255Met Cys Asn Val Tyr Val Pro
Pro Asp Cys Ser Thr Ile Asn Val Pro 260 265 270Tyr Ala Asn Ile Asp
Ala Gly Ile Gly Gly Gln 275 280202086DNAArtificial sequencegamma
gliadin DNA M36999 20gcatgcattg tcaaagtttg tgaagtagaa ttaataacct
tttggttatt gatcactgta 60tgtatcttag atgtcccgta gcaacggtaa gggcattcac
ctagtactag tccaatatta 120attaataact tgcacagaat tacaaccatt
gacataaaaa ggaaatatga tgagtcatgt 180attgattcat gttcaacatt
actacccttg acataaaaga agaatttgac gagtcgtatt 240agcttgttca
tcttaccatc atactatact gcaagctagt ttaaaaaaga atyaaagtcc
300agaatgaaca gtagaatagc ctgatctatc tttaacaaca tgcacaagaa
tacaaattta 360gtcccttgca agctatgaag atttggttta tgcctaacaa
catgataaac ttagatccaa 420aaggaatgca atctagataa ttgtttgact
tgtaaagtcg ataagatgag tcagtgccaa 480ttataaagtt ttcgccactc
ttagatcata tgtacaataa aaaggcaact ttgctgacca 540ctccaaaagt
acgtttgtat gtagtgccac caaacacaac acaccaaata atcagtttga
600taagcatcga atcactttaa aaagtgaaag aaataatgaa aagaaaccta
accatggtag 660ctataaaaag cctgtaatat gtacactcca taccatcatc
catccttcac acaactagag 720cacaagcatc aaatccaagt aagtattagt
taacgcaaat ccaccatgaa gaccttactc 780atcctaacaa tccttgcgat
ggcaacaacc atcgccaccg ccaatatgca agtcgacccc 840agcggccaag
tacaatggcc acaacaacaa ccattccccc agccccaaca accattctgc
900cagcaaccac aacgaactat tccccaaccc catcaaacat tccaccatca
accacaacaa 960acatttcccc aaccccaaca aacatacccc catcaaccac
aacaacaatt tccccagacc 1020caacaaccac aacaaccatt tccccagccc
caacaaacat tcccccaaca accccaacta 1080ccatttcccc aacaacccca
acaaccattc ccccagcctc agcaacccca acaaccattt 1140ccccagtcac
aacaaccaca acaacctttt ccccagcccc aacaacaatt tccgcagccc
1200caacaaccac aacaatcatt cccccaacaa caacaaccgg cgattcagtc
atttctacaa 1260caacagatga acccctgcaa gaatttcctc ttgcagcaat
gcaaccatgt gtcattggtg 1320tcatctctcg tgtcaataat tttgccacga
agtgattgcc aggtgatgca gcaacaatgt 1380tgccaacaac tagcacaaat
tcctcaacag ctccagtgcg cagccatcca cagcgtcgcg 1440cattccatca
tcatgcaaca agaacaacaa caaggcgtgc cgatcctgcg gccactattt
1500cagctcgccc agggtctggg tatcatccaa cctcaacaac cagctcaatt
ggaggggatc 1560aggtcattgg tattgaaaac tcttccaacc atgtgcaacg
tgtatgtgcc acctgactgc 1620tccaccatca acgtaccata tgccaacata
gacgctggca ttggtggcca atgaaaaatg 1680caagatcatc attgcttagc
tgatgcacca atcgttgtag cgatgacaaa taaagtggtg 1740tgcaccatca
tgtgtgaccc cgaccagtgc tagttcaagc ttgggaataa aagacaaaca
1800aagttcttgt ttgctagcat tgcttgtcac tgttacattc actttttatt
tcgatgttca 1860tccctaaccg caatcctagc cttacacgtc aatagctagc
tgcttgtgct ggcaggttac 1920tatataatct atcaattaat ggtcgaccta
ttaatccaag taataggcta ttgatagact 1980gctcccaagc cgaccgagca
cctatcagtt acggatttct tgaacattgc acactataat 2040aattcaacgt
atttcaacct ctagaagtaa agggcatttt agtagc 208621537DNAArtificial
sequencebeta zein AF371264 DNA 21atgaagatgg tcatcgttct cgtcgtgtgc
ctggctctgt cagctgccag cgcctctgca 60atgcagatgc cctgcccctg cgcggggctg
cagggcttgt acggcgctgg cgccggcctg 120acgacgatga tgggcgccgg
cgggctgtac ccctacgcgg agtacctgag gcagccgcag 180tgcagcccgc
tggcggcggc gccctactac gccgggtgtg ggcagccgag cgccatgttc
240cagccgctcc ggcaacagtg ctgccagcag cagatgagga tgatggacgt
gcagtccgtc 300gcgcagcagc tgcagatgat gatgcagctt gagcgtgccg
ctgccgccag cagcagcctg 360tacgagccag ctctgatgca gcagcagcag
cagctgctgg cagcccaggg tctcaacccc 420atggccatga tgatggcgca
gaacatgccg gccatgggtg gactctacca gtaccagctg 480cccagctacc
gcaccaaccc ctgtggcgtc tccgctgcca ttccgcccta ctactga
53722178PRTArtificial sequencebeta zein AF371264 protein 22Met Lys
Met Val Ile Val Leu Val Val Cys Leu Ala Leu Ser Ala Ala1 5 10 15Ser
Ala Ser Ala Met Gln Met Pro Cys Pro Cys Ala Gly Leu Gln Gly 20 25
30Leu Tyr Gly Ala Gly Ala Gly Leu Thr Thr Met Met Gly Ala Gly Gly
35 40 45Leu Tyr Pro Tyr Ala Glu Tyr Leu Arg Gln Pro Gln Cys Ser Pro
Leu 50 55 60Ala Ala Ala Pro Tyr Tyr Ala Gly Cys Gly Gln Pro Ser Ala
Met Phe65 70 75 80Gln Pro Leu Arg Gln Gln Cys Cys Gln Gln Gln Met
Arg Met Met Asp 85 90 95Val Gln Ser Val Ala Gln Gln Leu Gln Met Met
Met Gln Leu Glu Arg 100 105 110Ala Ala Ala Ala Ser Ser Ser Leu Tyr
Glu Pro Ala Leu Met Gln Gln 115 120 125Gln Gln Gln Leu Leu Ala Ala
Gln Gly Leu Asn Pro Met Ala Met Met 130 135 140Met Ala Gln Asn Met
Pro Ala Met Gly Gly Leu Tyr Gln Tyr Gln
Leu145 150 155 160Pro Ser Tyr Arg Thr Asn Pro Cys Gly Val Ser Ala
Ala Ile Pro Pro 165 170 175Tyr Tyr23453DNAArtificial sequencedelta
zein AF371266 23atggcagcca agatgcttgc attgttcgct ctcctagctc
tttgtgcaag cgccactagt 60gcgacgcata ttccagggca cttgccacca gtcatgccat
tgggtaccat gaacccatgc 120atgcagtact gcatgatgca acaggggctt
gccagcttga tggcgtgtcc gtccctgatg 180ctgcagcaac tgttggcctt
accgcttcag acgatgccag tgatgatgcc acagatgatg 240acgcctaaca
tgatgtcacc attgatgatg ccgagcatga tgtcaccaat ggtcttgccg
300agcatgatgt cgcaaatgat gatgccacaa tgtcactgcg acgccgtctc
gcagattatg 360ctgcaacagc agttaccatt catgttcaac ccaatggcca
tgacgattcc acccatgttc 420ttacagcaac cctttgttgg tgctgcattc tag
45324150PRTArtificial sequencedelta zein AF371266 protein 24Met Ala
Ala Lys Met Leu Ala Leu Phe Ala Leu Leu Ala Leu Cys Ala1 5 10 15Ser
Ala Thr Ser Ala Thr His Ile Pro Gly His Leu Pro Pro Val Met 20 25
30Pro Leu Gly Thr Met Asn Pro Cys Met Gln Tyr Cys Met Met Gln Gln
35 40 45Gly Leu Ala Ser Leu Met Ala Cys Pro Ser Leu Met Leu Gln Gln
Leu 50 55 60Leu Ala Leu Pro Leu Gln Thr Met Pro Val Met Met Pro Gln
Met Met65 70 75 80Thr Pro Asn Met Met Ser Pro Leu Met Met Pro Ser
Met Met Ser Pro 85 90 95Met Val Leu Pro Ser Met Met Ser Gln Met Met
Met Pro Gln Cys His 100 105 110Cys Asp Ala Val Ser Gln Ile Met Leu
Gln Gln Gln Leu Pro Phe Met 115 120 125Phe Asn Pro Met Ala Met Thr
Ile Pro Pro Met Phe Leu Gln Gln Pro 130 135 140Phe Val Gly Ala Ala
Phe145 1502519PRTArtificial sequencegamma zein signal peptide 25Met
Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1 5 10
15Ala Thr Ser2620PRTArtificial sequencealpha gliadin signal peptide
26Met Lys Thr Phe Leu Ile Leu Val Leu Leu Ala Ile Val Ala Thr Thr1
5 10 15Ala Thr Thr Ala 202721PRTArtificial sequencegamma gliadin
signal peptide 27Met Lys Thr Leu Leu Ile Leu Thr Ile Leu Ala Met
Ala Ile Thr Ile1 5 10 15Gly Thr Ala Asn Met 202825PRTArtificial
sequencePR10 signal sequence 28Met Asn Phe Leu Lys Ser Phe Pro Phe
Tyr Ala Phe Leu Cys Phe Gly1 5 10 15Gln Tyr Phe Val Ala Val Thr His
Ala 20 2529720DNAArtificial sequenceECFP DNA 29atggtgagca
agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa
acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
ctggcccacc 180ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc
gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc
gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta
caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca
tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
420aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa
gcagaagaac 480ggcatcaagg ccaacttcaa gatccgccac aacatcgagg
acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc
gacggccccg tgctgctgcc cgacaaccac 600tacctgagca cccagtccgc
cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt
tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
72030239PRTArtificial sequenceECFP protein 30Met Val Ser Lys Gly
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1 5 10 15Val Glu Leu Asp
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu
Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65 70 75
80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
Glu 100 105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
His Lys Leu Glu Tyr 130 135 140Asn Tyr Ile Ser His Asn Val Tyr Ile
Thr Ala Asp Lys Gln Lys Asn145 150 155 160Gly Ile Lys Ala Asn Phe
Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175Val Gln Leu Ala
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val
Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200
205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
Lys225 230 235311854DNAArtificial sequenceGUS 1381 DNA 31atggtagatc
tgactagttt acgtcctgta gaaaccccaa cccgtgaaat caaaaaactc 60gacggcctgt
gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg
120gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag gcagttttaa
cgatcagttc 180gccgatgcag atattcgtaa ttatgcgggc aacgtctggt
atcagcgcga agtctttata 240ccgaaaggtt gggcaggcca gcgtatcgtg
ctgcgtttcg atgcggtcac tcattacggc 300aaagtgtggg tcaataatca
ggaagtgatg gagcatcagg gcggctatac gccatttgaa 360gccgatgtca
cgccgtatgt tattgccggg aaaagtgtac gtatcaccgt ttgtgtgaac
420aacgaactga actggcagac tatcccgccg ggaatggtga ttaccgacga
aaacggcaag 480aaaaagcagt cttacttcca tgatttcttt aactatgccg
gaatccatcg cagcgtaatg 540ctctacacca cgccgaacac ctgggtggac
gatatcaccg tggtgacgca tgtcgcgcaa 600gactgtaacc acgcgtctgt
tgactggcag gtggtggcca atggtgatgt cagcgttgaa 660ctgcgtgatg
cggatcaaca ggtggttgca actggacaag gcactagcgg gactttgcaa
720gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc tctatgaact
gtgcgtcaca 780gccaaaagcc agacagagtg tgatatctac ccgcttcgcg
tcggcatccg gtcagtggca 840gtgaagggcc aacagttcct gattaaccac
aaaccgttct actttactgg ctttggtcgt 900catgaagatg cggacttacg
tggcaaagga ttcgataacg tgctgatggt gcacgaccac 960gcattaatgg
actggattgg ggccaactcc taccgtacct cgcattaccc ttacgctgaa
1020gagatgctcg actgggcaga tgaacatggc atcgtggtga ttgatgaaac
tgctgctgtc 1080ggctttcagc tgtctttagg cattggtttc gaagcgggca
acaagccgaa agaactgtac 1140agcgaagagg cagtcaacgg ggaaactcag
caagcgcact tacaggcgat taaagagctg 1200atagcgcgtg acaaaaacca
cccaagcgtg gtgatgtgga gtattgccaa cgaaccggat 1260acccgtccgc
aaggtgcacg ggaatatttc gcgccactgg cggaagcaac gcgtaaactc
1320gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca
caccgatacc 1380atcagcgatc tctttgatgt gctgtgcctg aaccgttatt
acggatggta tgtccaaagc 1440ggcgatttgg aaacggcaga gaaggtactg
gaaaaagaac ttctggcctg gcaggagaaa 1500ctgcatcagc cgattatcat
caccgaatac ggcgtggata cgttagccgg gctgcactca 1560atgtacaccg
acatgtggag tgaagagtat cagtgtgcat ggctggatat gtatcaccgc
1620gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc
cgattttgcg 1680acctcgcaag gcatattgcg cgttggcggt aacaagaaag
ggatcttcac tcgcgaccgc 1740aaaccgaagt cggcggcttt tctgctgcaa
aaacgctgga ctggcatgaa cttcggtgaa 1800aaaccgcagc agggaggcaa
acaagctagc caccaccacc accaccacgt gtga 185432617PRTArtificial
sequenceGUS 1381 protein 32Met Val Asp Leu Thr Ser Leu Arg Pro Val
Glu Thr Pro Thr Arg Glu1 5 10 15Ile Lys Lys Leu Asp Gly Leu Trp Ala
Phe Ser Leu Asp Arg Glu Asn 20 25 30Cys Gly Ile Asp Gln Arg Trp Trp
Glu Ser Ala Leu Gln Glu Ser Arg 35 40 45Ala Ile Ala Val Pro Gly Ser
Phe Asn Asp Gln Phe Ala Asp Ala Asp 50 55 60Ile Arg Asn Tyr Ala Gly
Asn Val Trp Tyr Gln Arg Glu Val Phe Ile65 70 75 80Pro Lys Gly Trp
Ala Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val 85 90 95Thr His Tyr
Gly Lys Val Trp Val Asn Asn Gln Glu Val Met Glu His 100 105 110Gln
Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile 115 120
125Ala Gly Lys Ser Val Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn
130 135 140Trp Gln Thr Ile Pro Pro Gly Met Val Ile Thr Asp Glu Asn
Gly Lys145 150 155 160Lys Lys Gln Ser Tyr Phe His Asp Phe Phe Asn
Tyr Ala Gly Ile His 165 170 175Arg Ser Val Met Leu Tyr Thr Thr Pro
Asn Thr Trp Val Asp Asp Ile 180 185 190Thr Val Val Thr His Val Ala
Gln Asp Cys Asn His Ala Ser Val Asp 195 200 205Trp Gln Val Val Ala
Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala 210 215 220Asp Gln Gln
Val Val Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln225 230 235
240Val Val Asn Pro His Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu
245 250 255Leu Cys Val Thr Ala Lys Ser Gln Thr Glu Cys Asp Ile Tyr
Pro Leu 260 265 270Arg Val Gly Ile Arg Ser Val Ala Val Lys Gly Gln
Gln Phe Leu Ile 275 280 285Asn His Lys Pro Phe Tyr Phe Thr Gly Phe
Gly Arg His Glu Asp Ala 290 295 300Asp Leu Arg Gly Lys Gly Phe Asp
Asn Val Leu Met Val His Asp His305 310 315 320Ala Leu Met Asp Trp
Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr 325 330 335Pro Tyr Ala
Glu Glu Met Leu Asp Trp Ala Asp Glu His Gly Ile Val 340 345 350Val
Ile Asp Glu Thr Ala Ala Val Gly Phe Gln Leu Ser Leu Gly Ile 355 360
365Gly Phe Glu Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala
370 375 380Val Asn Gly Glu Thr Gln Gln Ala His Leu Gln Ala Ile Lys
Glu Leu385 390 395 400Ile Ala Arg Asp Lys Asn His Pro Ser Val Val
Met Trp Ser Ile Ala 405 410 415Asn Glu Pro Asp Thr Arg Pro Gln Gly
Ala Arg Glu Tyr Phe Ala Pro 420 425 430Leu Ala Glu Ala Thr Arg Lys
Leu Asp Pro Thr Arg Pro Ile Thr Cys 435 440 445Val Asn Val Met Phe
Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu 450 455 460Phe Asp Val
Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser465 470 475
480Gly Asp Leu Glu Thr Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala
485 490 495Trp Gln Glu Lys Leu His Gln Pro Ile Ile Ile Thr Glu Tyr
Gly Val 500 505 510Asp Thr Leu Ala Gly Leu His Ser Met Tyr Thr Asp
Met Trp Ser Glu 515 520 525Glu Tyr Gln Cys Ala Trp Leu Asp Met Tyr
His Arg Val Phe Asp Arg 530 535 540Val Ser Ala Val Val Gly Glu Gln
Val Trp Asn Phe Ala Asp Phe Ala545 550 555 560Thr Ser Gln Gly Ile
Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe 565 570 575Thr Arg Asp
Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg 580 585 590Trp
Thr Gly Met Asn Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln 595 600
605Ala Ser His His His His His His Val 610 615332041DNAArtificial
sequenceGUS 1391Z DNA 33atggtagatc tgagggtaaa tttctagttt ttctccttca
ttttcttggt taggaccctt 60ttctcttttt atttttttga gctttgatct ttctttaaac
tgatctattt tttaattgat 120tggttatggt gtaaatatta catagcttta
actgataatc tgattacttt atttcgtgtg 180tctatgatga tgatgatagt
tacagaaccg acgactcgtc cgtcctgtag aacgtgaaat 240caaaaaactc
gacggcctgt gggcattcag tctggatcgc gaaaactgtg gaattgatca
300gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag
gcagttttaa 360cgatcagttc gccgatgcag atattcgtaa ttatgcgggc
aacgtctggt atcagcgcga 420agtctttata ccgaaaggtt gggcaggcca
gcgtatcgtg ctgcgtttcg atgcggtcac 480tcattacggc aaagtgtggg
tcaataatca ggaagtgatg gagcatcagg gcggctatac 540gccatttgaa
gccgatgtca cgccgtatgt tattgccggg aaaagtgtac gtatcaccgt
600ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga
ttaccgacga 660aaacggcaag aaaaagcagt cttacttcca tgatttcttt
aactatgccg gaatccatcg 720cagcgtaatg ctctacacca cgccgaacac
ctgggtggac gatatcaccg tggtgacgca 780tgtcgcgcaa gactgtaacc
acgcgtctgt tgactggcag gtggtggcca atggtgatgt 840cagcgttgaa
ctgcgtgatg cggatcaaca ggtggttgca actggacaag gcactagcgg
900gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc
tctatgaact 960gtgcgtcaca gccaaaagcc agacagagtg tgatatctac
ccgcttcgcg tcggcatccg 1020gtcagtggca gtgaagggcg aacagttcct
gattaaccac aaaccgttct actttactgg 1080ctttggtcgt catgaagatg
cggacttacg tggcaaagga ttcgataacg tgctgatggt 1140gcacgaccac
gcattaatgg actggattgg ggccaactcc taccgtacct cgcattaccc
1200ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga
ttgatgaaac 1260tgctgctgtc ggctttaacc tctctttagg cattggtttc
gaagcgggca acaagccgaa 1320agaactgtac agcgaagagg cagtcaacgg
ggaaactcag caagcgcact tacaggcgat 1380taaagagctg atagcgcgtg
acaaaaacca cccaagcgtg gtgatgtgga gtattgccaa 1440cgaaccggat
acccgtccgc aagtgcacgg gaatatttcg ccactggcgg aagcaacgcg
1500taaactcgac ccgacgcgtc cgatcacctg cgtcaatgta atgttctgcg
acgctcacac 1560cgataccatc agcgatctct ttgatgtgct gtgcctgaac
cgttattacg gatggtatgt 1620ccaaagcggc gatttggaaa cggcagagaa
ggtactggaa aaagaacttc tggcctggca 1680ggagaaactg catcagccga
ttatcatcac cgaatacggc gtggatacgt tagccgggct 1740gcactcaatg
tacaccgaca tgtggagtga agagtatcag tgtgcatggc tggatatgta
1800tcaccgcgtc tttgatcgcg tcagcgccgt cgtcggtgaa caggtatgga
atttcgccga 1860ttttgcgacc tcgcaaggca tattgcgcgt tggcggtaac
aagaaaggga tcttcactcg 1920cgaccgcaaa ccgaagtcgg cggcttttct
gctgcaaaaa cgctggactg gcatgaactt 1980cggtgaaaaa ccgcagcagg
gaggcaaaca agctagccac caccaccacc accacgtgtg 2040a
204134617PRTArtificial sequenceGUS 1391Z protein 34Met Val Asp Leu
Arg Val Asn Arg Arg Leu Val Arg Pro Val Glu Arg1 5 10 15Glu Ile Lys
Lys Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu 20 25 30Asn Cys
Gly Ile Asp Gln Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser 35 40 45Arg
Ala Ile Ala Val Pro Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala 50 55
60Asp Ile Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu Val Phe65
70 75 80Ile Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe Asp
Ala 85 90 95Val Thr His Tyr Gly Lys Val Trp Val Asn Asn Gln Glu Val
Met Glu 100 105 110His Gln Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val
Thr Pro Tyr Val 115 120 125Ile Ala Gly Lys Ser Val Arg Ile Thr Val
Cys Val Asn Asn Glu Leu 130 135 140Asn Trp Gln Thr Ile Pro Pro Gly
Met Val Ile Thr Asp Glu Asn Gly145 150 155 160Lys Lys Lys Gln Ser
Tyr Phe His Asp Phe Phe Asn Tyr Ala Gly Ile 165 170 175His Arg Ser
Val Met Leu Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp 180 185 190Ile
Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala Ser Val 195 200
205Asp Trp Gln Val Val Ala Asn Gly Asp Val Ser Val Glu Leu Arg Asp
210 215 220Ala Asp Gln Gln Val Val Ala Thr Gly Gln Gly Thr Ser Gly
Thr Leu225 230 235 240Gln Val Val Asn Pro His Leu Trp Gln Pro Gly
Glu Gly Tyr Leu Tyr 245 250 255Glu Leu Cys Val Thr Ala Lys Ser Gln
Thr Glu Cys Asp Ile Tyr Pro 260 265 270Leu Arg Val Gly Ile Arg Ser
Val Ala Val Lys Gly Glu Gln Phe Leu 275 280 285Ile Asn His Lys Pro
Phe Tyr Phe Thr Gly Phe Gly Arg His Glu Asp 290 295 300Ala Asp Leu
Arg Gly Lys Gly Phe Asp Asn Val Leu Met Val His Asp305 310 315
320His Ala Leu Met Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser His
325 330 335Tyr Pro Tyr Ala Glu Glu Met Leu Asp Trp Ala Asp Glu His
Gly Ile 340 345 350Val Val Ile Asp Glu Thr Ala Ala Val Gly Phe Asn
Leu Ser Leu Gly 355 360 365Ile Gly Phe Glu Ala Gly Asn Lys Pro Lys
Glu Leu Tyr Ser Glu Glu 370 375 380Ala Val Asn Gly Glu Thr Gln Gln
Ala His Leu Gln Ala Ile Lys Glu385 390 395 400Leu Ile Ala Arg Asp
Lys Asn His Pro Ser Val Val Met Trp Ser Ile 405 410 415Ala Asn Glu
Pro Asp Thr Arg Pro Gln Val His Gly Asn Ile Ser Pro 420 425
430Leu Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys
435 440 445Val Asn Val Met Phe Cys Asp Ala His Thr Asp Thr Ile Ser
Asp Leu 450 455 460Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly Trp
Tyr Val Gln Ser465 470 475 480Gly Asp Leu Glu Thr Ala Glu Lys Val
Leu Glu Lys Glu Leu Leu Ala 485 490 495Trp Gln Glu Lys Leu His Gln
Pro Ile Ile Ile Thr Glu Tyr Gly Val 500 505 510Asp Thr Leu Ala Gly
Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu 515 520 525Glu Tyr Gln
Cys Ala Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg 530 535 540Val
Ser Ala Val Val Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala545 550
555 560Thr Ser Gln Gly Ile Leu Arg Val Gly Gly Asn Lys Lys Gly Ile
Phe 565 570 575Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu
Gln Lys Arg 580 585 590Trp Thr Gly Met Asn Phe Gly Glu Lys Pro Gln
Gln Gly Gly Lys Gln 595 600 605Ala Ser His His His His His His Val
610 6153534PRTArtificial sequenceSalmon calcitonin BAC57417 35Lys
Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu1 5 10
15Leu His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr
20 25 30Pro Gly36102DNAArtificial sequenceSalmon calcitonin
BAC57417 DNA 36aagtgctcca acctctctac ctgcgttctt ggtaagctct
ctcaggagct tcacaagctc 60cagacttacc ctagaaccaa cactggttcc ggtacccctg
gt 1023753PRTArtificial sequencehEGF based on AAF85790 37Asn Ser
Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp
Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25
30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys
35 40 45Trp Trp Glu Leu Arg 5038162DNAArtificial sequencehEGF based
on AAF85790 DNA 38aactctgatt cagaatgccc actcagtcac gacggatatt
gtcttcacga tggggtatgc 60atgtacatcg aggccttgga caagtacgca tgtaattgtg
tagtgggata cattggtgaa 120cgctgtcagt atcgagactt gaaatggtgg
gagcttaggt ga 16239191PRTArtificial sequencehGH based on P01241
39Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg1
5 10 15Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe
Glu 20 25 30Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln
Asn Pro 35 40 45Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro
Ser Asn Arg 50 55 60Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu
Arg Ile Ser Leu65 70 75 80Leu Leu Ile Gln Ser Trp Leu Glu Pro Val
Gln Phe Leu Arg Ser Val 85 90 95Phe Ala Asn Ser Leu Val Tyr Gly Ala
Ser Asp Ser Asn Val Tyr Asp 100 105 110Leu Leu Lys Asp Leu Glu Glu
Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125Glu Asp Gly Ser Pro
Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140Lys Phe Asp
Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr145 150 155
160Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe
165 170 175Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly
Phe 180 185 19040576DNAArtificial sequencehGH based on P01241
40ttcccaacca ttcccttatc caggcttttt gacaacgcta tgctccgcgc ccatcgtctg
60caccagctgg cctttgacac ctaccaggag tttgaagaag cctatatccc aaaggaacag
120aagtattcat tcctgcagaa cccccagacc tccctctgtt tctcagagtc
tattccgaca 180ccctccaaca gggaggaaac acaacagaaa tccaacctag
agctgctccg catctccctg 240ctgctcatcc agtcgtggct ggagcccgtg
cagttcctca ggagtgtctt cgccaacagc 300ctggtgtacg gcgcctctga
cagcaacgtc tatgacctcc taaaggacct agaggaaggc 360atccaaacgc
tgatggggag gctggaagat ggcagccccc ggactgggca gatcttcaag
420cagacctaca gcaagttcga cacaaactca cacaacgatg acgcactact
caagaactac 480gggctgctct actgcttcag gaaggacatg gacaaggtcg
agacattcct gcgcatcgtg 540cagtgccgct ctgtggaggg cagctgtggc ttctga
5764153PRTArtificial sequencehEGF protein 41Asn Ser Asp Ser Glu Cys
Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met
Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly
Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu
Leu Arg 5042162DNAArtificial sequencehEGF DNA 42aactctgatt
cagaatgccc actcagtcac gacggatatt gtcttcacga tggggtatgc 60atgtacatcg
aggccttgga caagtacgca tgtaattgtg tagtgggata cattggtgaa
120cgctgtcagt atcgagactt gaaatggtgg gagcttaggt ga
1624333DNAArtificial sequence22aZ5'DNA 43gaggatccgc atggctacca
agatattagc cct 334448DNAArtificial sequence22aZ3' DNA 44cattcatgat
tccgccacct ccaccaaaga tggcacctcc aacgatgg 484542DNAArtificial
sequenceRice13ProI-5' 45gagtcgacgg atccatgaag atcattttcg tctttgctct
cc 424651DNAArtificial sequenceRice13ProI-3' 46catccatggt
tccgccacct ccacccaaga caccgccaag ggtggtaatg g 51477PRTArtificial
sequencewheat prolamin storage protein 47Pro Gln Gln Pro Phe Pro
Gln1 5488PRTArtificial sequencewheat prolamin storage protein 48Pro
Gln Gln Gln Pro Pro Phe Ser1 5495PRTArtificial sequencewheat
prolamin storage protein 49Pro Gln Gln Pro Gln1 55034DNAArtificial
sequencePrimer GFP5' 50aattcatgag cagtaaagga gaagaacttt tcac
345135DNAArtificial sequencePrimer GFP3' 51attggatcct cattatttgt
atagttcatc catgc 355234DNAArtificial sequencePrimer RTB5
52aattcatgag cagtaaagga gaagaacttt tcac 345326DNAArtificial
sequencePrimer RTB3 53ttaccattat tttgataccc gggaag
265439DNAArtificial sequencePrimer SPfor 54cagtcgacac catgagggtg
ttgctcgttg ccctcgctc 395530DNAArtificial sequencePimer RX3ECFP3'
55ggtggatccc tagaatccat ggtctggcac 305644DNAArtificial
sequencePrimer RX3G5ECFP3' 56ggtggatccc tagagccacc gccacctcca
tccatggtct ggca 44571580DNAArtificial sequenceHindII/XbaI DNA
fragment 57aagcttcgaa ttctgcagtc gacaacatgg ctaccaagat attagccctc
cttgcgcttc 60ttgccctttt tgtgagcgca acaaatgcgt tcattattcc acaatgctca
cttgctccta 120gtgccattat accacagttc ctcccaccag ttacttcaat
gggcttcgaa cacctagctg 180tgcaagccta caggctacaa caagcgcttg
cggcaagcgt cttacaacaa ccaattaacc 240aattgcaaca acaatccttg
gcacatctaa ccatacaaac catcgcaacg caacagcaac 300aacagttcct
accagcactg agccaactag atgtggtgaa ccctgtcgcc tacttgcaac
360agcagctgct tgcatccaac ccacttgctc tggcaaacgt agctgcatac
caacaacaac 420aacaattgca gcagtttctg ccagcgctca gtcaactagc
catggtgaac cctgccgcct 480acctacaaca gcaacaactg ctttcatcta
gccctctcgc tgtgggtaat gcacctacat 540acctgcaaca acaattgctg
caacagattg taccagctct gactcagcta gctgtggcaa 600accctgctgc
ctacttgcaa cagctgcttc cattcaacca actgactgtg tcgaactctg
660ctgcgtacct acaacagcga caacagttac ttaatccact agaagtgcca
aacccattgg 720tcgctgcctt cctacagcag caacaattgc taccatacag
ccagttctct ttgatgaacc 780ctgccttgtc gtggcagcaa cccatcgttg
gaggtgccat ctttggtgga ggtggcggaa 840tcatggtgag caagggcgag
gagctgttca ccggggtggt gcccatcctg gtcgagctgg 900acggcgacgt
aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
960acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
ccctggccca 1020ccctcgtgac caccctgacc tggggcgtgc agtgcttcag
ccgctacccc gaccacatga 1080agcagcacga cttcttcaag tccgccatgc
ccgaaggcta cgtccaggag cgcaccatct 1140tcttcaagga cgacggcaac
tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 1200tggtgaaccg
catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
1260acaagctgga gtacaactac atcagccaca acgtctatat caccgccgac
aagcagaaga 1320acggcatcaa ggccaacttc aagatccgcc acaacatcga
ggacggcagc gtgcagctcg 1380ccgaccacta ccagcagaac acccccatcg
gcgacggccc cgtgctgctg cccgacaacc 1440actacctgag cacccagtcc
gccctgagca aagaccccaa cgagaagcgc gatcacatgg 1500tcctgctgga
gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
1560aaagcggccg cgactctaga 15805829DNAArtificial sequenceECFP NcoI
oligonucleotide 58gtaccatggt gagcaagggc gaggagctg
295948DNAArtificial sequenceECFPN1 BamNotSac oligonucleotide
59gcagagctcg cggccgcgga tccttacttg tacagctcgt ccatgccg
486024DNAArtificial sequencemCherry RcaI 5' template 60atcatgatgg
tgagcaaggg cgag 246127DNAArtificial sequenceRX3STOP3' fragment
61tcggatcctt ctagaatcat caggtct 276230DNAArtificial sequenceprimer
5'DNAb 62agccatggcg cgagtccgga gctatctctg 306323DNAArtificial
sequenceprimer 3' DNAb 63gttgtgtaca atgatgtcat tcg
236443DNAArtificial sequenceDNAbhGH 64gaatgacatc attgtacaca
acttcccaac cattccctta tcc 436540DNAArtificial sequence3'hGH primer
65atggtaccac gcgtcttatc agaagccaca gctgccctcc 406636DNAArtificial
sequenceprimer IM-for 66atcattgtac acgccttccc aaccattccc ttatcc
366735DNAArtificial sequenceprimer IM-rev 67tcaggatcct tatcagaagc
cacagctgcc ctcca 356845DNAArtificial sequenceCasp forward fragment
68gactcatgat cgatgaggtg gacatggaga acactgaaaa ctcag
456949DNAArtificial sequenceCasp3 reverse fragment 69ctgggtacca
tgtctagatc attagtgata aaaatagagt tcttttgtg 497043DNAArtificial
sequenceCasp2 for fragment 70gactcatgat cgatgagcac gacggtcctc
tctgccttca ggt 437143DNAArtificial sequenceCasp2 reverse fragment
71ctgggtacca tgtctagata atcatgtggg agggtgtcct ggg
43721148DNAArtificial sequenceDNA fragment 72gctagcgttt aaacgggccc
tctagactcg acaccatgag ggtgttgctc gttgccctcg 60ctctcctggc tctcgctgcg
agcgccacct ccacgcatac aagcggcggc tgcggctgcc 120agccaccgcc
gccggttcat ctaccgccgc cggtgcatct gccacctccg gttcacctgc
180cacctccggt gcatctccca ccgccggtcc acctgccgcc gccggtccac
ctgccaccgc 240cggtccatgt gccgccgccg gttcatctgc cgccgccacc
atgccactac cctactcaac 300cgccccggcc tcagcctcat ccccagccac
acccatgccc gtgccaacag ccgcatccaa 360gcccgtgcca aaggcgcgcc
ggtggaggcg gaggtaccat gattgagggt aggattgttg 420gtggaagtga
ttcccgtgaa ggtgcttggc cttgggttgt ggctctttat ttcgatgatc
480agcaagtttg tggagcctcc cttgtttcta gagattggct tgtgtctgct
gcacattgcg 540tgtatggaag aaatatggaa ccaagtaagt ggaaggcagt
tcttggattg catatggctt 600caaatcttac aagtccacag attgaaactc
gtctcatcga tcaaattgtt atcaacccac 660actataacaa gaggagaaaa
aacaatgata ttgctatgat gcatcttgag atgaaagtga 720actacacaga
ttacattcag ccaatttgtc ttccagagga aaaccaagtt ttcccacctg
780gaaggatttg ttctattgcc ggttggggag cacttatcta tcaaggatca
actgcagatg 840ttcttcaaga agcagatgtt ccacttttgt caaatgagaa
atgccaacag caaatgcctg 900agtataacat tactgagaat atggtgtgtg
ctggatacga ggcaggaggt gtggattctt 960gtcagggaga ttctggaggt
cctcttatgt gccaggagaa taacagatgg cttttagccg 1020gagttacttc
tttcggatac caatgcgcat tgccaaatag acctggtgtg tatgctagag
1080ttccaaggtt tacagagtgg attcaatcat ttctacattg ataaggatcc
gagctcggta 1140ccaagctt 11487318DNAArtificial sequencebGH rev
primer 73cctcgactgt gccttcta 187437DNAArtificial sequencebGH rev2
primer 74cctctagact cgacccatgg tgagcaaggg cgaggag 37
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