U.S. patent application number 14/721478 was filed with the patent office on 2015-11-26 for production of biologically active proteins.
The applicant listed for this patent is ERA BIOTECH, S.A.. Invention is credited to Miriam BASTIDA VIRGILI, Roser Pallisse BERGWERF, Peter Bernard HEIFETZ, Blanca LLOMPART ROYO, Maria Immaculada LLOP TOUS, Maria Dolores LUDEVID M GICA, Pablo MARZ BAL LUNA, Kevin James O'CONNER, Margarita TORRENT QUETGLAS.
Application Number | 20150335720 14/721478 |
Document ID | / |
Family ID | 38134862 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150335720 |
Kind Code |
A1 |
HEIFETZ; Peter Bernard ; et
al. |
November 26, 2015 |
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; Maria 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; Maria Immaculada; (St. Feliu de Llobregat,
ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ERA BIOTECH, S.A. |
Cerdanyola del Valles-Barcelona |
|
ES |
|
|
Family ID: |
38134862 |
Appl. No.: |
14/721478 |
Filed: |
May 26, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13166579 |
Jun 22, 2011 |
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14721478 |
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11709527 |
Feb 22, 2007 |
8163880 |
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13166579 |
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60776391 |
Feb 23, 2006 |
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Current U.S.
Class: |
424/192.1 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 9/6475 20130101; C12N 15/8257 20130101; A61P 37/04 20180101;
A61K 39/00 20130101; C07K 14/425 20130101; A61K 9/0019 20130101;
C07K 14/61 20130101; C12N 9/6424 20130101; C12P 21/02 20130101;
C12N 2799/026 20130101; C12Y 304/21009 20130101; A61K 9/0053
20130101; C12N 15/62 20130101; C07K 14/485 20130101; C07K 14/43504
20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/425 20060101 C07K014/425; C07K 14/435 20060101
C07K014/435; A61K 9/00 20060101 A61K009/00 |
Claims
1.-35. (canceled)
36. A method for inducing an immune response in a host animal
against an immunogenic polypeptide which comprises administering a
pharmaceutical composition comprising recombinant protein body-like
assemblies (RPBLAs), wherein the RPBLAs comprise a recombinant
fusion protein, and wherein said recombinant fusion protein
comprises two sequences linked together in which one sequence is a
protein body-inducing sequence (PBIS) and the other is an
immunogenic polypeptide.
37. The method according to claim 36, wherein said fusion protein
further includes a linker sequence between the protein
body-inducing sequence and the sequence of the immunogenic
polypeptide.
37. The method according to claim 36, wherein the PBIS comprises a
prolamin sequence.
38. The method according to claim 37, wherein the prolamin sequence
is gamma-zein, alpha-zein, gamma-gliadin, or rice prolamin.
39. The method according to claim 38, wherein the prolamin sequence
is the gamma-zein RX3 sequence.
40. The method according to claim 36, wherein said RPBLAs improve
antigen delivery to antigen-presenting cells.
41. The method according to claim 36, wherein said RPBLAs improve
antigen processing and presentation to antigen presenting
cells.
42. The method according to claim 36, wherein the pharmaceutical
composition is administered orally.
43. The method according to claim 36, wherein the pharmaceutical
composition is administered parenterally.
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 decade. Introduction of heterologous genes having a desired
nucleotide sequence leads to expression of a polypeptide or protein
having the corresponding desired amino acid residue sequence or
primary structure. In many instances, however, the protein or
polypeptide expressed has had the amino acid residue sequence of
the naturally-produced material, but has lacked the biological
activity of that material.
[0004] Biological activity, given the proper primary structure of
the expressed product, can be a function of the product having the
proper folding and internal hydrogen, Van der Waals, ionic and
disulfide bonding, and also having proper post-translational
modification, as for instance glycosylation. For example, disulfide
bond formation occurs spontaneously in the lumen of the endoplasmic
reticulum (ER), but not in the cytosol of prokaryotes, which makes
bacterial cells such as E. coli cells 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 were PDI-like proteins are functional
(Fernandez, et al., 2001. Mol. Microbiol. April 40(2):332-346),
however the oxi-redox system is not very efficient.
[0005] A particular case in point relates to erythropoietin (EPO),
a protein that stimulates red blood cell production. Recombinant
EPO is disclosed in U.S. Pat. No. 4,703,008 to Lin. The patent
discloses activities for EPO protein expressed from E. coli, S.
cerevisiae, and mammalian Chinese hamster ovary (CHO) and African
green monkey kidney (COS-1) cells. Although anti-EPO antisera
immunoreacted with EPO expressed by each cell type, only the
proteins expressed from mammalian cells exhibited substantial in
vivo biological activity as EPO, and similar concentrations by
antibody assay, in vitro and in vivo assays. The
mammalian-expressed protein is that used to treat humans.
[0006] It is believed that those differences in biological activity
were a function of glycosylation in that E. coli, a prokaryote,
cannot glycosylate its expressed proteins. Yeast cells are
eukaryotes, but their glycosylation pattern for secreted proteins
is different from a mammal's. On the other hand, the CHO and COS-1
cells used to provide protein of substantial biological activity
were mammalian and the protein expressed therefrom was useful.
Published studies of glycosylated and unglycosylated EPO indicate
that glycosylation plays a critical role in stabilizing
erythropoietin to 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 of the molecule.
[0007] Eukaryotic cells are therefore greatly preferred for
recombinant production of therapeutic, industrial and other useful
proteins of eukaryotic origin. Different eukaryotic cells and
organisms have been shown to be able to produce active
protein-based therapeutics. Unfortunately, the high costs
frequently derived from low recombinant protein production levels
and/or from protein isolation and purification procedures, can
invalidate their industrial application. Active research is done to
improve both production levels and purification procedures by
different approaches.
[0008] 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. Inclusion bodies
are amorphous protein deposits found in bacteria. Structural
characterization studies showed that the insoluble nature of the
inclusion bodies may be due to the hydrophobic intermolecular
interactions of non-native folded proteins (Seshadri et al., 1999,
Methods Enzymol. 309:559-576). The general strategy used to recover
active proteins from inclusion bodies requires the solubilization
of the protein to disrupt the random aggregates followed by one or
more chemical refolding steps. This is an important problem to be
solved because the renaturing efficiency of denaturated proteins
can be limited, mostly if the protein contains disulfide-bonds
(Clarc, Ed., April 2001 Curr. Opin. Biotechnol. 12(2):202-207).
[0009] More particularly, strong denaturants such as high
concentration of chaotropic agents (i.e. urea and guanidinium
hydrochloride) are used to solubilize unfolded proteins that
accumulate in aggregates. The denaturants are thereafter dialyzed
away in an attempt to refold the protein in a natural conformation.
Biological activity of such refolded proteins is usually much less
than that of the native-formed protein.
[0010] 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. Protein bodies (PBs) share
some of the characteristics of the inclusion bodies from bacteria.
They are dense, and contain a high quantity of aggregated 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]. Moreover, the presence of
a large quantity of disulfide-bonds in some of the PBIS, 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 to produce a biologically
active, native-folded protein, particularly a protein that contains
cysteine residues.
[0011] 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.
[0012] A new technology based on the fusion of a plant seed storage
protein domain with the 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 stably accumulate in protein
bodies (Galili et al., 1993, Trends Cell Biol 3:437-442).
[0013] The storage proteins are inserted into the lumen of the
endoplasmic reticulum (ER) via a signal peptide and are assembled
either in the endoplasmic reticulum developing specific organelles
called ER-derived protein bodies (ER-PBs) or in protein storage
vacuoles (PSV) (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 described to assemble
in PB-like organelles in non-plant host systems as Xenopus
oocytes.
[0014] Expression of cereal prolamins (the most abundant cereal
storage proteins) has been described in Xenopus oocytes after
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).
[0015] Zeins, the complex group of maize prolamins, have also been
produced in yeast with various objectives. 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 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 the
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 not a good model to study zein interactions because zeins, by
themselves, were poorly accumulated in transformed yeast. The yeast
cells were also used as a model to study the mechanisms that
control the transport and protein body deposition of the wheat
storage proteins called gliadins (Rosenberg et al., 1993, Plant
Physiol 102:61-69).
[0016] 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 adjuvanting immunomodulatory
components associated with attenuated or killed vaccines often
results in weaker immunogenicity for such vaccines.
[0017] Immunologic adjutants are agents that enhance specific
immune responses to vaccines. 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.
[0018] Phagocytosis involves the entry of large particles, such us
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 fulfil 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).
[0019] 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.
[0020] 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).
[0021] 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
specialised APC, with 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 specialized for clearing
necrotic and apoptotic material. Macrophages can act not only as
APC, but can also perform either pro- or anti-inflammatory roles,
dependent on the means by which they are activated.
[0022] Considering that APC plays a central role in the induction
and regulation of the adaptive immunity (humoral and cellular), the
recognition and phagocytosis of the 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).
[0023] 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).
[0024] 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
[0025] 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 (often
collectively referred to herein as a polypeptide or target) of
interest 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.
[0026] 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).
[0027] 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. 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 that causes the host cell to form protein body-like
assemblies or RPBLAs.
[0028] 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.
[0029] 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.
[0030] 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).
[0031] 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.
[0032] In a further embodiment, the method of the invention further
comprises the isolation and purification of the biologically active
fusion protein.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] In another embodiment, the biologically active polypeptide
contains at least two N-linked glycosylation sequences.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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 is
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.
[0041] 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.
[0042] The present invention has several benefits and
advantages.
[0043] One benefit is that use of the invention enables relatively
simple and rapid expression of a desired recombinant biologically
active protein in an eukaryotic cell of choice.
[0044] 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 the expression in
RPBLAs.
[0045] Another benefit of the invention is that the fusion
protein-containing RPBLAs can be used for delivery of vaccines,
including oral delivery vaccine.
[0046] 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.
[0047] Another advantage of the present invention is that RPBLAs
can be used as insulators, membrane bond 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.
[0048] Still further benefits and advantages will be apparent to
the skilled worker from the discussion that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] In the drawings forming a portion of this disclosure,
[0050] FIG. 1A 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
(22KDa) fused to ECFP. On the bottom, the pcDNA3.1(-) (Invitrogen)
based vectors are represented along with several constructs
discussed hereinafter.
[0051] FIG. 1B 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.CMW"=human cytomegalovirus
promoter; "P.sub.PH"=Polyhedrin promoter; "P.sub.SV40"=SV40 early
promoter; "CaMV35S x2"=Double cauliflower mosaic virus promoter;
"P.sub.cbh1"=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; "cbh1 ter"=major cellulase
polyadenylation signal; "Kana/Neo"=kanamycin/neomycin resistance
gene; "Amp R"=Ampicilin resistance gene; "Gentamicine"=Gentamicin
resistance gene "SP.sub.cbh1"=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 from New England Biolabs; "mInt"=mutated
version of Ssp DNAb intein (Asp154.fwdarw.Ala substitution).
[0052] FIG. 2A shows immunoblots from subcellular fractionation
studies of CHO cells transfected with pRX3-ECFP, pRX3-G-ECFP and
pECFP-N1 as a control. 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.
[0053] FIG. 2B shows shows immunoblots from subcellular
fractionation studies of CHO cells transfected with 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. In FIG. 2B the immunoblot from subcellular
fractionation studies of tobacco plants agroinfiltrated with
pB-RX3-RTB are also shown. 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.
[0054] FIG. 2C 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.
[0055] FIG. 3A is a confocal microscopy photograph showing the
localization of the fusion protein RX3-ECFP in RPBLAs within
transfected CHO cells. Some of the RPBLA structures containing the
active (fluorescent) fusion proteins are indicated by arrows.
[0056] FIG. 3B is a confocal microscopy photograph showing the
localization of the fusion protein RX3-Gx5-ECFPin RPBLAs within
transfected CHO cells. Some of the RPBLA structures containing the
active (fluorescent) fusion proteins are indicated by arrows.
[0057] FIG. 3C shows the localization of the ECFP in the cytosol
and the nucleus (panel C) in CHO cells transfected by pECFP-N1,
shown as a control. "N"=nucleus.
[0058] FIG. 3D is a confocal microscopy photograph showing the
localization of the fusion protein 22aZ-ECFPin RPBLAs within
transfected CHO cells. Some of the RPBLA structures containing the
active (fluorescent) fusion proteins are indicated by arrows.
[0059] FIG. 3E is a confocal microscopy photograph showing the
localization of the fusion protein RX3-GFPin RPBLAs within
transfected CHO cells. Some of the RPBLA structures containing the
active (fluorescent) fusion proteins are indicated by arrows.
[0060] FIG. 3F is a confocal microscopy photograph showing the
localization of the fusion protein RX3-DsRED in RPBLAs within
transfected CHO cells. Some of the RPBLA structures containing the
active (fluorescent) fusion proteins are indicated by arrows.
[0061] FIG. 4A is a confocal microscopy photograph showing the
localization of fluorescent RX3 fusion proteins in 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.
Some of the RPBLA structures containing the active (fluorescent)
fusion proteins are indicated by arrows.
[0062] FIG. 4B is a confocal microscopy photograph showing the
localization of fluorescent RX3 fusion proteins in different hosts.
On the right, the merging of the RX3-GFP fluorescence and the
contrast phase shows the high percentage of transiently transfected
cells. Some of the RPBLA structures containing the active
(fluorescent) fusion proteins are indicated by arrows.
[0063] FIG. 4C is a confocal microscopy photograph showing the
localization of fluorescent RX3 fusion proteins in different hosts.
The projection of optical sections of SF9 insect cells infected
with pF-RX3-DsRED is shown in FIG. 4C. Some of the RPBLA structures
containing the active (fluorescent) fusion proteins are indicated
by arrows.
[0064] FIG. 4D is a confocal microscopy photograph showing the
localization of fluorescent RX3 fusion proteins in different hosts.
One micrometer optical sections of fat tissue from insect larvae
infected with pF-RX3-DsRED are shown in FIG. 4D. Some of the RPBLA
structures containing the active (fluorescent) fusion proteins are
indicated by arrows.
[0065] FIG. 5A is a photograph showing the localization of RX3
fusion proteins inside RPBLAs in CHO cells, four days after their
transfection. Optical microscopy was used to show CHO cells
expressing RX3-hGH immunolocalized by using anti-RX3 serum. The
endoplasmic reticulum (ER) and the RPBLAs are indicated.
[0066] FIG. 5B is a photograph showing the localization of RX3
fusion proteins inside RPBLAs in CHO cells, four days after their
transfection. Optical microscopy was used to show CHO cells
expressing RX3-hGH immunolocalized by using anti-hGH serum. The
endoplasmic reticulum (ER) and the RPBLAs are indicated.
[0067] FIG. 5C shows RX3 protein immunolocalization with RX3
antiserum. The endoplasmic reticulum (ER) and the RPBLAs are
indicated.
[0068] FIG. 5D shows the use of anti-hGH serum to immunolocalize
the RX3-I-hGH fusion protein. The endoplasmic reticulum (ER) and
the RPBLAs are indicated.
[0069] FIG. 5E shows the incubation of CHO cells expressing RX3-GUS
fusion protein with RX3 antiserum. The endoplasmic reticulum (ER)
and the RPBLAs are indicated.
[0070] FIG. 5F shows that smaller RPBLAs were observed in CHO cells
expressing RX3-EK, incubated with anti-RX3 serum. The endoplasmic
reticulum (ER) and the RPBLAs are indicated.
[0071] FIG. 6A shows western blots that illustrate the induction of
Ssp DNAb intein self-cleavage after RX3-I-hGH fusion protein
solubilization from a RPBLAs preparation by low speed
centrifugation. FIG. 6A illustrates 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. The full length fusion proteins are
indicated with white arrowheads and the products of the Ssp DNAb
intein self-cleavage (RX3-I) are indicated with black arrowheads.
"S"=Soluble fraction; "U"=insoluble fraction.
[0072] FIG. 6B shows western blots that illustrate the induction of
Ssp DNAb intein self-cleavage after RX3-I-hGH fusion protein
solubilization from a RPBLAs preparation by low speed
centrifugation. FIG. 6B illustrates 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-hGH serum. The full length fusion proteins are
indicated with white arrowheads and the products of the Ssp DNAb
intein self-cleavage (hGH) are indicated with black arrowheads.
"S"=Soluble fraction; "U"=insoluble fraction.
[0073] FIG. 6C illustrates the comparison of RX3-I-hGH fusion
protein self-cleavage efficiency after 0.1% SDS (S1) and biphasic
(S2) solubilization. Equivalent volumes of the samples were loaded
per lane, except T0 that was overloaded 4-folds. 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.
[0074] FIG. 7A shows the uptake and processing of RX3-DsRED RPBLAs
from insect larvae by macrophages. In FIG. 7A, 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 shown the
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 observation of
the nucleus (N) in this optical section indicates that the RPBLAs
have been taken up and are now intracellular.
[0075] FIG. 7B shows the uptake and processing of RX3-DsRED RPBLAs
from insect larvae by macrophages. In FIG. 7B B shows the 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, the 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.
[0076] FIG. 8A shows the uptake of RX3-DsRED RPBLAs from insect
larvae by dendritic cells. The photographs in FIG. 8A correspond to
dendritic cells incubated with RPBLAs over time (2, 5 and 10
hours). In the upper of each panel the phase contrast shows 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.
[0077] FIG. 8B shows the uptake of RX3-DsRED RPBLAs from insect
larvae by dendritic cells. The photographs in FIG. 8B correspond to
dendritic cells incubated with membrane-less RPBLAs over time (2, 5
and 10 hours). In the upper of each panel the phase contrast shows
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.
DETAILED DESCRIPTION OF THE INVENTION
[0078] 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 endomenbrane 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.
[0079] 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.
[0080] 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.
[0081] These biological activities can be readily determined and
quantified using standard techniques for determining 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.
[0082] 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.
[0083] Most protein bodies have 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.
[0084] The recombinant protein body-like assemblies have a
predetermined density that can differ among different fusion
proteins, but is known 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 assemble as
multimers 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.
[0085] The polypeptide portion of the fusion protein is believed to
obtain its biological activity from folding within the ER and in
some instances from glycosylation in the ER. Interestingly, most
plants, animals such as mammals and single celled eukaryotes such
as fungi, N-glycosylate proteins in the same pattern based upon the
tripeptide glycosylation sequence Asn-X-Ser or Asn-X-Thr, where "X"
is any amino acid residue but proline. Thus, a
Glc.sub.3Man.sub.9(G1cNAc).sub.2 N-linked polypeptide is formed
initially, and is trimmed back after formation to a
Man.sub.7-9(G1cNAc).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 such as host-specificterminal
glycosylation can occur in the Golgi for proteins not maintained in
RPBLAs as are the fusion proteins contemplated here
[0086] In this method, recombinant protein body-like assemblies
(RPBLAs) are provided that frequently comprise a membrane-enclosed
fusion protein ordered assembly, 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 as are
will known in biochemistry for solubilizing lipids.
[0087] 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.
[0088] 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 and
solubilized as a fusion protein exhibited significant activity and
also as a cleaved polypeptide exhibited activities substantially
similar to that of the native polypeptide.
[0089] In yet other embodiments, for 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 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
[0090] 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.
[0091] 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.
[0092] 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 Dec. 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.
[0093] 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).
[0094] 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 (111 aa).
[0095] 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), 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
(W02004003207A1) 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.
[0096] 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.
[0097] Inasmuch as protein bodies 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).
[0098] 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 no 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 protein body formation.
[0099] 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.)
[0100] 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. group 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.)
[0101] Illustrative other 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
[0102] 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:
RX3 query (SEQ ID NO: 2)
Alpha-zein (SEQ ID NO: 3)
[0103] Rice prolamin query (SEQ ID NO: 4)
[0104] 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.
[0105] 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 (SEQ ID NO: 5)
Protein Sequence (SEQ ID NO: 6)
RX3
DNA Sequence (SEQ ID NO: 7)
Protein Sequence (SEQ ID NO: 8)
R3
DNA Sequence (SEQ ID NO: 9)
Protein Sequence (SEQ ID NO: 10)
P4
DNA Sequence (SEQ ID NO: 11)
Protein Sequence (SEQ ID NO: 12)
X10
DNA Sequence (SEQ ID NO: 13)
Protein Sequence (SEQ ID NO: 14)
[0106] 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 (SEQ ID NO: 15)
DNA Sequence (SEQ ID NO: 16)
[0107] 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) (SEQ ID NO: 17) DNA
Sequence (full length) (SEQ ID NO: 18) Gamma-Gliadin
precursor--AAA34272--Scheets et al., 1988 Plant Sci.
57:141-150.
Protein Sequence (SEQ ID NO: 19)
DNA Sequence (M36999) (SEQ ID NO:20)
[0108] Beta zein--AF371264--Woo et al., (2001) Plant Cell 13 (10),
2297-2317.
DNA (SEQ ID NO: 21)
Protein (SEQ ID NO: 22)
[0109] Delta zein 10 kD--AF371266--Woo et al., (2001) Plant Cell
(10), 2297-2317. and Kirihara et al., (1988) Gene. November 30;
71(2):359-70.
DNA (SEQ ID NO:23)
Protein (SEQ ID NO:24)
Signal Peptides
Gamma-Zein (SEQ ID NO:25)
Alpha-Gliadin (SEQ ID NO:26)
Gamma-Gliadin (SEQ ID NO:27)
PR10 (SEQ ID NO:28)
Proteins of Interest
[0110] Examples of polypeptides or proteins of interest (targets)
include any protein having therapeutic, nutraceutical,
agricultural, biocontrol, 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 as 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. August 15;
10(16):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.
[0111] Assays for the bioactivity of such expressed polypeptides
are well known in the art and are available in one or more
publications. For example, the ECFP (enhanced cyan fluorescent
protein) 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.
[0112] The EGF is a growth factor involved in fibroblasts
proliferation. The 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]
[0113] 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,
biocontrol, 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, biocontrol, 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.
[0114] Illustrative DNA and amino acid residue sequences for
illustrative proteins of interest are provided below.
ECFP
DNA (SEQ ID NO:29)
[0115] protein (SEQ ID NO:30)
GUS1381
DNA (SEQ ID NO:31)
[0116] protein (SEQ ID NO:32)
GUS1391Z
DNA (SEQ ID NO:33)
[0117] protein (SEQ ID NO:34) Salmon calcitonin BAC57417 Protein
sequence (SEQ ID NO: 35) DNA sequence (SEQ ID NO: 36)
hEGF--Construction based in the AAF85790 without the signal peptide
Protein sequence (SEQ ID NO: 37) DNA sequence (SEQ ID NO: 38)
hGH--Construction based in the P01241 without the signal peptide
Protein sequence (SEQ ID NO: 39) DNA sequence (SEQ ID NO:40)
[0118] 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.
[0119] 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, 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.
[0120] In a further embodiment, the nucleic acid sequence used for
transformation 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
[0121] In a preferred embodiment, the fusion proteins are prepared
according to a method that comprises transforming an 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.
[0122] 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
recombinant protein body-like assemblies (RPBLAs). Upon expression,
the resulting fusion protein accumulates in the transformed
host-system as high density recombinant protein body-like
assemblies. 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.
[0123] 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.
[0124] 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.
[0125] 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).
[0126] 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.
[0127] 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.
[0128] 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).
[0129] 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.
[0130] 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.
[0131] A recombinant nucleic acid molecule such as a DNA molecule,
comprising a vector 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 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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".
[0136] 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.
[0137] 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.
[0138] 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).
[0139] 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. E. 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.
[0140] 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: A Laboratory 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.
[0141] 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.
[0142] 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.
[0143] 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.
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.
[0144] 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.
[0145] 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.
[0146] A similar root-enhanced promoter, that 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 radical
of seed, a tissue destined to become a root. That promoter is also
useful herein.
[0147] Another useful root-enhanced promoter is the -1616 to -1
promoter of the oil seed rape (Brassica napes 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, GB
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.
[0148] 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-XbalI fragment.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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 (Lel) 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.
[0155] 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.
[0156] Still further higher plant organ-enhanced and organ-specific
promoter are disclosed in Benfey et al. (1988) Science,
244:174-181.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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
[0165] In yet another embodiment of the invention, RPBLAs are used
as the immunogen of an inoculum or vaccine in a human patient or
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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] The amount of RPBLA immunogen utilized in each immunization
is referred to as an immunogenically effective amount and can vary
widely, depending inter alia, upon the RPBLA immunogen, patient
immunized, and the presence of an adjuvant in the vaccine, as
discussed below.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] "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".
[0178] 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).
[0179] 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
[0180] 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 CHO mammal cultured
cells by the Lipofectamine-based transfection method (Invitrogen).
CHO cells transfected with plasmid pECFP-N1 (Clontech) containing
the gene sequence of the cytosolic ECFP, were used as controls.
[0181] 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
(FIG. 2A). The results shown in that figure indicate that RX3-ECFP
and RX3-Gx5-ECFP appeared 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 the 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 fusion proteins just synthesized that had not
assembled.
[0182] Contrarily, 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
[0183] To determine if the fusion proteins RX3-ECFP, RX3-Gx5-ECFP
and 22aZ-ECFP are active inside the RPBLAs, confocal microscopic
analyses were performed in CHO cells transfected with the
constructs that code for them (FIG. 1A). Cyan fluorescent images
were collected at 458 nm excitation with the argon ion laser by
using 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 the endoplasmic
reticulum, indicating that the gamma-zein and the alpha-zein signal
peptide is functional in mammal cells where it mediates the
translocation of the fusion protein into the ER.
[0184] It is important to note that the fusion proteins
surprisingly appear preferentially accumulated large and dense
spherical structures that strongly resembled both natural PBs of
cereal seed and RPBLAs in the heterologous systems visualised by
immunodetection. The intense fluorescence observed in these
structures indicates that the fusion protein remains properly
folded, and therefore active, in spite of being highly 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
cysteines 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.
[0185] 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
[0186] 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 inside the
RPBLAs and bioactive. It is important to note that DsRED shares no
homology to ECFP, which implies a completely different folding
mechanism. Micrographs 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
at 488 nm excitation with the Argon ion laser by using an emission
window set at 495-535 nm. Red fluorescent images were collected
after 543 nm excitation with a HeNe laser and emission window
550-600. Optical sections were 0.5 .mu.m thick.
[0187] 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
RPBLAs.
Example 4
Subcellular Localization of Fluorescent RX3 Fusion Proteins in
Plants and Insects
[0188] 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) showed the
presence of a large amount of fluorescent RPBLAs. Similar results
were obtained when transformed tobacco mesophyll cells were
analyzed.
[0189] Similar results were obtained when Spodoptera SF9 insect
cells or insect larvae (Trichoplusia ni) were infected with
baculovirus coding for the fusion protein RX3-DsRED. As shown in
FIG. 4C the projection of optical sections of infected insect cells
accumulated a large amount of fluorescent RPBLAs about 0.5
micrometers in diameter containing the active RX3-DsRED fusion
protein. Confocal analysis of infected larvae also showed an
impressive amount of fluorescent RPBLAs in whatever tissue
analyzed. In FIG. 4D, fat 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
[0190] Studies were undertaken to determine the activity of human
growth hormone (hGH) produced in RPBLAs. The hGH was chosen because
this molecule contains 2 disulphide bonds that are important for
the proper folding of the protein. 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.
[0191] 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,
permebealized and incubated with anti-RX3 or an anti-hGH antiserum
(FIGS. 5A and 5B, respectively) and the secondary antibody
conjugated to Alexa Fluor 488 (Invitrogen). The presence of large
RPBLAs (1-3 micrometers) containing the RX3-hGH fusion protein was
observed by optical microscopy analysis independently of the
primary antibody used.
[0192] In order to corroborate that the RPBLAs were dense
organelles as was described previously, CHO cells expressing
RX3-hGH were homogenized, and the homogenates loaded on a density
step gradient and centrifuged as described elsewhere. 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).
[0193] This 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 80000.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.
[0194] This same sample was applied to a bioactivity 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 to a hGH
binding protein provided by the kit, this interaction being
dependent on a functional conformation of the hGH. The sample gave
a positive result at 24 ng/ml of bioactive protein. The hGH
proteins evidently 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.
[0195] In determining the activity of hGH fused to RX3, the fusion
protein was 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%, 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 quantify and 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 is 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.
[0196] 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 with EKmax as described by the manufacturer
(Invitrogen). After that, free hGH was isolated from the uncleaved
fusion protein (insoluble) by centrifugation at 16000.times.g at
4.degree. C. for 1 hour. The soluble hGH was recovered from the
supernatant and applied to the quantification and bioactivity
assays from Diagnostic Systems Laboratories. Surprisingly, the
results from both these kits gave the same value of 90 ng/ml 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 determined to be bioactive.
[0197] Summary table for the quantification and bioactivity of the
hGH protein in all the formulations is presented below:
TABLE-US-00002 Quantification Bioactivity Formulation Amount ng/ml
Amount ng/ml Intact RPBLAs 14 25 Membrane removed RPBLAs 35 45
Soluble RX3-hGH 250 70 Cleaved hGH 90 90
[0198] 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, the expression of RX3 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
[0199] 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).
[0200] 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 big round-shaped RPBLA, 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 in the
high density structures.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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
[0206] 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 U200S--IKA 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).
[0207] 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).
Proliferation of MDA-MB231 Cells
TABLE-US-00003 [0208] % 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
[0209] 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 145%, respectively).
Unexpectedly, 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 EGF (Promega).
Example 8
Activity of RX3-GUS Assembled in RPBLAs in CHO Cells
[0210] 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 a challenge, mainly by the presence of 9 cysteine
amino acid residues, and also because it is a large protein (about
70 kDa).
[0211] 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.
[0212] 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 afterwards 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).
[0213] 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 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).
[0214] GUS activity test 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). Fifty .mu.I of the
solubilized RX3-GUS fusion protein (around 0.25 ng of
RX3-GUS/.mu.L) were incubated in the presence of MUG at room
temperature, and the appearance of 4-MU was carried out in a
fluorimeter (excitation wavelength 355 nm; emission wavelength 420
nm). To rule out the possibility of measuring endogenous GUS-like
activity present in the RPBLAs preparation from CHO cells, RPBLAs
from CHO cells transfected with p3.1-RX3 were isolated, and once
the RX3 protein was solubilized, this sample was included in the
activity test as a control. The table below summarizes the results
obtained:
Absorbance at 420 nm
TABLE-US-00004 [0215] 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
[0216] 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-1 of RX3-GUS. No
significant endogenous GUS-like activity was observed when the RX3
preparation was analyzed.
Example 9
Activity of RX3-EK Assembled in RPBLAs in CHO Cells
[0217] Bos taurus enterokinase (enteropeptidase) is a
membrane-bound serine protease of the duodenal mucosa, involved in
the processing of the trypsinogen to trypsin (DDDK1) 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 whole 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.
[0218] 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).
[0219] This construct was used in CHO cells transfection by the
lipofectamine method (Invitrogen). Immunochemistry analysis of
those transfected cells with anti-RX3 antiserum revealed the
presence of a large quantity of small RPBLAs. These organelles were
to be seen all along the cytoplasm of the transfected cells, but
the size usually did not exceed 0.5 micrometers (FIG. 5F).
[0220] 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
F56 fraction (FIG. 2B). The high density of the RX3-EK fusion
protein assemblies suggests that this fusion protein accumulates in
dense RPBLAs. It is important to note that no fusion protein was
detected in the supernatant, meaning that almost all RX3-EK is
assembled in dense structures (RPBLAs). Interestingly, the
molecular weight of the RX3-EK fusion protein was estimated at 58
KDa, about 15 KDa higher than the theoretical molecular weight.
This result suggests that the EK in the RPBLAs is highly
glycosylated, as has been described for the natural protein
(LaVallie et al., 1993 J. Biol. Chem. 268(31):23311-23317).
[0221] 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 SDS was added. Afterwards, the
sample was centrifuged at 5000.times.g at room temperature for 10
minutes, and the supernatant containing the soluble disassembled
RX3-EK fusion protein was dialyzed at 4.degree. C. against a 50 mM
Tris pH 8 solution over night (about 18 hours). Then, the fusion
protein was digested by FXa as described by the manufacturer
(Quiagen), and the EK activity was measured 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
[0222] Studies were undertaken to determine the activity of
caspases produced in RPBLAs.
[0223] Caspases are a family of cysteine proteases that cleave with
high specificity after an aspartic acid of a consensus sequence.
They are the main executioners of the highly regulated process of
apoptosis.
[0224] 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. 115:2665-2671). 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 the
heterotetramer.
[0225] 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.
[0226] The accumulation of both RX3-caspases fusion proteins in the
different fractions was analyzed by immunoblot (FIG. 2B). As it 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.
[0227] In the immunoblot presented in FIG. 2B, only the full length
fusion protein was shown, but bands of different molecular weight
are present in this fraction. These bands being 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.
[0228] 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.
[0229] 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
[0230] 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).
[0231] 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 in
fractions F42 and F56 (FIG. 2B), suggesting that the fusion protein
self-assembles and accumulates 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 can be
glycosylated in RPBLAs.
[0232] 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 was sonicated at 50%
amplitude and 50% cycle for 1 minute, repeated 5 times (Ikasonic
U200S--IKA Labortechnik). Afterwards, the sample was centrifuged at
5000.times.g at room temperature for 10 minutes, and the
supernatant containing the soluble disassembled RX3-RTB was
analyzed by ELISA for binding to the glycoprotein fetuin treated
with sialydase to expose galactose-terminated glycans. The RX3-RTB
binds to it.
Example 12
Plasmid Construction for Plant Transformation
[0233] 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.
hEGF protein (SEQ ID NO:41) hEGF DNA (SEQ ID NO:42)
[0234] 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.
[0235] Synthetic hEGF cDNA was purified from agarose gel (Amersham)
and cloned into 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.
[0236] 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).
[0237] The cDNA encoding the alpha-zein of 22 kD (22aZ) and the
rice prolamin of 13 kD (rP13) 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:
22aZ-5' (SEQ ID NO:43) 22aZ-3' (SEQ ID NO:44)
Rice13Prol-5' (SEQ ID NO:45)
Rice13Prol-3' (SEQ ID NO:46)
[0238] 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.
[0239] 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:
GFP 5' (SEQ ID NO:50)
GFP 3' (SEQ ID NO:51)
[0240] 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)
[0241] 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:
RTB5 (SEQ ID NO:52)
RTB3 (SEQ ID NO:53)
Plant Material
[0242] 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.
[0243] 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
[0244] 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.
[0245] 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
[0246] Vacuum Agroinfiltration
[0247] 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.
[0248] 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/1) and rifampicine (100
mg/1) at 28.degree. C. with shaking (250 rpm) overnight (about 18
hours). Agrobacteria were then inoculated in 30 ml of LB also
supplemented with kanamycin (50 mg/1) and rifampicin (100 mg/1).
After overnight culture at 28.degree. C. (about 18 hours),
agrobacterial 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/1 and sucrose 30 g/1 at pH 5.8.
Bacterial culture was adjusted to a final OD.sub.600 of 0.1 for
agroinfiltration. Then, cell culture was supplemented with
acetosyringone to a final concentration of 0.2 mM and incubated for
90 minutes at 28.degree. C.
[0249] For agroinfiltration, the 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
extraction analyzed by immunoblot using anti-gamma-zein
antibody.
[0250] Agroinfiltration by Syringe
[0251] Agrobacterium tumefaciens strain EHA 105 was grown at
28.degree. C. in L-broth supplemented with 50 .mu.g mL.sup.-1
kanamycin and 50 .mu.g mL.sup.-1 rifampycin to stationary phase.
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 h at room temperature.
Individual Agrobacterium cultures carrying the RX3 constructs and
the HC-Pro silencing supressor 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
[0252] The gene coding for RX3-EGF gamma-zein derived fusion
proteins was introduced in tobacco plants via Agrobacterium
tumefaciens. Transformed plants were analyzed by immunoblot to
determine those plants with higher recombinant protein expression.
The predominant lower bands of immunoblots correspond to the
monomer form of fusion proteins and the higher bands to the dimers.
The fusion proteins usually accumulate as multimers and the amount
of monomers and oligomers detected in the immunoblots depends on
the disulfide bond reduction level.
[0253] 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 show a density higher
than 1.3163 g/cm.sup.3.
[0254] These novel RPBLAs formed in tobacco leaves exhibit
densities in the range of the natural maize protein bodies (Ludevid
et al., 1984 Plant Mol. Biol. 3:227-234; Lending et al., 1989 Plant
Cell 1:1011-1023), or are even more dense.
[0255] 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.
[0256] 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, the fractions in which, 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.
[0257] 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 a
RPBLAs 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 Dry Plant
Tissues
[0258] An important point in molecular farming is the presence of
an easy means to store plant biomass. In this context, drying can
provide a convenient method to lessen storage volume and preserve
the product. Nevertheless, drying frequently promotes the
degradation of the proteins of interest. The use of desiccated
plants to isolate RPBLAs containing recombinant proteins would be
of great interest for industrial purposes.
[0259] Transformed tobacco leaves accumulating RX3-EGF fusion
protein as described above were dried as also discussed above.
After 5 months of dry storage, the stability of recombinant
proteins was analyzed. Protein extracts from equivalent amounts of
wet (fresh) and dry leaf tissue were analyzed by immunoblot. The
RX3-EGF protein was stable in desiccated transformed plants, the
amount recovered in wet and dry plants being similar.
[0260] 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.
[0261] Thus, recombinant proteins can be purified from dried
tissues via isolation of RPBLAs thereby illustrating that
transgenic plant collection and recombinant protein extraction and
purification can be independent in time. In keeping with these
results, gamma-zein fusion proteins were also accumulated in RPBLAs
in rice seeds.
Example 15
Recombinant Protein Recovery by Isolation of RPBLAs from
Transiently Transformed Tobacco Plantlets
[0262] The transient expression systems can be a convenient tool to
test the accumulation behavior of recombinant proteins in 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
[0263] 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 simply centrifuged at low speed
centrifugation (i.e. 1000-2500.times.g for 10 minutes).
[0264] In agreement with the previously described results, the
RX3-EGF protein was recovered in high yields (more than 90%) in the
pellets obtained after centrifugation through 1.1868 g/cm3 sucrose
cushions. In addition, the purification of RX3-EGF protein was very
high in that contaminant tobacco endogenous proteins were barely
detected in the corresponding pellet.
[0265] 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.
[0266] 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.
[0267] 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. It is noted that the amount as
well the pattern of proteins noted in this study is similar to
those obtained after washing the pellet obtained after
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 to scale up.
[0268] Transgenic tobacco plants expressing fusion proteins that
include EGF linked to rice prolamin or alpha-zein rather than RX3,
rP13-EGF and the 22aZ-EGF, were produced by Agrobacterium
tumefasciens transformation. The best expressers where determined
by immunoblot using an antibody against the 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.
[0269] Taking all these results together, it is clear that
prolamins are able to induce high density RPBLAs, even when they
are fused to other proteins. That is an unexpected result, mainly
when almost no homology is observed between them. Moreover, there
are some data suggesting that the prolamins interact to stabilize
the protein bodies, and that some of them are not stable when
expressed in vegetative tissue alone, as for instance alpha-zein
(Coleman et al., 1996 Plant Cell 8:2335-2345)
Example 17
Extraction of Recombinant Proteins from Isolated RPBLAs
[0270] It has been demonstrated that the isolation of dense
recombinant PB-like assemblies is an advantageous method to recover
recombinant proteins with high yield and high purification level
from transgenic organisms. Here it is shown that these recombinant
proteins can be extracted from the storage organelles.
[0271] After an overnight (about 18 hours) incubation of RPBLAs
fractions at 37.degree. C. in a buffer containing a detergent and
reducing agents (SB buffer that contained sodium borate 12.5 mM pH
8, 0.1% SDS and 2% .beta.-mercaptoethanol; treatment), RX3-EGF
protein was solubilized. The extracted fusion protein was recovered
in its soluble form. Afterwards, as a function of their
application, the extracted proteins can be submitted to further
purification or used as partially purified extracts.
Example 18
Plasmid Construction for Animal Cell Transformation
[0272] 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)
opened by the same enzymes to obtain pRX3-ECFP and pRX3-G-ECFP
plasmids, respectively.
Primers:
SPfor (SEQ ID NO:54)
RX3ECFP3' (SEQ ID NO:55)
RX3G5ECFP3' (SEQ ID NO:56)
[0273] The p22aZ-ECFP vector corresponds to the following
HindIII/XbaI DNA fragment in pEGFP-N1 plasmid (Clontech) (SEQ ID
NO:57)
[0274] The GFP was obtained by PCR amplification of the plasmid
pEGFP-N1 (Clontech) with specific oligonucleotides containing
enzyme restriction sites for further cloning:
ECFP NcoI 5' (SEQ ID NO:58)
ECFPN1 BamNotSac 3''(SEQ ID NO:59)
[0275] 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)
[0276] A construct containing the coding sequence of an improved
monomeric DS Red protein (mCherry; Shaner et al., 2004 Nat.
Biotechnol. 22:1567-1572) was a template in a PCR reaction (mCherry
RcaI 5'/ECFPN1 BamNotSac 3').
mCherry RcaI 5' (SEQ ID NO:60)
[0277] 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)
[0278] 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.
RX3STOP3'(SEQ ID NO:61)
[0279] 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.
[0280] 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:
5'DNAb (SEQ ID NO:62)
3'DNAb (SEQ ID NO:63)
DNAb-hGH: (SEQ ID NO:64)
3'hGH (SEQ ID NO:65)
[0281] As negative control of cleavage induction, an uncleavable
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:
IM-for (SEQ ID NO:66)
IM-rev (SEQ ID NO:67)
[0282] Full length cDNAs of human caspase-2 (IRAUp969A0210D6) and
caspase-3 (IRATp970B0521D6) were acquired from RZPD GmbH (Berlin),
from an original reference based at the Nacional Lawrence Livermore
Library.
[0283] By PCR, the caspase-3 and the caspase-2 specific cleavage
(DEVD and DEHD, respecively) site were added at 5' termini of the
corresponding caspase sequence. It is important to note that
amplified fragment corresponding to caspase-2 did not contain the
pro-domain.
Casp3 forward (SEQ ID NO:68) Casp3 reverse (SEQ ID NO:69)
Casp2 for (SEQ ID NO:70)
[0284] Casp2 reverse (SEQ ID NO:71)
[0285] 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).
[0286] The pUC18RX3hGH (US2006123509 (A1)) vector was digested by
HindIII/EcoRI, and the liberated insert cloned in 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 by BstEII, then filling in
the overhang with klenow and finally digesting by SalI. This
fragment was cloned in pcDNA3.1(-) digested by XhoI/EcoRV to obtain
the p3.1-RX3-GUS clone.
[0287] The p3.1-RX3-EK corresponds to the following NheI/HindIII
DNA fragment in pcDNA3.1(-) (Invitrogen) (SEQ ID NO:72)
Example 19
Plasmid Construction for Insect Infection
[0288] The RX3-DsRED fragment from p3.1-RX3-DsRED was digested by
XbaI/HindIII and cloned in pFastBacl (Invitrogen) digested also by
these two enzymes in order to obtain pF-RX3-DsRED vector.
[0289] The DsRED cDNA was amplified by PCR from pF-RX3-DsRED by
using the following primers:
bGH rev (SEQ ID NO:73)
[0290] bGH rev2 (SEQ ID NO:74) To obtain the pF-DsRED vector, the
PCR-amplified DNA fragment was digested by XbaI/HindIII and cloned
in pFastBacl (Invitrogen) also digested by XbaI/HindIII.
Example 20
Insect Cell and Larvae Infection
[0291] Baculovirus and Larvae
[0292] The baculoviral expression vector system (pFastBac,
Invitrogen), was used as the basis vector for this work. The
recombinant virus was produced and amplified as described by the
manufacturer. Cabbage looper, Trichoplusia ni, eggs were obtained
from Entopath, Inc. (Easton, Pa.). The eggs were hatched according
to the directions provided by the manufacturer; and fourth instar
larvae were used for infection.
[0293] Larvae Infection
[0294] 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.
[0295] SF9 Infection
Spodoptera
[0296] Sf9 cells were obtained from Invitrogen (San Diego, Calif.,
U.S.A.) and cultured as previously described (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 viruses were 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-titre
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 (107 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. Commonly cells were used at 30-36 hours after
infection.
Example 21
RPBLAs Preparation from Mammal Cells and Insect Larvae
[0297] Homogenization
[0298] Mammal Cells
[0299] 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 taken 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.
[0300] Insect Larvae
[0301] 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 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.
[0302] RPBLAs Isolation by Density
[0303] 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
[0304] Cell homogenates were diluted with PBS and centrifuged at
16,000.times.g for 15 minutes. The supernatant was removed and the
pellet dried. It was added 2 ml of ice cold Solubilisation Buffer
(50 mM Tris pH7, 5% Triton X-114, 20 mM TCEP, 20 mM NDSB195 and 100
mM MgCl.sub.2) to the pellet, and afterwards 1 ml of PBS containing
1M Urea, 10% Glycerol and 100 mM MgCl.sub.2.
[0305] This composition was incubated on ice for 15 minutes 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 the cold temperature. The suspension was then
incubated at 37.degree. C. for 15 minutes to form the 2 phases.
Three milliliters of 10% PEG were added to the lower hydrophobic
layer (Triton X-114 rich) and the composition was incubated on ice
for 20 minutes. Then, the solution was incubated at 37.degree. C.
for 15 minutes to form the 2 phases again. The upper phase (4 ml)
was recovered and stored for analysis.
Example 23
Immunolocalization
[0306] Immunocytochemistry using a fluorescent microscope (Vertical
Eclipse Microscope Nikon E600A). Between 2 to 4 days after
transfection, cells were fixed for 30 minutes in 1%
paraformaldehyde solution and after washing with phosphate saline
buffer, incubated for 45 minutes with the antibody against: (i) hGH
(dilution 1/150), (ii) EK (dilution 1/500), (iii) RX3 (dilution
1/700). In order to detect the antigen-antibody reaction, an
incubation for 45 minutes with anti-rabbit conjugated to Alexa
Fluor 488(Invitrogen)
[0307] Confocal analysis were performed in a Confocal laser
scanning microscope (Leica TCS SP, Heidelberg, Germany) fitted with
spectrophotometers for emission band wavelength selection. Green
fluorescent images were collected at 488 nm excitation with the
Argon ion laser by using an emission window set at 495-535 nm. Red
fluorescent images were collected after 543 nm excitation with a
HeNe laser and emission window 550-600. Optical sections were 0.5
to 1 .mu.m thick.
Example 24
Activity Assays
[0308] EGF Activity Assay
[0309] MDA-MB231 cells (breast cancer cells that overexpress EGF
receptor) are seeded in 96-well plates at 5,500 cells/well. Cells
were allowed
to adhere for 8 hours in growth medium with 10% FCS (Fetal calf
serum) and then starved overnight in medium supplemented with 0.1%
of FCS. Afterwards, the media is removed and the EGF (positive
control) from Promega or the corresponding sample (solubilyzed
RX3-EGF) is added at different concentrations. Then, the
radioactive timidine is added to a final concentration of 0.5
.mu.Ci. Proliferation is studied at 48 hours after stimulation at
37.degree. C. Then, the cells are washed twice with cold PBS, and
the cells are kept on ice to stop the cell metabolism. A 10%
trichloroacetic acid (TCA) solution is added, and the cells are
incubated for 20 minutes at 4.degree. C. Once the TCA solution is
removed, the plates are washed twice with Ethanol at 70%, and the
cells are incubated for 20 minutes at 37.degree. C. in 0.5 mL the
lysis solution (2% CO3Na2, 0.1N NaOH and 10% SDS). Plates are mixed
by vortex agitation and the sample is not measured before 12 hours
to avoid undesired chemo-luminiscent phenomena.
[0310] EK Activity Assay
[0311] 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 CaC12, 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 min. The activity was calculated as
change in fluorescence over time.
[0312] GUS Activity Assay
[0313] 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 R A, 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 pH7, 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).
[0314] 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-1*mg-1)=(.lamda.em(T1)-(.lamda.em(T0))/(k*(T1-T0)).
"K"=ratio (Units of fluorescence)/(pmol 4-MU).
[0315] RTB Activity Assay
[0316] (Asialofetuin-Binding ELISA)
[0317] 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, Pittsburgh, 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 h at
RT. The wells were then washed three times with 200 ml mPBS, 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 h at RT.
The wells were washed three times as described above and 100 ml
pNPP (pnitrophenyl phosphate disodium salt) substrate (Pierce,
Rockford, Ill.) was applied. The reaction was stopped after 15
minutes by the addition of 50 .mu.l of 2 N NaOH. Absorbance (A405)
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
[0318] 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.
[0319] 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 1
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).
[0320] It has been reported that antigen presentation by the
antigen presentation cells (APC) such as the macrophages and
dendritic cells is a key process necessary to induce the immune
response (Greenberg et al, Current Op. Immunology (2002),
14:136-145). In this process, the APC phagocytoses the antigen,
which is subsequently cleaved in small peptides in the
phagolysosome. These peptides interact with the MHCII 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).
[0321] 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 (M-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
TABLE-US-00005 Percentage of Fluorescent Macrophages 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
[0322] 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 can
not impair significantly the RPBLAs 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.
[0323] When the amount of fluorescence associated with the
macrophages was analyzed over time of incubation, the result was
even more surprising. In any of the conditions analyzed (ratio
RPBLAs/cells or presence of absence of M-CSF) no saturation effect
on 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 to the macrophages. This result
indicates that, the macrophages are phagocytosing a large quantity
of fluorescent RPBLAs particles.
TABLE-US-00006 Time Dependent Macrophage Fluorescence 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
[0324] To demonstrate that RPBLAs containing the RX3-DsRED fusion
protein were inside the macrophages and not simply adsorbed to
plasma membrane, confocal microscopy analysis were performed. FIG.
7A (left panel) shows some of those 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 RPBLAs particles (FIG. 7A, black
arrowhead) in the same optical section indicated that the RPBLAs
had been taken up inside the cells by phagocytosis.
[0325] Another important factor to be analyzed is the degradation
of the immunogen once it has been phagocytosed by the macrophage.
Antigen degradation is needed to produce the antigenic peptides
that are presented on the MHCII receptor. The analysis of the DsRED
fluorescent pattern of the macrophages over the time showed that
the RPBLAs particles were actively digested.
[0326] Another set of micrographs shows that after 1 hour of
incubation, the RPBLA particles were 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 all
along the cells, 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
[0327] Dendritic cells plays 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 ability both to instigate and regulate immune
reactivity (Lau A H et al Gut 2003 52:307-314). To asses the
capacity of those cells to phagocyte 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
before and (ii) the same RPBLAs through fully washed in 50 mM Tris
pH 8, 1% Triton X-100, in order to remove the ER 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.
TABLE-US-00007 Percentage of Fluorescent Dendritic cells % of
fluorescent dendritic cells Membrane- Time RPBLAs 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
TABLE-US-00008 Time Dependent Dendritic cells Fluorescence
Fluorescence associated ot dendritic cells Membrane- Time RPBLAs
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
[0328] 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 to the macrophages over time was
analyzed.
[0329] 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 those cells is
not saturated, as have been observed with macrophages.
[0330] 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.
[0331] 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).
[0332] It is important to note that even at 10 hours of incubation
with RPBLAs or membrane-less RPBLAs, most of the phagocytosed
particles remain 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
[0333] Macrophages
[0334] Macrophages were obtained from marrow of mice Balb/C. 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 macrophage culture of 99% purity was obtained
after 7 days of incubation at 37.degree. C.
[0335] The differentiated macrophages were cultivated in complete
medium to give 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
done with 100 or 1000 particles: 1 cell. The number of particles
(RPBLAs) was counted by Coulter Epics XL FACS using the 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 check the flowing.
[0336] After different times (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 fixed by PBS with 2% of
paraformaldehyde. The treated macrophages were stored at 4.degree.
C. and the fluorescence was analyzed by FACS (with the same program
used for the counting).
[0337] To verify that RX3-DsRED particles were been phagocitated
inside the cells an experiment of immunocitochemistry was done. 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 with 2% formaldehyde
for 15 minutes. Treated cells were analyzed by confocal
microscopy.
[0338] Dendritic Cells
[0339] The marrow from Balb/C mice was cultivated with complete
medium (DMEM, 10% FCS, 5 ng/m1 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
incubated for 2 days to obtain immature denditric cells. Denditric
cells were incubated with 100:1 particles of RX3-DsRED for 1, 5 and
10 hours. After treatments, cells were fixed with 2%
paraformaldehyde, stored at 4.degree. C. and the fluorescence was
analyzed by FACS.
[0340] 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.
[0341] 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 SequenceSynthetic Construct 1Pro Pro Pro Val His
Leu 1 5 253PRTArtificial SequenceSynthetic Construct 2Pro Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro 1 5 10 15 Val
His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu Pro 20 25
30 Pro Pro Val His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His
35 40 45 Leu Pro Pro Pro Pro 50 365PRTArtificial SequenceSynthetic
Construct 3Gln Gln Gln Gln Gln Phe Leu Pro Ala Leu Ser Gln Leu Asp
Val Val 1 5 10 15 Asn Pro Val Ala Tyr Leu Gln Gln Gln Leu Leu Ala
Ser Asn Pro Leu 20 25 30 Ala Leu Ala Asn Val Ala Ala Tyr Gln Gln
Gln Gln Gln Leu Gln Gln 35 40 45 Phe Leu Pro Ala Leu Ser Gln Leu
Ala Met Val Asn Pro Ala Ala Tyr 50 55 60 Leu 65 470PRTArtificial
SequenceSynthetic Construct 4Gln Gln Val Leu Ser Pro Tyr Asn Glu
Phe Val Arg Gln Gln Tyr Gly 1 5 10 15 Ile Ala Ala Ser Pro Phe Leu
Gln Ser Ala Thr Phe Gln Leu Arg Asn 20 25 30 Asn Gln Val Trp Gln
Gln Leu Ala Leu Val Ala Gln Gln Ser His Cys 35 40 45 Gln Asp Ile
Asn Ile Val Gln Ala Ile Ala Gln Gln Leu Gln Leu Gln 50 55 60 Gln
Phe Gly Asp Leu Tyr 65 70 5672DNAArtificial SequenceSynthetic
Construct 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 6726224PRTArtificial SequenceSynthetic Construct
6Met 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 7339DNAArtificial
SequenceSynthetic Construct 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 SequenceSynthetic Construct 8Met 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 9240DNAArtificial
SequenceSynthetic Construct 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 SequenceSynthetic Construct 10Met 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 11213DNAArtificial SequenceSynthetic Construct
11atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg
60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctgcc gccgccacca
120tgccactacc ctacacaacc gccccggcct cagcctcatc cccagccaca
cccatgcccg 180tgccaacagc cgcatccaag cccgtgccag acc
2131271PRTArtificial SequenceSynthetic Construct 12Met 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 13180DNAArtificial
SequenceSynthetic Construct 13atgagggtgt tgctcgttgc cctcgctctc
ctggctctcg ctgcgagcgc cacctccacg 60catacaagcg gcggctgcgg ctgccaatgc
cactacccta ctcaaccgcc ccggcctcag 120cctcatcccc agccacaccc
atgcccgtgc caacagccgc atccaagccc gtgccagacc 1801460PRTArtificial
SequenceSynthetic Construct 14Met 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 Cys His Tyr 20 25 30 Pro Thr Gln Pro Pro
Arg Pro Gln Pro His Pro Gln Pro His Pro Cys 35 40 45 Pro Cys Gln
Gln Pro His Pro Ser Pro Cys Gln Tyr 50 55 60 15150PRTArtificial
SequenceSynthetic Construct 15Met Lys Ile Ile Phe Val Phe Ala Leu
Leu Ala Ile Ala Ala Cys Ser 1 5 10 15 Ala Ser Ala Gln Phe Asp Val
Leu Gly Gln Ser Tyr Arg Gln Tyr Gln 20 25 30 Leu Gln Ser Pro Val
Leu Leu Gln Gln Gln Val Leu Ser Pro Tyr Asn 35 40 45 Glu Phe Val
Arg Gln Gln Tyr Gly Ile Ala Ala Ser Pro Phe Leu Gln 50 55 60 Ser
Ala Thr Phe Gln Leu Arg Asn Asn Gln Val Trp Gln Gln Leu Ala 65 70
75 80 Leu Val Ala Gln Gln Ser His Cys Gln Asp Ile Asn Ile Val Gln
Ala 85 90 95 Ile Ala Gln Gln Leu Gln Leu Gln Gln Phe Gly Asp Leu
Tyr Phe Asp 100 105 110 Arg Asn Leu Ala Gln Ala Gln Ala Leu Leu Ala
Phe Asn Val Pro Ser 115 120 125 Arg Tyr Gly Ile Tyr Pro Arg Tyr Tyr
Gly Ala Pro Ser Thr Ile Thr 130 135 140 Thr Leu Gly Gly Val Leu 145
150 16450DNAArtificial SequenceSynthetic Construct 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
SequenceSynthetic Construct 17Met Ala Thr Lys Ile Leu Ala Leu Leu
Ala Leu Leu Ala Leu Phe Val 1 5 10 15 Ser Ala Thr Asn Ala Phe Ile
Ile Pro Gln Cys Ser Leu Ala Pro Ser 20 25 30 Ala Ile Ile Pro Gln
Phe Leu Pro Pro Val Thr Ser Met Gly Phe Glu 35 40 45 His Leu Ala
Val Gln Ala Tyr Arg Leu Gln Gln Ala Leu Ala Ala Ser 50 55 60 Val
Leu Gln Gln Pro Ile Asn Gln Leu Gln Gln Gln Ser Leu Ala His 65 70
75 80 Leu Thr Ile Gln Thr Ile Ala Thr Gln Gln Gln Gln Gln Phe Leu
Pro 85 90 95 Ala Leu Ser Gln Leu Asp Val Val Asn Pro Val Ala Tyr
Leu Gln Gln 100 105 110 Gln Leu Leu Ala Ser Asn Pro Leu Ala Leu Ala
Asn Val Ala Ala Tyr 115 120 125 Gln Gln Gln Gln Gln Leu Gln Gln Phe
Leu Pro Ala Leu Ser Gln Leu 130 135 140 18432DNAArtificial
SequenceSynthetic Construct 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 SequenceSynthetic Construct 19Asn Met Gln Val
Asp Pro Ser Gly Gln Val Gln Trp Pro Gln Gln Gln 1 5 10 15 Pro Phe
Pro Gln Pro Gln Gln Pro Phe Cys Gln Gln Pro Gln Arg Thr 20 25 30
Ile Pro Gln Pro His Gln Thr Phe His His Gln Pro Gln Gln Thr Phe 35
40 45 Pro Gln Pro Gln Gln Thr Tyr Pro His Gln Pro Gln Gln Gln Phe
Pro 50 55 60 Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln
Gln Thr Phe 65 70 75 80 Pro Gln Gln Pro Gln Leu Pro Phe Pro Gln Gln
Pro Gln Gln Pro Phe 85 90 95 Pro Gln Pro Gln Gln Pro Gln Gln Pro
Phe Pro Gln Ser Gln Gln Pro 100 105 110 Gln Gln Pro Phe Pro Gln Pro
Gln Gln Gln Phe Pro Gln Pro Gln Gln 115 120 125 Pro Gln Gln Ser Phe
Pro Gln Gln Gln Gln Pro Ala Ile Gln Ser Phe 130 135 140 Leu Gln Gln
Gln Met Asn Pro Cys Lys Asn Phe Leu Leu Gln Gln Cys 145 150 155 160
Asn His Val Ser Leu Val Ser Ser Leu Val Ser Ile Ile Leu Pro Arg 165
170 175 Ser Asp Cys Gln Val Met Gln Gln Gln Cys Cys Gln Gln Leu Ala
Gln 180 185 190 Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Val
Ala His Ser 195 200 205 Ile Ile Met Gln Gln Glu Gln Gln Gln Gly Val
Pro Ile Leu Arg Pro 210 215 220 Leu Phe Gln Leu Ala Gln Gly Leu Gly
Ile Ile Gln Pro Gln Gln Pro 225 230 235 240 Ala Gln Leu Glu Gly Ile
Arg Ser Leu Val Leu Lys Thr Leu Pro Thr 245 250 255 Met Cys Asn Val
Tyr Val Pro Pro Asp Cys Ser Thr Ile Asn Val Pro 260 265 270 Tyr Ala
Asn Ile Asp Ala Gly Ile Gly Gly Gln 275 280 20 2086DNAArtificial
SequenceSynthetic Construct 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 SequenceSynthetic Construct 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 SequenceSynthetic
Construct 22Met Lys Met Val Ile Val Leu Val Val Cys Leu Ala Leu Ser
Ala Ala 1 5
10 15 Ser Ala Ser Ala Met Gln Met Pro Cys Pro Cys Ala Gly Leu Gln
Gly 20 25 30 Leu Tyr Gly Ala Gly Ala Gly Leu Thr Thr Met Met Gly
Ala Gly Gly 35 40 45 Leu Tyr Pro Tyr Ala Glu Tyr Leu Arg Gln Pro
Gln Cys Ser Pro Leu 50 55 60 Ala Ala Ala Pro Tyr Tyr Ala Gly Cys
Gly Gln Pro Ser Ala Met Phe 65 70 75 80 Gln Pro Leu Arg Gln Gln Cys
Cys Gln Gln Gln Met Arg Met Met Asp 85 90 95 Val Gln Ser Val Ala
Gln Gln Leu Gln Met Met Met Gln Leu Glu Arg 100 105 110 Ala Ala Ala
Ala Ser Ser Ser Leu Tyr Glu Pro Ala Leu Met Gln Gln 115 120 125 Gln
Gln Gln Leu Leu Ala Ala Gln Gly Leu Asn Pro Met Ala Met Met 130 135
140 Met Ala Gln Asn Met Pro Ala Met Gly Gly Leu Tyr Gln Tyr Gln Leu
145 150 155 160 Pro Ser Tyr Arg Thr Asn Pro Cys Gly Val Ser Ala Ala
Ile Pro Pro 165 170 175 Tyr Tyr 23453DNAArtificial
SequenceSynthetic Construct 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 SequenceSynthetic Construct
24Met Ala Ala Lys Met Leu Ala Leu Phe Ala Leu Leu Ala Leu Cys Ala 1
5 10 15 Ser Ala Thr Ser Ala Thr His Ile Pro Gly His Leu Pro Pro Val
Met 20 25 30 Pro Leu Gly Thr Met Asn Pro Cys Met Gln Tyr Cys Met
Met Gln Gln 35 40 45 Gly Leu Ala Ser Leu Met Ala Cys Pro Ser Leu
Met Leu Gln Gln Leu 50 55 60 Leu Ala Leu Pro Leu Gln Thr Met Pro
Val Met Met Pro Gln Met Met 65 70 75 80 Thr Pro Asn Met Met Ser Pro
Leu Met Met Pro Ser Met Met Ser Pro 85 90 95 Met Val Leu Pro Ser
Met Met Ser Gln Met Met Met Pro Gln Cys His 100 105 110 Cys Asp Ala
Val Ser Gln Ile Met Leu Gln Gln Gln Leu Pro Phe Met 115 120 125 Phe
Asn Pro Met Ala Met Thr Ile Pro Pro Met Phe Leu Gln Gln Pro 130 135
140 Phe Val Gly Ala Ala Phe 145 150 2519PRTArtificial
SequenceSynthetic Construct 25Met Arg Val Leu Leu Val Ala Leu Ala
Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser 2620PRTArtificial
SequenceSynthetic Construct 26Met Lys Thr Phe Leu Ile Leu Val Leu
Leu Ala Ile Val Ala Thr Thr 1 5 10 15 Ala Thr Thr Ala 20
2721PRTArtificial SequenceSynthetic Construct 27Met Lys Thr Leu Leu
Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile 1 5 10 15 Gly Thr Ala
Asn Met 20 2825PRTArtificial SequenceSynthetic Construct 28Met Asn
Phe Leu Lys Ser Phe Pro Phe Tyr Ala Phe Leu Cys Phe Gly 1 5 10 15
Gln Tyr Phe Val Ala Val Thr His Ala 20 25 29720DNAArtificial
SequenceSynthetic Construct 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
SequenceSynthetic Construct 30Met Val Ser Lys Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val
Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu
Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70
75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile
Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Ile Ser His Asn Val
Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala
Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195
200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
Tyr Lys 225 230 235 311854DNAArtificial SequenceSynthetic Construct
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
SequenceSynthetic Construct 32Met Val Asp Leu Thr Ser Leu Arg Pro
Val Glu Thr Pro Thr Arg Glu 1 5 10 15 Ile Lys Lys Leu Asp Gly Leu
Trp Ala Phe Ser Leu Asp Arg Glu Asn 20 25 30 Cys Gly Ile Asp Gln
Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg 35 40 45 Ala Ile Ala
Val Pro Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp 50 55 60 Ile
Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile 65 70
75 80 Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe Asp Ala
Val 85 90 95 Thr His Tyr Gly Lys Val Trp Val Asn Asn Gln Glu Val
Met Glu His 100 105 110 Gln Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val
Thr Pro Tyr Val Ile 115 120 125 Ala Gly Lys Ser Val Arg Ile Thr Val
Cys Val Asn Asn Glu Leu Asn 130 135 140 Trp Gln Thr Ile Pro Pro Gly
Met Val Ile Thr Asp Glu Asn Gly Lys 145 150 155 160 Lys Lys Gln Ser
Tyr Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His 165 170 175 Arg Ser
Val Met Leu Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile 180 185 190
Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala Ser Val Asp 195
200 205 Trp Gln Val Val Ala Asn Gly Asp Val Ser Val Glu Leu Arg Asp
Ala 210 215 220 Asp Gln Gln Val Val Ala Thr Gly Gln Gly Thr Ser Gly
Thr Leu Gln 225 230 235 240 Val Val Asn Pro His Leu Trp Gln Pro Gly
Glu Gly Tyr Leu Tyr Glu 245 250 255 Leu Cys Val Thr Ala Lys Ser Gln
Thr Glu Cys Asp Ile Tyr Pro Leu 260 265 270 Arg Val Gly Ile Arg Ser
Val Ala Val Lys Gly Gln Gln Phe Leu Ile 275 280 285 Asn His Lys Pro
Phe Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala 290 295 300 Asp Leu
Arg Gly Lys Gly Phe Asp Asn Val Leu Met Val His Asp His 305 310 315
320 Ala Leu Met Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr
325 330 335 Pro Tyr Ala Glu Glu Met Leu Asp Trp Ala Asp Glu His Gly
Ile Val 340 345 350 Val Ile Asp Glu Thr Ala Ala Val Gly Phe Gln Leu
Ser Leu Gly Ile 355 360 365 Gly Phe Glu Ala Gly Asn Lys Pro Lys Glu
Leu Tyr Ser Glu Glu Ala 370 375 380 Val Asn Gly Glu Thr Gln Gln Ala
His Leu Gln Ala Ile Lys Glu Leu 385 390 395 400 Ile Ala Arg Asp Lys
Asn His Pro Ser Val Val Met Trp Ser Ile Ala 405 410 415 Asn Glu Pro
Asp Thr Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro 420 425 430 Leu
Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys 435 440
445 Val Asn Val Met Phe Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu
450 455 460 Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val
Gln Ser 465 470 475 480 Gly Asp Leu Glu Thr Ala Glu Lys Val Leu Glu
Lys Glu Leu Leu Ala 485 490 495 Trp Gln Glu Lys Leu His Gln Pro Ile
Ile Ile Thr Glu Tyr Gly Val 500 505 510 Asp Thr Leu Ala Gly Leu His
Ser Met Tyr Thr Asp Met Trp Ser Glu 515 520 525 Glu Tyr Gln Cys Ala
Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg 530 535 540 Val Ser Ala
Val Val Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala 545 550 555 560
Thr Ser Gln Gly Ile Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe 565
570 575 Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys
Arg 580 585 590 Trp Thr Gly Met Asn Phe Gly Glu Lys Pro Gln Gln Gly
Gly Lys Gln 595 600 605 Ala Ser His His His His His His Val 610 615
332041DNAArtificial SequenceSynthetic Construct 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
SequenceSynthetic Construct 34Met Val Asp Leu Arg Val Asn Arg Arg
Leu Val Arg Pro Val Glu Arg 1 5 10 15 Glu Ile Lys Lys Leu Asp Gly
Leu Trp Ala Phe Ser Leu Asp Arg Glu 20 25 30 Asn Cys Gly Ile Asp
Gln Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser 35 40 45 Arg Ala Ile
Ala Val Pro Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala 50 55 60 Asp
Ile Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu Val Phe 65 70
75 80 Ile Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe Asp
Ala 85 90 95 Val Thr His Tyr Gly Lys Val Trp Val Asn Asn Gln Glu
Val Met Glu 100 105 110 His Gln Gly Gly Tyr Thr Pro Phe Glu Ala Asp
Val Thr Pro Tyr Val 115 120 125 Ile Ala Gly Lys Ser Val Arg Ile Thr
Val Cys Val Asn Asn Glu Leu 130 135 140 Asn Trp Gln Thr Ile Pro Pro
Gly Met Val Ile Thr Asp Glu Asn Gly 145 150 155 160 Lys Lys Lys Gln
Ser Tyr Phe His Asp Phe Phe Asn Tyr Ala Gly Ile 165 170 175 His Arg
Ser Val Met Leu Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp
180 185 190 Ile Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala
Ser Val 195 200 205 Asp Trp Gln Val Val Ala Asn Gly Asp Val Ser Val
Glu Leu Arg Asp 210 215 220 Ala Asp Gln Gln Val Val Ala Thr Gly Gln
Gly Thr Ser Gly Thr Leu 225 230 235 240 Gln Val Val Asn Pro His Leu
Trp Gln Pro Gly Glu Gly Tyr Leu Tyr 245 250 255 Glu Leu Cys Val Thr
Ala Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro 260 265 270 Leu Arg Val
Gly Ile Arg Ser Val Ala Val Lys Gly Glu Gln Phe Leu 275 280 285 Ile
Asn His Lys Pro Phe Tyr Phe Thr Gly Phe Gly Arg His Glu Asp 290 295
300 Ala Asp Leu Arg Gly Lys Gly Phe Asp Asn Val Leu Met Val His Asp
305 310 315 320 His Ala Leu Met Asp Trp Ile Gly Ala Asn Ser Tyr Arg
Thr Ser His 325 330 335 Tyr Pro Tyr Ala Glu Glu Met Leu Asp Trp Ala
Asp Glu His Gly Ile 340 345 350 Val Val Ile Asp Glu Thr Ala Ala Val
Gly Phe Asn Leu Ser Leu Gly 355 360 365 Ile Gly Phe Glu Ala Gly Asn
Lys Pro Lys Glu Leu Tyr Ser Glu Glu 370 375 380 Ala Val Asn Gly Glu
Thr Gln Gln Ala His Leu Gln Ala Ile Lys Glu 385 390 395 400 Leu Ile
Ala Arg Asp Lys Asn His Pro Ser Val Val Met Trp Ser Ile 405 410 415
Ala Asn Glu Pro Asp Thr Arg Pro Gln Val His Gly Asn Ile Ser Pro 420
425 430 Leu Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr
Cys 435 440 445 Val Asn Val Met Phe Cys Asp Ala His Thr Asp Thr Ile
Ser Asp Leu 450 455 460 Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly
Trp Tyr Val Gln Ser 465 470 475 480 Gly Asp Leu Glu Thr Ala Glu Lys
Val Leu Glu Lys Glu Leu Leu Ala 485 490 495 Trp Gln Glu Lys Leu His
Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val 500 505 510 Asp Thr Leu Ala
Gly Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu 515 520 525 Glu Tyr
Gln Cys Ala Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg 530 535 540
Val Ser Ala Val Val Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala 545
550 555 560 Thr Ser Gln Gly Ile Leu Arg Val Gly Gly Asn Lys Lys Gly
Ile Phe 565 570 575 Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu
Leu Gln Lys Arg 580 585 590 Trp Thr Gly Met Asn Phe Gly Glu Lys Pro
Gln Gln Gly Gly Lys Gln 595 600 605 Ala Ser His His His His His His
Val 610 615 3534PRTArtificial SequenceSynthetic Construct 35Lys Cys
Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu 1 5 10 15
Leu His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr 20
25 30 Pro Gly 36102DNAArtificial SequenceSynthetic Construct
36aagtgctcca acctctctac ctgcgttctt ggtaagctct ctcaggagct tcacaagctc
60cagacttacc ctagaaccaa cactggttcc ggtacccctg gt
1023753PRTArtificial SequenceSynthetic Construct 37Asn Ser Asp Ser
Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10 15 Asp Gly
Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30
Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35
40 45 Trp Trp Glu Leu Arg 50 38162DNAArtificial SequenceSynthetic
Construct 38aactctgatt cagaatgccc actcagtcac gacggatatt gtcttcacga
tggggtatgc 60atgtacatcg aggccttgga caagtacgca tgtaattgtg tagtgggata
cattggtgaa 120cgctgtcagt atcgagactt gaaatggtgg gagcttaggt ga
16239191PRTArtificial SequenceSynthetic Construct 39Phe Pro Thr Ile
Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His
Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30
Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35
40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn
Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg
Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln
Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala
Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu
Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro
Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp
Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165
170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
180 185 190 40576DNAArtificial SequenceSynthetic Construct
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 SequenceSynthetic Construct 41Asn Ser Asp Ser
Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10 15 Asp Gly
Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30
Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35
40 45 Trp Trp Glu Leu Arg 50 42162DNAArtificial SequenceSynthetic
Construct 42aactctgatt cagaatgccc actcagtcac gacggatatt gtcttcacga
tggggtatgc 60atgtacatcg aggccttgga caagtacgca tgtaattgtg tagtgggata
cattggtgaa 120cgctgtcagt atcgagactt gaaatggtgg gagcttaggt ga
1624333DNAArtificial SequenceSynthetic Construct 43gaggatccgc
atggctacca agatattagc cct 334448DNAArtificial SequenceSynthetic
Construct 44cattcatgat tccgccacct ccaccaaaga tggcacctcc aacgatgg
484542DNAArtificial SequenceSynthetic Construct 45gagtcgacgg
atccatgaag atcattttcg tctttgctct cc 424651DNAArtificial
SequenceSynthetic Construct 46catccatggt tccgccacct ccacccaaga
caccgccaag ggtggtaatg g 51477PRTArtificial SequenceSynthetic
Construct 47Pro Gln Gln Pro Phe Pro Gln 1 5 488PRTArtificial
SequenceSynthetic Construct 48Pro Gln Gln Gln Pro Pro Phe Ser 1 5
495PRTArtificial SequenceSynthetic Construct 49Pro Gln Gln Pro Gln
1 5 5034DNAArtificial SequenceSynthetic Construct 50aattcatgag
cagtaaagga gaagaacttt tcac 345135DNAArtificial SequenceSynthetic
Construct 51attggatcct cattatttgt atagttcatc catgc
355234DNAArtificial SequenceSynthetic Construct 52aattcatgag
cagtaaagga gaagaacttt tcac 345326DNAArtificial SequenceSynthetic
Construct 53ttaccattat tttgataccc gggaag 265439DNAArtificial
SequenceSynthetic Construct 54cagtcgacac catgagggtg ttgctcgttg
ccctcgctc 395530DNAArtificial SequenceSynthetic Construct
55ggtggatccc tagaatccat ggtctggcac 305644DNAArtificial
SequenceSynthetic Construct 56ggtggatccc tagagccacc gccacctcca
tccatggtct ggca 44571580DNAArtificial SequenceSynthetic Construct
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 SequenceSynthetic Construct
58gtaccatggt gagcaagggc gaggagctg 295948DNAArtificial
SequenceSynthetic Construct 59gcagagctcg cggccgcgga tccttacttg
tacagctcgt ccatgccg 486024DNAArtificial SequenceSynthetic Construct
60atcatgatgg tgagcaaggg cgag 246127DNAArtificial SequenceSynthetic
Construct 61tcggatcctt ctagaatcat caggtct 276230DNAArtificial
SequenceSynthetic Construct 62agccatggcg cgagtccgga gctatctctg
306323DNAArtificial SequenceSynthetic Construct 63gttgtgtaca
atgatgtcat tcg 236443DNAArtificial SequenceSynthetic Construct
64gaatgacatc attgtacaca acttcccaac cattccctta tcc
436540DNAArtificial SequenceSynthetic Construct 65atggtaccac
gcgtcttatc agaagccaca gctgccctcc 406636DNAArtificial
SequenceSynthetic Construct 66atcattgtac acgccttccc aaccattccc
ttatcc 366735DNAArtificial SequenceSynthetic Construct 67tcaggatcct
tatcagaagc cacagctgcc ctcca 356845DNAArtificial SequenceSynthetic
Construct 68gactcatgat cgatgaggtg gacatggaga acactgaaaa ctcag
456949DNAArtificial SequenceSynthetic Construct 69ctgggtacca
tgtctagatc attagtgata aaaatagagt tcttttgtg 497043DNAArtificial
SequenceSynthetic Construct 70gactcatgat cgatgagcac gacggtcctc
tctgccttca ggt 437143DNAArtificial SequenceSynthetic Construct
71ctgggtacca tgtctagata atcatgtggg agggtgtcct ggg
43721148DNAArtificial SequenceSynthetic Construct 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
SequenceSynthetic Construct 73cctcgactgt gccttcta
187437DNAArtificial SequenceSynthetic Construct 74cctctagact
cgacccatgg tgagcaaggg cgaggag 37
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