U.S. patent application number 10/792491 was filed with the patent office on 2004-07-29 for expression of recombinant human acetylcholinesterase in transgenic plants.
Invention is credited to Arntzen, Charles J., Mason, Hugh S., Mor, Tsafrir S., Soreq, Hermona.
Application Number | 20040148657 10/792491 |
Document ID | / |
Family ID | 22701367 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040148657 |
Kind Code |
A1 |
Mor, Tsafrir S. ; et
al. |
July 29, 2004 |
Expression of recombinant human acetylcholinesterase in transgenic
plants
Abstract
Briefly stated, the invention includes a method of making a
transgenic plant that is capable of expressing a physiologically
active human acetylcholinesterase, comprising the steps of
introducing into at least one plant cell a polynucleotide that
encodes a human acetylcholinesterase, and regenerating from the
plant cell a transgenic plant that is capable of expressing a
physiologically active human acetylcholinesterase in at least one
tissue type of the transgenic plant. Another embodiment of the
invention includes a method of making a physiologically active
human acetylcholinesterase, comprising the steps of introducing
into at least one plant cell a polynucleotide that encodes a human
acetylcholinesterase, regenerating from the plant cell a transgenic
plant that is capable of expressing a physiologically active human
acetylcholinesterase in at least one tissue type of the transgenic
plant, and isolating or purifying from the transgenic plant or a
part thereof a physiologically active human
acetylcholinesterase.
Inventors: |
Mor, Tsafrir S.; (Tempe,
AZ) ; Soreq, Hermona; (Jerusalem, IL) ;
Arntzen, Charles J.; (Ithaca, NY) ; Mason, Hugh
S.; (Itahca, NY) |
Correspondence
Address: |
BROWN & MICHAELS, PC
400 M & T BANK BUILDING
118 NORTH TIOGA ST
ITHACA
NY
14850
US
|
Family ID: |
22701367 |
Appl. No.: |
10/792491 |
Filed: |
March 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10792491 |
Mar 3, 2004 |
|
|
|
09810861 |
Mar 16, 2001 |
|
|
|
60190440 |
Mar 17, 2000 |
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Current U.S.
Class: |
800/288 ;
435/196; 435/419; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/18 20130101; C12N
15/8257 20130101; C12N 15/8242 20130101 |
Class at
Publication: |
800/288 ;
435/069.1; 435/196; 435/419; 536/023.2 |
International
Class: |
A01H 001/00; C12N
009/16; C12N 015/82; C12N 005/04; C07H 021/04 |
Claims
What is claimed is:
1. A method of making a physiologically active human
acetylcholinesterase, comprising the steps of: a) introducing into
at least one plant cell a polynucleotide that encodes a human
acetylcholinesterase; b) regenerating from said plant cell a
transgenic plant that is capable of expressing said physiologically
active human acetylcholinesterase in at least one tissue type of
said transgenic plant; and c) isolating or purifying from said
transgenic plant or a part thereof said physiologically active
human acetylcholinesterase.
2. A method of treating a victim of acetylcholinesterase poisoning,
comprising the step of administering a therapeutic amount of a
physiologically active human acetylcholinesterase expressed in
plant tissue.
3. An isolated polynucleotide comprising a nucleic acid molecule
including a sequence selected from the group consisting of: a) SEQ
ID NO:1; b) SEQ ID NO:2; c) SEQ ID NO:3; d) SEQ ID NO:4; and e) SEQ
ID NO:5.
4. A transformed cell comprising the polynucleotide of claim 3.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional patent application of copending
application Ser. No. 09/810,861, filed Mar. 16, 2001, entitled
"EXPRESSION OF RECOMBINANT HUMAN ACETYLCHOLINESTERASE IN TRANSGENIC
PLANTS", which claims an invention that was disclosed in
Provisional Application Serial Number 60/190,440, filed Mar.
17,2000, entitled "EXPRESSION OF RECOMBINANT HUMAN
ACETYLCHOLINESTERASE IN TRANSGENIC TOMATOES," now expired. The
aforementioned applications are hereby incorporated herein by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention pertains to the field of transgenic plants.
More particularly, the invention pertains to the expression of a
recombinant form of human acetylcholinesterase in transgenic
plants.
[0004] 2. Description of the Related Art
[0005] Acetylcholine (ACh) is one of the major signaling molecules
in metazoans, functioning mostly as a neurotransmitter in chemical
synapses between neurons and in neuromuscular junctions. To ensure
a discrete "all-or-none" response across the synapse, the release
of ACh is tightly controlled and the neurotransmitter is
efficiently removed by the hydrolyzing enzyme, acetylcholinesterase
(AChE). In humans, AChE is encoded by a single gene which yields,
through alternative splicing of its pre-mRNA, three polypeptide
isoforms having distinct C-termini. See Soreq et al., Proc. Nat.
Acad. Sci. U.S.A. 87: 9688-9692 (1990); Ben Aziz-Aloya et al.,
Proc. Natl. Acad. Sci. U.S.A. 90: 2471-5 (1993); GenBank Accession
No. M55040; and U.S. Pat. No. 5,595,903. The complete disclosure of
each of the foregoing references is hereby incorporated herein by
reference.
[0006] Various compounds are well known to inhibit the hydrolyzing
activity of ACHE. Exposure to such anti-AChE agents leads to
over-stimulation of cholinergic pathways, causing muscular tetany,
autonomous dysfunction and potentially death. While some naturally
occurring AChE inhibitors are very potent, human exposure to them
is rare. However, man-made anti-AChE compounds, especially
organophosphates (OPs), are widely used as pesticides and pose a
substantial occupational and environmental risk. Even more ominous
is the fear of deliberate use of OPs as chemical warfare agents
against individuals or populations.
[0007] Current medical interventions, in the case of acute exposure
to anticholinesterase agents, include use of the muscarinic
receptor antagonist, atropine, and oximes to reactivate the
OP-modified ACHE. The reversible carbamate, pyridostigmine bromide,
is also used as a prophylactic. However, these conventional
treatments have limited effectiveness and have serious short and
long-term side effects. In fact, the routine treatments, while
successfully decreasing anticholinesterase-induced lethality,
rarely alleviate post-exposure delayed toxicity, which may result
in significant performance deficits, and even permanent brain
damage.
[0008] A different approach in treatment and prevention of
anti-AChE toxicity seeks to mimic one of the physiological lines of
defense against such agents present in mammals.
Butyrylcholinesterase (BuChE) is a serum cholinesterase with a
broad hydrolytic spectrum that provides protection against a
variety of ACHE inhibitors. A similar end may be achieved by a
variant of AChE found on the membranes of erythrocytes. Both
enzymes are believed to serve as circulating scavengers for
anti-AChE agents in protection of the vital synaptic ACHE.
Therefore, administration of cholinesterases could boost their
natural potential to counter-act the toxic effects of
anti-cholinergic agents. The efficacy of this treatment to protect
against a challenge of OPs was tested in a variety of animal models
such as mice, rats, guinea pigs, and primates, and was found to be
comparable to or better than the currently-used drug regimens in
preventing OP-induced mortality without any detrimental
side-effects.
[0009] Enzyme therapy has the additional benefit of the relatively
long half-life time (several days) of the injected enzymes in the
blood stream, making it especially useful for prophylaxis. In the
foregoing experiments, cholinesterases purified from human or
animal blood were used. To be effective, the stoichiometry of
cholinesterase to inhibitor must be close to unity. Hence, large
amounts of pure, properly folded, stable enzymatic preparations
that are free of mammalian pathogens are needed, if enzyme therapy
is to be feasible.
[0010] Genetically engineered plants have recently been recognized
as one of the most cost-effective means for the production of
useful recombinant proteins and pharmaceuticals. Therefore, we
examined the use of transgenic plants as a cost-effective and safe
alternative to the production of human acetylcholinesterase (hAChE)
from blood or cell cultures, herein providing the first
demonstration of the expression in plants of a key protein
component of the nervous system of humans.
SUMMARY OF THE INVENTION
[0011] Briefly stated, the invention includes one or more plant
cells comprising a polynucleotide that encodes a human
acetylcholinesterase.
[0012] An embodiment of the invention includes a method of making a
transgenic plant that is capable of expressing a physiologically
active human acetylcholinesterase, comprising the steps of
introducing into at least one plant cell a polynucleotide that
encodes a human acetylcholinesterase, and regenerating from the
plant cell a transgenic plant that is capable of expressing a
physiologically active human acetylcholinesterase in at least one
tissue type of the transgenic plant.
[0013] Another embodiment of the invention includes a method of
making a physiologically active human acetylcholinesterase,
comprising the steps of introducing into at least one plant cell a
polynucleotide that encodes a human acetylcholinesterase,
regenerating from the plant cell a transgenic plant that is capable
of expressing a physiologically active human acetylcholinesterase
in at least one tissue type of the transgenic plant, and isolating
or purifying from the transgenic plant or a part thereof a
physiologically active human acetylcholinesterase.
[0014] Another embodiment of the invention includes a method of
treating a victim of acetylcholinesterase poisoning, comprising the
step of administering a therapeutic amount of a physiologically
active human acetylcholinesterase expressed in plant tissue.
BRIEF DESCRIPTION OF THE DRAWING
[0015] FIG. 1 shows a graphic map of pTMO36, the pGPTVkan
derivative construct used in the generation of transgenic tomato
plants that constitutively express hAChE-E4.
[0016] FIG. 2 shows a bar graph depicting high activity of hAChE in
transgenic tomato lines.
[0017] FIG. 3 shows substrate inhibition of recombinant hAChE
obtained from transgenic plants.
[0018] FIG. 4 shows an inhibition profile of ACHE obtained from
transgenic plants (diamonds), human erythrocytes (circles) and
transgenic mice (squares).
[0019] FIG. 5A shows a graph of data indicating that a recombinant
hAChE derived from transgenic plants is labile at relatively high
temperatures.
[0020] FIG. 5B shows a graph of data indicating that a
plant-derived hAChE is relatively stable at room temperature.
DETAILED DESCRIPTION OF THE INVENTION
[0021] DNA Constructs and Plant Transformation
[0022] A cDNA encoding human AChE exons II, III and IV was
amplified from the plasmid pAChE-E4 (see Stemfeld et al., J.
Neurosci. 18: 1240-1249 (1990), the complete disclosure of which is
hereby incorporated herein by reference) via the polymerase chain
reaction (PCR), according to standard methods, which are well known
in the art, using the following primers:
1 AChE-Nco-- (5'-GATATCTGCAGCCATGgctAGGCCCCCGC) (SEQ ID NO: 1) AChE
-Kpn-- (5'-CggtaccTATCAGGTaGCGCTGAGCAA- TTTG) (SEQ ID NO: 2)
[0023] The lower case letters in the foregoing primer sequences
represent bases that were introduced to create restriction sites
for cloning the gene into plant expression vectors. The PCR product
was cloned and sequenced using well known methods. An Nco I-Kpn I
fragment from a partial digest of pAChE-E4 was cloned into
pIBT210.1 (see Haq et al., Science 268: 714-716 (1995), the
complete disclosure of which is hereby incorporated herein by
reference) behind a CaMV 35S promoter and the 5' UTR of Tobacco
Etch Virus, and in front of the 3' UTR of the soy bean vspB gene to
form pTM034 (FIG. 1, SEQ ID NO:3), according to standard methods,
which are well known in the art. A Hind III-Eco RI fragment
containing the plant expression cassette was then cloned into the
T.sub.i plasmid derivative pGPTV-Kan to form pTMO36 (SEQ ID NO:4),
using standard methods that are well known in the art. This plasmid
was then transferred to Agrobacterium tumefaciens strain EHA105,
and was used in the subsequent transformation of the Lycopersicum
esculentum cultivar referred to as "Micro-Tom," as described by
Meissner et al. in Plant J. 12: 1465-1472 (1997), the complete
disclosure of which is hereby incorporated herein by reference.
[0024] Genomic PCR, DNA and RNA Blot Analysis
[0025] Screening by genomic PCR was performed on 0.8 .mu.g total
DNA isolated from kanamycin resistant plants, using the AChE-Nco
and AChE-Kpn primers, according to well known methods. For DNA blot
analysis, total DNA was prepared, digested with Nco I, and the
digested DNA (.about.20 .mu.g) was resolved by electrophoresis,
transferred to a nylon hybridization membrane, and hybridized to a
digoxigenin-labeled probe, according to standard methods, which are
well known in the art. The digoxigenin-labeled probe was
synthesized using the following primers:
2 AChE585for (5'-CGAGAGGACTGTGCTGGTGTC) AChE1374rev
(5'-GTCGCCCACCACATCGCTC)
[0026] Hybridization and detection were performed according to well
known methods. Total RNA was isolated and 5 .mu.g samples were
resolved by denaturing formaldehyde gel electrophoresis and
transferred to nylon hybridization membranes, according to well
known methods.
[0027] Acetylcholinesterase Assays and Protein Determination
[0028] Plant samples were homogenized in the presence ice-cold
extraction buffer (100 mM NaCl, 25 mM Tris, 0.1 mM EDTA, 10
.mu.g/ml leupeptin, pH 7.4, 3 ml per 1 g tissue) using ceramic
beads in a bead-beater, and cleared supernatants were collected
followed by centrifugation (14,000 rpm). Scaled-down microtiter
plate Ellman assays were performed, according to standard methods,
which are well known in the art. Cleared extracts (.about.20 .mu.l)
were incubated for 30 minutes at room temperature with 80 .mu.l
assay buffer (0.1 M phosphate buffer, pH 7.4) with or without
2.times.10.sup.-5 M 1,5-bis(allyldimethylammiumphenyl)pen- tan-3one
dibromide (BW284c51), which is a specific inhibitor of mammalian
AChE. At the end of the 30 minute incubation period, 100 .mu.l of 1
mM 5-5'-dithio-bis(2-nitrobenzoate) (Ellman's reagent) and 2 mM
acetylthiocholine in assay buffer was added. Hydrolysis was
monitored by measuring optical density at 405 nm at 5 minute
intervals for 30 minutes, using a microtiter plate
spectrophotometer, plotted against time, and initial rates were
calculated from the slope of the linear portion of the graph. Net
hydrolysis rates were calculated by subtracting the rates measured
in the presence of BW284c51 from those obtained in its absence. To
determine the K.sub.m, the concentration of the acetylthiocholine
substrate in the Ellman's reagent was varied in the range of
0.05-50 mM.
[0029] Inhibition curves were obtained by performing the Ellman
assay with 1 mM acetylthiocholine in the presence of the indicated
concentrations of diethyl p-nitrophenyl phosphate (paraoxon),
neostigmine, phehylmethylsulfonyl fluoride (PMSF) or tetraisopropyl
pyrophosphoramide (Iso-OMPA). To determine K.sub.1 of BW284c51,
assays were performed in the presence of 1, 0.33 and 0.25 mM
acetylthiocholine, and the inhibitor at 10.sup.-4 to 10.sup.-10 M.
Results were then analyzed according to the method of Ordentlich et
al. (see Ordentlich et al., J. Biol. Chem. 268: 17083-17095 (1993),
the complete disclosure of which is hereby incorporated herein by
reference). In these experiments, acetylcholinesterase from human
erythrocytes was used.
[0030] To evaluate the heat stability of the enzyme, plant extracts
were incubated for 30 minutes at the indicated temperatures and
then assayed as described above. Stability of the enzymatic
activity was determined at 4 degrees C. and at 25 degrees C. by
incubating plant extracts at the respective temperatures and
assaying samples at the indicated time points.
[0031] A cDNA encoding exons 2-4 of the human AChE29 gene was
inserted into a plant expression cassette driven by the
constitutive cauliflower mosaic virus 35S promoter. Referring now
to FIG. 1, a graphic map is shown of pTM036, the pGPTVkan
derivative construct used in the generation of transgenic tomato
plants that constitutively express hAChE-E4. Empty arrowheads
denote positions of the PCR primers AChE-Nco and AChE-Kpn used for
amplification of the full length coding region of hAChE-E4. Filled
arrowheads denote the positions of the PCR primers AChE585for and
ACHEE1374rev used for the generation of DIG-labeled probe.
[0032] We used Agrobacterium tumefaciens to construct the tomato
explants, and regenerated 27 kanamycin resistant tomato lines. We
screened the transformants for the insertion of the recombinant
human gene AChE-E4 by PCR. Twelve out of 17 plants tested were
positive for the appropriate gene insertion event. The product of
the AChE-E4 construct was previously demonstrated to be a monomeric
soluble protein, which is fully active in acetylcholine hydrolysis.
Therefore, we screened the putative transgenic plants for the
expression of specific acetylcholinesterase activity in the soluble
protein fraction of leaf extracts of kanamycin-resistant lines.
[0033] Referring now to FIG. 2, kanamycin-resistant lines were
assayed for specific esterase activity (i.e., total minus activity
in the presence of the inhibitor BW284c51) in leaves by the method
of Ellman, using acetylthiocholine as a substrate. Protein samples
from the indicated transgenic plant lines (AChE-53, 54, 62, 68 and
83), untransformed plant (UT) and a commercially available
preparation of ACHE from human erythrocytes (E5) were resolved on a
non-denaturing gel which was then stained for AChE activity.
Plant-derived AChE migrates as a discrete band in non-denaturing
gel electrophoresis. On a per soluble protein basis, high activity,
comparable to a third of the activity present in mammalian brain
and five times more than that present in muscles, was registered in
several of the lines, including AChE-53, AChE-54, AChE-62 and
AChE-68. In these lines, activity was on the order of 100 mU/g leaf
tissue (fresh weight). Acetylcholinesterase present in the
transgenic lines appeared as a discrete band in non-denaturing
polyacrylamide gels stained for cholinesterase activity. This
result demonstrates the apparent uniformity of the protein produced
by the plants. No activity was detected in the untransformed line,
or in line AChE-83. Unexpectedly, in contrast to the sharp bands of
the plant derived recombinant enzyme, the activity of the
commercially available preparation of ACHE from human erythrocytes
appeared as a diffuse smear.
[0034] DNA blot analysis revealed that three of the lines that
express high levels of activity, AChE-54, AChE-62 and AChE-68, each
have one copy of the hAChE-E4 gene inserted in their genomes. Total
DNA was isolated from the indicated lines, digested with Nco I,
resolved by agarose gel electrophoresis, blotted to nylon membrane
and probed with digoxigenin-labeled probe, according to well known
methods. Referring to FIG. 2, AChE-83, a transgenic line that does
not exhibit ACHE activity, has at least two copies of the gene
inserted in its genome. However, in this line, the mRNA encoding
hAChE-E4 failed to accumulate to detectable levels, as demonstrated
by RNA blot analysis, suggesting that transgene silencing in this
line might have occurred. RNA blot analysis of several
kanamycin-resistant tomato lines indicated that mRNA accumulated to
similar levels in all the other lines that were tested. Total RNA
was isolated from the indicated lines, resolved by agarose gel
electrophoresis, blotted to nylon membrane and stained with
methylene blue. The membrane was then probed with AChE specific
DIG-labeled probe.
[0035] Kinetic Properties of the Plant-Produced Recombinant
Enzyme
[0036] We calculated the K.sub.m of the plant-derived enzyme for
four of our expressing lines to be 0.44.+-.0.10 mM (FIG. 3, inset).
This value is similar to that reported for the same molecular form
of the enzyme expressed in injected oocytes of Xenopus laevis and
also to those reported for other forms of the human enzyme.
Hydrolysis was inhibited by the presence of substrate at high
concentration (FIG. 3), as previously reported for native and
recombinant AChE. Enzyme activity was assayed in the presence of
acetylthiocholine at 0.05-50 mM, and hydrolysis in the presence of
the inhibitor BW284c51 was subtracted at each concentration. A
representative high expression line (AChE-54) is shown in FIG. 3.
The inset of FIG. 3 shows Lineweaver-Burk analysis for the
determination of the K.sub.m for four lines: AChE-53 (squares),
AChE-54 (diamonds), AChE-62 (triangles) and AChE-68 (crosses).
[0037] AChE inhibitors of various classes, including the reversible
inhibitors neostigmine (a carbamate), BW284C51 (an AChE-specific
bisquatemary inhibitor), as well as the irreversible inhibitors
paraoxon (an organophosphate, the activated form of the pesticide
parathion) and PMSF (a general serine hydrolase inhibitor) can
inhibit the plant derived recombinant ACHE (rAChE), and the
inhibition profile is very similar to that of a commercially
available preparation of human AChE derived from erythrocytes (FIG.
4). The K.sub.1 calculated for BW284c51 is 16 nM, which is in close
agreement with the values for the recombinant human synaptic enzyme
transiently expressed in mammalian cell cultures (10 nM) and for
the erythrocyte form (5 nM). As expected, the
butyrylcholinesterase-specific organophosphate, Iso-OMPA, had no
effect on either the plant-derived or the erythrocyte-derived
enzyme preparations (up to 100 .mu.M), and only partial inhibition
was registered at 10 mM (FIG. 4). The plant-derived E4 enzyme was
somewhat less susceptible to paraoxon than an equivalent
recombinant enzyme obtained from transgenic mice (FIG. 4).
[0038] The plant-derived hAChE in total soluble protein extracts
retained 50% of its initial activity after incubation at 42 degrees
for at least 30 minutes (FIG. 5A). Crude leaf extracts were
incubated at the indicated temperatures for 30 minutes and then
subjected to Ellman's ACHE assay. Incubation of plant extracts at
room temperature (.about.25 degrees C.) resulted in gradual loss of
ACHE activity, with 20% residual activity remaining after 25 hours
(FIG. 5B). The activity was very stable at 4 degrees C., with only
20% loss after 24 hours (FIG. 5B). Crude leaf extracts were
incubated at 4 degrees C. or at 25 degrees C. for the indicated
time periods and then assayed for AChE activity.
[0039] Types of Cholinesterases that can be Expressed in Plants
[0040] Traditionally, cholinesterases are classified as either
acetylcholinesterase (EC 3.1.1.7, ACHE) or as butyrylcholine
hydrolases (EC 3.1.1.8, BChE, formerly referred to as
pseudo-acetylcholinesterase) on the basis of their substrate
specificity. While BChE can efficiently hydrolyze substrates with a
longer acyl group, the catalytic efficiency of AChE is limited to
acetylcholine and, to a lesser degree, propionylcholine. More
recently inhibitors have been identified that can selectively
inhibit the two types of cholinesterases.
[0041] The genes encoding AChE and BChE from several mammals,
including humans, have been cloned. Cholinesterases from
non-vertebrates and lower vertebrates, even when possessing several
different genes, have mixed characteristics. A further complication
of the molecular picture is presented by the alternative splicing
that the transcript of the AChE gene can undergo leading, in
mammals, to three distinct isoforms. These isoforms share a common
N-terminal catalytic domain, but diverge in their C-termini, which
impact their quaternary structure and membrane association.
[0042] The catalytic distinction between the enzymes is not
restricted to acyl-choline substrates but to other types of esters.
Thus, BCHE can catalyze the hydrolysis of cocaine whereas AChE
cannot. On the other hand, it was recently demonstrated that the
erythrocyte form of ACHE can hydrolyze heroin (3,6-diacylmorphine)
to morphine, while BCHE can hydrolyze heroin only to the
intermediate 6-NAM (6-monoacetylmorphine). Interestingly, the
synaptic isoform of AChE cannot hydrolyze heroin, making heroin
hydrolysis the first reported catalytic distinction between the
different isoforms of AChE.
[0043] The literature on the non-cholinergic functions of
cholinesterases, and especially of AChE, is becoming richer all the
time. These proteins apparently play important roles in the
developing nervous system and its maintenance, especially in
directing the growth of neurons and establishing synaptic
connections. The different isoforms have distinct roles through
their different C-termini. For example, addition of the synaptic
isoform of ACHE to cultured neurons has a marked activation effect
on neurite outgrowth, and a similar effect has been noted in
transgenic frog embryos. In contrast, frog embryos expressing
soluble forms of the enzyme do not exhibit such effects.
[0044] These small nuances make all of these different isoforms
valuable, and we anticipate that plant production of them will be
useful for many different ends, including, but not limited to, the
following: 1) scavengers of anticholinesterase agents including
organophosphates; 2) the hydrolysis of cocaine and heroin in
treatment of cases of overdose intoxication by drug abusers; and 3)
regeneration of damaged neuronal tissue.
[0045] Optimization of the Coding Sequence of hAChE-E4 for
Expression in Plants
[0046] In most cases, the accumulation of foreign proteins in
transgenic proteins is a desirable objective, as it tends to
maximize yield and reduce costs of production. Accumulation of
proteins is a complex function of many factors that effect
synthesis and degradation. By "synthesis" we mean all the
biochemical steps which lead to the formation of a mature protein,
from transcription of a gene, accumulation of mRNA, translation of
messages, localization of products, and many co- and
post-translational modifications. Not all of these steps can easily
be controlled directly (some, for example, are inherent to the
polypeptide in question) and, as yet, not all can be manipulated to
enhance accumulation. However, experience has shown that certain
optimization measures can have a profound effect on the overall
levels of foreign protein accumulation in plants.
[0047] Up to the translation stage, the expression of a gene is
dependent on the nucleotide sequence not only of the control
elements, such as promoter, enhancer elements and 3' sequences, but
also on the coding region as well. Molecular cues are encoded by
the nucleic acid sequence to allow molecular events, such as
termination of transcription, splicing of intervening sequences,
rapid turnover of mRNA, and its translatability. Many of these
features are common to many different types of organisms, while
others are specific for plants, including, for example, intron
splice sites, plant-specific RNA stabilizing sequences, and even
plant specific biases in codon usage. Thus, optimization of gene
sequences entails conforming the coding sequence to those of plant
genes.
[0048] Numerous methods for the optimization of DNA sequences for
increased expression in plants are well known in the art. For
example, see U.S. Pat. Nos. 6,180,774; 6,166,302; 6,121,014;
6,110,668; 6,075,185; 6,051,760; 6,043,415; 6,015,891; 6,013,523;
5,994,526; 5,952,547; 5,880,275; 5,877,306; 5,866,421; 5,859,347;
5,859,336; 5,689,052; 5,633,446; 5,625,136; 5,567,862; 5,567,600;
5,545,817; 5,500,365; and 5,380,831, the disclosures of each of
which are hereby incorporated herein by reference.
[0049] Analysis of the cDNA of hAChE-E4 to Assess its Suitability
for Expression in Plants
[0050] Although we present herein an example wherein the hAChE gene
is expressed in tomato plants, the tomato serves here only as a
model organism. The expression of hAChE in all major crop plants is
intended to be within the scope of the present invention, including
(but not restricted to): dicotyledonous plants, such as, for
example, tomato, potato, tobacco, legumes (i.e., soybean, peanut,
alfalfa), and sweet potato. These plants are typically engineered
by Agrobacterium transformation, various suitable methods for which
are well known in the art. Monocotyledonous plants are also
intended to be within the scope of the present invention, including
(but not restricted to): maize, rice, wheat, and barley. These
plants are typically engineered by biollistic transformation,
various suitable methods for which are well known in the art.
[0051] The hAChE-E4 nucleotide sequence includes a total of 574
codons and has an A+T content of 34.8%. Codon use in hAChE-E4
generally is unfavorable for expression in dicots, but acceptable
for expression in monocots. In summary, 3.6% of the codons are
monocot-unfavorable (including Arg--17.5%, Lys--42.9% and
Ser--15.6%), while 12.7% are dicot-unfavorable (including
Arg--72.5%, Gly--32.8, Pro--17.6% and Thr--32%), when favorability
is defined as making up less than 10% of codon choice for a
particular amino acid. Monocot and Dicot preferences were analyzed
separately, so as to reveal any potential monocot vs. dicot
problems. Tables I and II below summarize total codon use:
3TABLE I Dicot AA DNA Unfavorable Total % Ala GCG 5 53 9.5 Arg CGA
7 40 72.5 CGC 8 CGG 14 Gly GGG 19 58 32.8 Leu CTA 1 67 1.5 Pro CGG
9 51 17.6 Ser TCG 2 32 6.3 Thr ACG 8 25 32 Other Total 73 574
12.7
[0052]
4TABLE II Monocot AA DNA Unfavorable Total % Arg CGA 7 40 17.5 Ile
ATA 0 9 0 Leu CTA 1 67 1.5 TTA 0 Lys AAA 3 7 42.9 Ser AGT 5 32 15.6
Val GTA 5 53 9.4 STOP TAA 0 0 0 Other Total 21 574 3.6
[0053] Based on the data in the foregoing tables, it is evident
that some optimization of the native hAChE-E4 nucleotide sequence
is desirable, particularly if the gene is to be expressed in
dicots. Thus, we present herein an example of a synthetic DNA
sequence encoding human acetylcholinesterase that is optimized for
expression in plants, referred to herein as SEQ ID NO:5.
[0054] Purification of Plant-Produced Cholinesterases
[0055] While for some of the potential applications of
cholinesterases, no purification would be necessary (e.g., in-vivo
bioremediation), and for other applications only partially purified
preparations of the enzymes would be necessary (e.g., certain
industrial uses, oral administration, topical applications in
creams, etc.), for other applications relatively pure enzymes are
preferable, and may be required. This is especially true for
treating individuals by intra-venous or intramuscular injections of
cholinesterases.
[0056] Several published procedures for the purification of
acetylcholinesterase are known in the art. See, for example,
Fischer et al., Biotechnol. Appl. Biochem. 21: 295-311 (1995) and
Heim et al., Biochim. Biophys. Acta 1396: 306-319 (1998), the
complete disclosures of which are hereby incorporated herein by
reference. A large scale purification protocol for
butyrylcholinesterase based on ammonium sulfate fractionation
followed by an batch affinity chromatography should be applicable
also for acetylcholinesterase with minor modifications. See, for
example, Grunwald et al., J. Biochem. Biophys. Methods 34: 123-135
(1997), the complete disclosure of which is hereby incorporated
herein by reference.
[0057] Additional purification schemes, which are well known in the
art, involve engineering a tag to the recombinant enzyme by
creating translational fusions. Commercially available plasmids
directing such fusions exist mainly for bacterial expression, but
can easily be adapted to expression in plants. For example, well
known tags that can be used include histidine tags (whereby
purification is typically conducted by a nickel-based affinity
chromatography); intein-chitin binding tags (whereby purification
is conducted by chitin based affinity chromatography and cleavage
by a reducing agent, such as dithiothreitol or
beta-mercaptoethanol); cellulose binding domains (whereby
purification involves affinity chromatography with cellulose). The
latter is likely the most useful approach, as it can be done
without the addition of any exogenous affinity matrix, since the
cholinesterase-CBD fusion binds to cell walls of the plant extract.
Release is then mediated by either addition of cellobiose or brief
acidification. Cleavage is also possible. For some applications,
the cellulose immobilized enzyme-CBD will be extremely useful as a
catalytic platform for filters, cellulose based cleaning aids
etc.
[0058] Further Examples of Applications of the Invention
[0059] Administration of exogenous cholinesterases is an
efficacious and safe treatment for the prevention of anti-AChE
toxicity. In fact, a single pre-treatment injection of either AChE
or BuChE may be sufficient for full protection without any
post-exposure treatment. However, to ensure maximum protection
against a high dosage (equal to several LD50) of OPs, large amounts
of the enzymes are required to satisfy the 1:1 stoichiometry
required between the enzyme and the inhibitors in the blood. The
enzymes can be purified from human or animal blood, or
alternatively, they can be expressed in a variety of cell cultures.
However, these systems inherently suffer from high costs and risks
of contamination with human pathogens. Recombinant cholinesterases
of various sources have been expressed in Escherichia coli,
however, the enzymes thus produced must be denatured and refolded
to obtain even partial activity. In addition, they are very labile
as compared to the native enzymes. Production by fermentation of
yeast cell cultures is also possible, but costs are high and
scaling up is expensive.
[0060] Therefore, we introduced transgenic plants as a novel
production system for human acetylcholinesterase, a key component
of cholinergic synapses. Some of the transgenic tomato lines
obtained express high levels of AChE activity, with accumulation
levels (on a fresh weight basis) as reported for the yeast-derived
enzyme (FIGS. 1 and 2). This activity represents authentic human
acetylcholinesterase activity, as judged by its enzymatic
properties (FIGS. 3 and 4). The plant-derived enzyme is also very
stable in the crude plant extract (FIGS. 5A and 5B). Expression
levels of the gene product can be increased further by optimizing
the coding sequence of the human gene for expression in plants,
according to methods that are well known in the art, and by
regulating and restricting expression of the gene product to
certain tissues.
[0061] In humans, ACHE is encoded by a single gene which yields,
through alternative splicing of its pre-mRNA, three polypeptide
isoforms with distinct C-termini. We expressed the engineered
AChE-E4 form, encoded by exons 2-4 of the human gene. However, one
of ordinary skill in the art will appreciate that the other
isoforms can be used as well. AChE-E4 consists of the globular
N-terminal domain shared among the three physiological variants of
ACHE. Expressed by itself, the soluble AChE-E4 polypeptide is a
fully competent acetylcholine hydrolase with kinetic properties
which are similar to those of the natural forms. This recombinant
AChE-E4 variant is especially suited for application as a
protective decoy for the neutralization of ACHE inhibitors, because
its kinetic properties are practically identical to those of
synaptic ACHE (unlike BuChE). Because it is soluble, it may be
cleared more slowly from circulation (unlike the membrane bound
ACHE forms, which are cleared 50 times faster than soluble BuChE).
Because it has the same amino acid sequence as the human enzyme,
the plant-derived recombinant hAChE-E4 isoform is expected to be
less immunogenic than the heterologous cholinesterases used in
previous studies. There are three potential glycosylation sites in
human AChE, and glycosylation, which does not affect the enzymatic
properties of the enzyme, is important for both the stability of
ACHE and its pharmacokinetics and its immunogenic properties. As
eukaryotes, plants offer the advantage of all forms of
post-translational modification, including glycosylation, which,
however, differs in details from that in mammals.
[0062] Use of the inexpensively produced enzyme is not limited to
application by injection, as efficacy of other routes of entry into
the body (e.g., orally, inhalation) is expected as well. Lastly,
cholinesterases can be incorporated into cleansing preparations,
protective skin-creams, filtration devices, and biosensors. For
these purposes, the plant-derived enzyme is especially useful, due
to lower costs of partial purification and its higher
stability.
[0063] The extensive use of anticholinesterase pesticides and the
concurrent accidental poisoning, the unfortunate threat of OP
chemical warfare agents by terrorists and rogue governments, as
well as environmental concerns, are the driving force for the
development of effective, inexpensive and safe countermeasures and
bioremediation solutions. Plant-derived recombinant human AChE is
an important step in this direction.
[0064] Accordingly, it is to be understood that the embodiments of
the invention herein described are merely illustrative of the
application of the principles of the invention. Reference herein to
details of the illustrated embodiments is not intended to limit the
scope of the claims, which themselves recite those features
regarded as essential to the invention.
* * * * *