Note: Descriptions are shown in the official language in which they were submitted.
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
A METHOD OF EXTRACTION OF AN ENZYME FROM PLANT OR ANIMAL
TISSUE
FIELD OF THE INVENTION
The present invention relates to techniques for extraction, purification, and
detection
of enzymes from living tissue, and is further related to extraction,
purification, and detection
of heterologous enzymes from transgenic plant tissue.
BACKGROUND
Enzymes are used to process a variety of agricultural products such as wood,
fruits and
vegetables, starches, juices, and the like. Typically, processing enzymes are
produced and
recovered on an industrial scale from various sources, such as microbial
fermentation
(Bacillus a-amylase), or isolation from plants (coffee P-galactosidase or
papain from plant
parts). Enzyme preparations are used in different processing applications by
mixing the
enzyme and the substrate under the appropriate conditions of moisture,
temperature, time,
and mechanical mixing such that the enzymatic reaction is achieved in a
commercially viable
manner. One area where enzymes play an important role is in the area of corn
milling.
Today corn is milled to obtain cornstarch and other corn-milling co-products
such as corn
gluten feed, corn gluten meal, and corn oil. The starch obtained from the
process is often
further processed into other products such as derivatized starches and sugars,
or fermented to
make a variety of products including alcohols or lactic acid.
The process of starch recovery from corn grain is well known and involves a
wet-milling
process. Corn wet-milling involves many time consuming and costly steps, which
include
steeping the corn kernel, grinding the corn kernel, and separating the
components of the
kernel. Dry-mill processes that make fermentable sugars (and then ethanol, for
example)
from cornstarch facilitate efficient contacting of exogenous enzymes with
starch. These
processes are less capital intensive than wet-milling but significant cost
advantages are still
desirable, as often the co-products derived from these processes are not as
valuable as those
derived from wet-milling.
- 1 -
CA 2907491 2017-05-04
'Ibus, for dry milling, there is a need for a method that improves the
efficiency of the process
and/or increases the value of the co-products. For wet milling, there is a
need for a method of
processing starch that does not require the equipment necessary for prolonged
steeping,
grinding, milling, and/or separating the components of the kernel. For
example, there is a
need to modify or eliminate the steeping step in wet milling as this would
reduce the amount
of waste water requiring disposal, thereby saving energy and time, and
increasing mill
capacity (kernels would spend less time in steep tanks). There is also a need
to eliminate or
improve the process of separating the starch-containing endosperm from the
embryo.
The present invention relates to a transgenic corn (Zea mays) plant, event
3272 (Johnson et
al, U.S. Patent 7,635,799) that has incorporated into its genome a
synthetic a-amylase gene (amy797E), encoding a thermostable Amy797E a-amylase
capable
of processing starch in plants. a-amylase enzymes act on starch and related
polysaccharides
and oligosaccharides in a random manner, performing endohydrolysis of (1->4)-a-
D-
glucosidic linkages in polysaccharides containing three or more (1->4)-a-
linked D-glucose
units, such as those found in starch. Upon expression and activation of the
Amy797E a-
amylase, the enzyme processes the starch substrate found within the plant or
plant tissue. This
processing results in an altered composition which facilitates plant and grain
processing for
milling, thereby making a significant improvement in processing corn plants or
plant parts for
fermentation compared to corn plants which do not express amy797E (see, for
example,
Lanahan et al., US Patent 7,102,057 and Batie et al., US Patent 7,914,993).
A highly sensitive method for detecting the Amy797E a-amylase from transgenic
events is
necessary for use in environmental monitoring, monitoring traits in crops in
the field, or
monitoring products derived from a crop harvest, as well as for use in
ensuring compliance of
parties subject to contractual terms. It is ideal to have a limit of detection
of Amy797E
protein of less than 0.1%. In other words, a method of detection is needed to
detect less than
one kernel of corn event 3272 per 1000 kernels.
Additionally, certain applications of milling of event 3272 require mixing
event 3272 corn or
corn seed with corn or corn seed that is not event 3272 (see, for example,
U.S. Patent
7,914,993). This mixing is required so that an optimal amount of Amy797E a-
amylase per
unit of corn or corn seed is achieved. A highly sensitive method of Amy979E
detection
_ =-) _
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
would also benefit users who need to measure the amount of Amy797E present in
a given
amount of event 3272 corn or corn seed.
It is standard in the art to detect the presence of a specific polypeptide of
interest using an
antibody specific to the polypeptide of interest in an ELISA (enzyme-linked
immunosorbent
assay). An ELISA is a method for detecting a polypeptide of interest in a
biological sample,
the method typically comprising: (a) extracting protein from a biological
sample; (b) assaying
the extracted protein using an immunological method comprising an antibody
specific for the
polypeptide of interest; and (c) detecting the binding of said antibody to the
polypeptide of
interest. Detection is typically measured via an enzyme which is linked to a
secondary
antibody. The enzyme can metabolize a colorless substrate into a colored
product. The
optical density is measured, and this is proportional to the amount of colored
product and to
the amount of the polypeptide of interest present in the sample.
The ELISA method described above is known to one skilled in the art for the
detection of
transgenic polypeptides in plants. However, the polypeptide of interest here,
Amy797E,
presents an unusual challenge because its native substrate, starch, is found
within the plant
cell, and Amy797E tends to be pre-bound to its substrate in an inactive form.
Starch is
typically insoluble and comes out in the insoluble fraction during standard
purification.
Using the insoluble fraction for ELISA is undesirable, because the insoluble
fraction
significantly reduces the availability of the polypeptides, enzymes or other
substrates bound
or otherwise associated thereto for ELISA. Although not all of Amy797E in the
plant cell is
pre-bound to insoluble starch, it is preferred to release this pre-bound
Amy797E to increase
the limit of quantification and the limit of detection in a standard ELISA.
The present
invention solves this problem.
SUMMARY OF THE INVENTION
The present invention is drawn to a novel method of extracting a polypeptide
of interest from
a biological sample. The present invention includes a method for extraction of
a polypeptide
of interest which binds to an insoluble substrate in the context of a cell.
This method of
extraction from the biological sample a polypeptide bound to an insoluble
substrate
comprises initially homogenizing the biological sample in the presence of
extraction buffer
and a solubility-promoting compound. It is preferred that the solubility-
promoting compound
- 3 -
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
be non-immunoreactive. The sample is incubated to allow for solubilization of
the
polypeptide of interest. Finally, the sample is centrifuged to separate the
soluble and
insoluble fractions; the polypeptide of interest separates into the soluble
fraction. Detection
of the polypeptide in the soluble fraction is by any means known in the art,
including by
immunoassay such as ELISA, sandwich ELISA, ELISA dipstick, Lateral Flow
Immunochromatographic Assay, magnetic immunoassay, radioimmunoassay, or
fluorescent
immunoassay.
The present invention is drawn to a novel method of extracting a polypeptide
of interest from
plant tissue. The present invention is a novel approach for extraction of a
polypeptide of
interest which binds to an insoluble substrate in the context of a plant cell.
This method of
extracting from plant tissue a polypeptide bound to an insoluble substrate
comprises initially
homogenizing the tissue sample in the presence of extraction buffer and a
solubility-
promoting compound. It is preferred that the solubility-promoting compound be
non-
immunoreactive. The sample is incubated to allow for solubilization of the
polypeptide.
Finally, the sample is centrifuged to separate the soluble and insoluble
fractions; the
polypeptide of interest separates into the soluble fraction. From here, the
soluble polypeptide
of interest is available for detection. Detection of the polypeptide in the
soluble fraction is by
any means known in the art, including by immunoassay such as ELISA, sandwich
ELISA,
ELISA dipstick, Lateral Flow Immunochromatographic Assay, magnetic
immunoassay,
radioimmunoassay, or fluorescent immunoassay.
One aspect of the invention is directed to extracting a polypeptide which
interacts with an
insoluble macromolecule within a living cell using a non-immunoreactive
solubility-
promoting compound.
One aspect of the invention is directed to extracting a heterologous enzyme
which interacts
with an insoluble macromolecule within a living cell using a non-
immunoreactive solubility-
promoting compound.
One aspect of the invention is directed to extracting a heterologous enzyme
bound to an
insoluble substrate within a living cell using a non-immunoreactive solubility-
promoting
compound.
- 4 -
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
One aspect of the invention is directed to extracting a heterologous enzyme
which binds to an
insoluble substrate within a living cell using a non-immunoreactive solubility-
promoting
compound which is a macromolecule-degrading enzyme.
One aspect of the invention is directed to extracting a heterologous enzyme
which binds to an
insoluble substrate within a living cell using a non-immunoreactive solubility-
promoting
compound which is a substrate-degrading enzyme.
One aspect of the invention is directed to extracting a heterologous amylase
using a non-
immunoreactive substrate-degrading enzyme.
One aspect of the invention is directed to plant tissue from the transgenic
corn event 3272,
which heterologously expresses the a-amylase Amy797E, using a non-
immunoreactive
substrate-degrading enzyme.
One aspect of the invention is directed to plant tissue from the transgenic
corn event 3272,
which heterologously expresses the a-amylase Amy797E, using a non-
immunoreactive
amylase.
One aspect of the invention is directed to plant tissue from the transgenic
corn event 3272,
and homogenizing the plant tissue sample in the presence of extraction buffer
and a non-
immunoreactive amylase. The sample is incubated to allow the non-
immunoreactive amylase
to degrade the starch substrate, thereby releasing Amy797E from its insoluble
substrate.
Finally, the sample is centrifuged to separate the soluble and insoluble
fractions; Amy797E
fractionates into the soluble fraction. From here, Amy797E is available for
detection by
immunoassay such as ELISA, sandwich ELISA, ELISA dipstick, Lateral Flow
Immunochromatographic Assay, magnetic immunoassay, radioimmunoassay, or
fluorescent
immunoassay.
One aspect of the invention is directed to plant tissue from the transgenic
corn event 3272,
and homogenizing the plant tissue sample in the presence of extraction buffer
and a non-
immunoreactive amylase. The sample is then incubated at 89-95 C for 15
minutes to allow
the non-immunoreactive amylase to degrade the starch substrate. Finally, the
sample is
centrifuged to separate the soluble and insoluble fractions; Amy797E
fractionates into the
- 5 -
CA 2907491 2017-05-04
soluble fraction. From here, Amy797E is available for detection by immunoassay
such as
ELISA, sandwich ELISA, ELISA dipstick, Lateral Flow Immunochromatographic
Assay,
magnetic immunoassay, radioimmunoassay, or fluorescent immunoassay.
DEFINITIONS
The following definitions and methods are provided to better define the
present invention and
to guide those of ordinary skill in the art in the practice of the present
invention. Unless
otherwise defined, all technical and scientific terms used herein have the
same meaning as
commonly understood by one of ordinary skill in the relevant art. The
terminology used in
the description of the invention herein is for the purpose of describing
particular
embodiments only and is not intended to be limiting of the invention.
The term "transgene" as used herein, refers to any nucleic acid sequence used
in the
transformation of a plant, animal, or other organism. Thus, a transgene can be
a coding
sequence, a non-coding sequence, a cDNA, a gene or fragment or portion
thereof, a genomic
sequence, a regulatory element and the like. A "transgenic" organism, such as
a transgenic
plant, transgenic microorganism, or transgenic animal, is an organism into
which a transgene
has been delivered or introduced and the transgene can be expressed in the
transgenic
organism to produce a product, the presence of which can impart an effect
and/or a phenotype
in the organism.
A transgenic "event" refers to a transgenic plant produced by transformation
and regeneration
of a single plant cell with heterologous DNA, such as an expression cassette
that includes a
gene of interest. The term "event" also refers to progeny produced by the
event.
The terms "polypeptide," "protein," and "peptide" refer to a chain of
covalently linked amino
acids. In general, the term "peptide" can refer to shorter chains of amino
acids (e.g., 2-50
amino acids); however, all three terms overlap with respect to the length of
the amino acid
chain. As used herein, the terms "protein" and "polypeptide" are used
interchangeably and
encompass peptides, unless indicated otherwise. Polypeptides, proteins, and
peptides may
- 6 -
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
comprise naturally occurring amino acids, non-naturally occurring amino acids,
or a
combination of both. Non-naturally occurring polypeptides, proteins, peptides,
amino acids
may comprise heterologous nucleic acids. The polypeptides, proteins, and
peptides may be
isolated from sources (e.g., cells or tissues) in which they naturally occur,
produced
recombinantly in cells in vivo or in vitro or in a test tube in vitro, or
synthesized chemically.
Such techniques are known to those skilled in the art. See, e.g., Sambrook et
al., Molecular
Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989); Ausubel
et al.
Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and
John Wiley &
Sons, Inc., New York). A "polypeptide," "protein," or "peptide" may comprise
an enzyme.
A "heterologous" nucleic acid sequence is a sequence that is not naturally
associated with the
host cell into which it is introduced, including non-naturally occurring
multiple copies of a
naturally occurring sequence. A "heterologous" or "heterologously expressed"
polypeptide
or enzyme is that which is encoded by the heterologous nucleic acid sequence
and expressed
by that host cell.
"Amy797E" refers to the thermostable 797GL3 a-amylase encoded by the amy797E
gene
(Lanahan et al., US Patent 7,102,057) and expressed in corn event 3272
(Johnson et al., US
Patent 7,635,799).
"Corn event 3272," "maize event 3272," or "event 3272" refer to the transgenic
maize event
described in Johnson et al., US Patent 7,635,799.
A "macromolecule" is a very large molecule, such as a polymer or protein,
consisting of
many smaller structural units linked together. A "macromolecule" can refer to
biopolymers
such as polynucleotides, polypeptides, and polysaccharides. A "macromolecule"
can also
refer to synthetic polymers, such as plastics, synthetic fibers, or carbon
nanotubes. A
macromolecule can include within its meaning a "substrate" molecule. A
macromolecule can
include molecules such as deoxyribonucleic acids (DNA), ribonucleic acids
(RNA),
polypeptides, cellulose, xylan, arabinoxylan, arabinogalactan, pectin,
hemicellulose, cutin,
and lignin.
A "substrate" is a molecule upon which an enzyme acts to produce a product.
Enzymes
catalyze chemical reactions involving substrates. A substrate can include
molecules such as
- 7 -
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
deoxyribonucleic acids (DNA). ribonucleic acids (RNA), polypeptides,
cellulose, xylan,
arabinoxylan, arabinogalactan, pectin, hemicellulose, cutin, and lignin.
A "non-immunoreactive" or "non-immunogenic" compound or polypeptide refers to
a
compound or polypeptide which is not recognized by the antibody raised against
a
polypeptide of interest. For example, a non-immunogenic compound is not
recognized by the
antibody used in the ELISA to detect a polypeptide of interest.
An "immunoassay" refers to a biochemical test that measures the presence or
concentration
of a macromolecule in solution through the use of an antibody or
immunoglobulin.
DETAILED DESCRIPTION OF THE INVENTION
This description is not intended to be a detailed catalog of all the different
ways in which the
invention may be implemented, or all the features that may be added to the
instant invention.
For example, features illustrated with respect to one embodiment may be
incorporated into
other embodiments, and features illustrated with respect to a particular
embodiment may be
deleted from that embodiment. In addition, numerous variations and additions
to the various
embodiments suggested herein will be apparent to those skilled in the art in
light of the
instant disclosure, which do not depart from the instant invention. Hence, the
following
descriptions are intended to illustrate some particular embodiments of the
invention, and not
to exhaustively specify all permutations, combinations and variations thereof.
The present invention includes a novel method of extracting a polypeptide of
interest from a
biological sample. The present invention includes a method for extraction of a
polypeptide of
interest bound to an insoluble substrate in the context of a cell. This method
of extracting
from the biological sample a polypeptide bound to an insoluble substrate
comprises initially
homogenizing the biological sample in the presence of extraction buffer and a
solubility-
promoting compound. It is preferred that the solubility-promoting compound be
non-
immunoreactive. The sample is incubated to allow for solubilization of the
polypeptide of
interest. Finally, the sample is centrifuged to separate the soluble and
insoluble fractions; the
polypeptide of interest separates into the soluble fraction. Detection of the
polypeptide in the
soluble fraction is by any means known in the art, including by immunoassay
such as ELISA,
- 8 -
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
sandwich ELISA, ELISA dipstick, Lateral Flow Immunocluomatographic Assay,
magnetic
immunoassay, radioimmunoas say, or fluorescent immunoassay.
The present invention is a method for extraction and detection of a
polypeptide expressed in
plant tissue. In particular, this method is directed toward polypeptides which
may be
interacting with an insoluble macromolecule found within the host cell,
including but not
limited to polysaccharides such as starch or plant cell wall components such
as cellulose,
xylan, arabinoxylan, arabinogalactan, pectin, hemicellulose, and lignin.
Examples of such
polypeptides include but are not limited to amylolytic enzymes such as a-
amylases and 3-
amylases, or cell wall degrading enzymes such as cellulases, xylanases,
pectinases, pectin
methylesterases, polygalacturonases, xylosidases, hemicellulases, glucanases,
galactanases,
or arabinosidases, or proteases, cutinases, or lignases.
The present invention further includes a method for extraction and detection
of a
heterologously expressed enzyme from transgenic plant tissue. The present
invention
includes a method directed toward heterologously expressed enzymes which may
be pre-
bound to an insoluble substrate found within the host cell, including but not
limited to starch
or plant cell wall components such as cellulose, xylan, arabinoxylan,
arabinogalactan, pectin,
hemicellulose, and lignin. Examples of potentially such enzymes include but
are not limited
to amylolytic enzymes such as a-amylases and [3-amylases, or cell wall
degrading enzymes
such as cellulases, xylanases, pectinases, pectin methylesterases,
polygalacturonases,
xylosidases, hemicellulases, glucanases, or arabinosidases, or proteases,
cutinases, or
lignases.
The present invention uses a solubility-promoting compound in the extraction
buffer to
increase the amount of heterologously expressed enzyme found in the soluble
fraction
following purification. It is preferred that this compound be non-immunogenic
so that it is
not recognized by the antibodies used to detect the polypeptide of interest,
for example in an
ELISA. Usage of a non-immunogenic compound allows the method to not require a
step for
the removal of a possible immunogenic compound prior to analysis of the sample
by ELISA.
Examples of non-immunogenic, solubility-promoting compounds include but are
not limited
to substrate-degrading compounds, such as an enzyme which can degrade
insoluble
macromolecules. Enzymes which can degrade insoluble macromolecules, including
but not
- 9 -
CA 2907491 2017-05-04
limited to polysaccharides, such as starch or plant cell wall components such
as cellulose,
xylan, arabinoxylan, arabinogalacian, pectin, hemicellulose, and lignin,
include but are not
limited to amylolytic enzymes such as amylases, or cell wall degrading enzymes
such as
cellulases, xylanases, pectinases, pectin methylesterases, polygalacturonases,
xylosidases,
hemicellulases, glucanases, or arabinosidases, or proteases, cutinases, or
lignases.
Although the following examples use ground kernels, the tissue from any part
of the maize
plant, including but not limited to leaf, root, kernel, or pollen, may be used
in this assay.
Additionally, this assay may be used for animal diet samples, including but
not limited to
Starter, Grower, or Finisher Broiler diets, or Rat diet. Additionally, this
assay may be used
for other biological samples, including but not limiting to tissue samples
from any part of any
plant, insect, or animal, including blood, serum, or cell culture.
Additionally, this assay may
be used for microorganismal cultures, including but not limited to such
microorganisms as
fungi, yeast, bacteria, or algae.
Example 1. Addition of amylase results in reduced lower limit of
quantification and higher
sensitivity
30 mg of ground kernels from maize event 3272 were mixed with 3 ml of 1X PBS-T
TM
(phosphate buffered saline with Tween) solution. The "Control" sample did not
have
amylase added; the "+amylase" sample had 0.05% of a commercially available,
non-
immunoreactive amylase added to the IX PBS-T solution. Four Control samples
were
prepared, and 9 +amylase samples were prepared. The samples were homogenized
using the
Omni-Prep Homogenizer at 30 K rpm for 30 seconds, twice. The samples were then
heat
treated for 15 minutes at 90 C for 15 minutes. Next, the samples were
centrifuged at 10,000
X g for 15 minutes at room temperature. The soluble fraction, which is the
supernatant, was
taken, dilutions were made, and ELISA was performed using a commercially
available kit
from EnviroLogix containing an antibody specific to Amy797E. The following
table
displays the Lower Limit of Quantification (LLOQ) for the ELISA from each
sample,
expressed for each sample as the dilution factor and as ii.tg Amy797E per g
tissue. The LLOQ
represents the limit at which the ELISA can accurately measure how much of the
polypeptide
of interest is present.
- 10 -
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
Sample Dilution factor LLOQ (p..g/g)
Control >128 not able to determine
+amylase 1 0.125
The Control samples, which do not have an amylase added, had a high dilution
factor of
greater than 128. This means that the samples were diluted 128-fold in an
effort to reduce
interference from cellular carbohydrates. This relatively high interference is
likely due to
relatively low amounts of Amy797E in the soluble fraction available for ELISA.
At a 128-
fold dilution, a minimum dilution factor still could not be determined.
Additionally, the
dilution factor was so great than an accurate LLOQ could not be calculated.
When amylase is
added to the samples, a dilution factor of only 1 is required, likely due to
the increase of
Amy797E now available in the soluble fraction. The relatively low LLOQ is also
indicative
of the sensitivity of the method.
Example 2. Detection of one maize event 3272 kernel in 1000 non-transgenic
kernels
In the following example, a positive sample was prepared by mixing one kernel
of maize
event 3272 with 1000 kernels of non-transgenic maize. The kernels were then
ground into a
powder. As a negative control, 1000 kernels of non-transgenic maize were also
ground into a
powder. 6 samples of positive and 6 samples of control ground kernels were
prepared.
Positive and control samples were randomized and a blind experiment was
performed.
For each sample, 30 mg of ground kernels were mixed with 3 ml of 1X PBS-T
(phosphate
buffered saline with Tween) solution and 0.05% of a commercially available,
non-
immunoreactive amylase; As in Example 1, the samples were homogenized, heat
treated, and
centrifuged. For each sample, the undiluted supernatant, which is the soluble
fraction, was
then taken and ELISA was performed in duplicate using a commercially available
kit from
EnviroLogix containing an antibody specific to Amy797E. A reference known to
contain
0.0313 ng/ml Amy797E, was also included in the analysis as the baseline, above
which the
- 11 -
CA 02907491 2015-08-10
WO 2014/126579
PCT/US2013/026303
results would be considered positive. Results are shown in the following
table. The optical
density (OD) at 450 nm was used to evaluate the positive signal to noise
ratio. Anything
above the reference OD of 0.065 was considered positive.
Positive/
Mean
Sample # Replicate Negative
OD
Controls
reference 0.065
1 1 0.038 Negative
2 0.036 Negative
2 1 0.434 Positive
0.821 Positive
3 1 0.034 Negative
2 0.036 Negative
4 1 0.673 Positive
2 2.664 Positive
1 0.223 Positive
2 0.080 Positive
6 1 0.037 Negative
0.035 Negative
7 1 0.041 Negative
2 0.033 Negative
8 1 0.23 Positive
2 0.145 Positive
9 1 0.035 Negative
0.036 Negative
1 0.037 Negative
2 0.039 Negative
11 1 0.120 Positive
2 0.131 Positive
12 1 0.123 Positive
2 0.435 Positive
5
The limit of detection (LOD) was calculated using the mean OD values from the
negative
controls plus three standard deviations. An OD of 0.043 or lower captures 99%
of negative
samples, indicating that the probability of a false negative is low. These
results demonstrate
10 the sensitivity of the methodology, which can reliably detect the
presence of the Amy797E
protein at a level of 1:1000 maize kernels.
- 12 -
CA 2907491 2017-05-04
WO 2014/126579
PCT/US2013/026303
All publications and patent applications mentioned in the specification are
indicative of the
level of skill of those skilled in the art to which this invention pertains.
Although the foregoing invention has been described in some detail by way of
illustration and
example for purposes of clarity of understanding, it will be obvious that
certain changes and
modifications may be practiced within the scope of the list of the foregoing
embodiments and
the appended claims.
- 13 -
