Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
METHODS FOR PRODUCING RECOMBINANT PROTEINS
FIELD OF THE INVENTION
The invention relates to the field of biotechnology, particularly to the
production of recombinant proteins in plants. The invention further relates to
methods for recovering the recombinant proteins from transgenic plants.
BACKGROUND OF THE INVENTION
One of the fundamental achievements of the field of the genetic engineering is
the ability to genetically manipulate an organism to produce a protein that
the
organism was not capable of making prior to human intervention. Typically, the
production of such a protein is brought about by facilitating the insertion of
a
recombinant DNA molecule into an organism. Nucleotide sequences within the
recombinant DNA molecule contain the necessary genetic information to direct
the
host organism to produce the desired recombinant protein. Using such an
approach,
genetic engineers have modified a variety of eukaryotic and prokaryotic
organisms,
including bacteria, fungi, animals, and plants, to produce a wide array of
recombinant
proteins.
Recombinant proteins have had a major impact on agriculture, particularly on
crop plants. Recombinant proteins have been used to provide new traits to crop
plants
which improve their performance in the field. Transgenic corn and cotton
plants that
have been genetically engineered to produce a bacterially derived insecticidal
protein
are now widely utilized by farmers. Genetic engineers have also provided the
agricultural community with a variety of genetically engineered crop plants
that
produce proteins which increase a crop plant's tolerance to certain
herbicides. Such
genetically engineered, herbicide-tolerant soybeans, corn, cotton, and canola
are now
routinely used in agriculture.
While genetic engineers have achieved resounding successes with the
development of such insect-resistant and herbicide-tolerant crop plants, they
have not
yet reached a similar level of achievement in their attempts to use plants,
particularly
crop plants, as synthesizers of recombinant proteins for uses such as
therapeutic
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
proteins, industrial enzymes, nutritional supplements, and animal food
additives.
Several problems have hindered the progress of genetic engineers including
insufficient levels of accumulation of recombinant proteins in desired plant
tissues
and economically inefficient protein extraction methods.
Currently, many recombinant proteins are produced commercially by
fermentation utilizing microorganisms. However, fermentative methods are
relatively
expensive. All inputs necessary for the growth of the microorganisms must be
provided including both reduced carbon and nitrogen. Additionally, the
microorganisms must be grown under closed, temperature-controlled conditions
designed to prevent contamination of the fermentation process with undesired
microorganisms.
The production of recombinant proteins in crop plants has the potential to be
more cost effective than fermentation, particularly for large-scale
recombinant protein
production. Because of photosynthesis, plants produce their own reduced
carbon,
using sunlight, carbon dioxide, and water. Furthermore, crop plant production
systems do not involve the expensive facilities that the fermentation of
microorganisms reqmre.
However, a major impediment prevents the widespread use of crop plants for
recombinant protein production. Difficulties encountered in the extraction of
recombinant proteins from plant tissues have made the production of
recombinant
proteins in plants, for the most part, uneconomical. While difficulties
encountered in
extracting recombinant proteins from plants may be due to a variety of
reasons, often
the recombinant protein is produced in cells or parts of cells that make
recombinant
protein extraction inefficient using current processing technologies. Thus,
new
methods are needed to take advantage of the potential efficiencies of
recombinant
protein production in crop plants.
SUMMARY OF THE INVENTION
Methods are provided for producing and recovering recombinant proteins
from plant tissues. The methods find use in the biotechnology industry as an
efficient
means for producing and isolating large quantities of recombinant proteins.
Such
recombinant proteins can be, for example, therapeutic proteins for humans and
other
animals, industrial enzymes, and food additives. The methods involve steeping
plant
2
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
tissues in a solution under conditions favorable for extraction of the
recombinant
proteins. The methods additionally involve genetically manipulating plants to
improve recovery of recombinant proteins from plant tissues by optimizing
nucleic
acid constructs which comprise a coding sequence of a recombinant protein.
Also provided are plants, plant cells, plant tissues, and seeds thereof that
are
optimized for the recovery of recombinant proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphical representation of the effect of steeping solution on
aprotinin and corn protein extraction from whole corn kernels after steeping
from 0 to
48 hours as described in Example 2. Panel (A) represents the aprotinin and
corn
protein in steep water. Panel (B) represents the aprotinin and corn protein
remaining
in the kernels after steeping.
DETAILED DESCRIPTION OF THE INVENTION
The invention is drawn to methods for producing recombinant proteins in
plant tissues and recovering the recombinant proteins from the plant tissues.
By
"recombinant protein" is intended a protein that is produced in an organism as
a result
of recombinant DNA. The methods find use in the biotechnology industry for
producing recombinant proteins such as, for example, industrial enzymes,
pesticidal
proteins, and proteins used as therapeutic agents, nutritional supplements and
food
additives for humans and/or animals. The methods of the invention are
particularly
well suited for use in conjunction with existing grain-processing streams such
as, for
example, those that make use of wet-milling methodologies. The methods of the
invention can be used alone or integrated into existing or newly developed
seed-
processing systems. Thus, the methods find further use in agriculture by
providing
producers and processors with a potential new source of income resulting from
the
production and recovery of recombinant proteins in transgenic crop plants.
The methods of the present invention involve producing and recovering
recombinant proteins from plant tissues. By "plant tissue" is intended a whole
plant,
or any part thereof, including, but not limited to, seeds, organs, and cells.
Preferred
plant tissues of the invention are plant tissues that produce, or are capable
of
producing, a recombinant protein therein. More preferred plant tissues are
seeds,
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
fruits, tubers, roots, shoots, leaves, petioles, stems. and flowers, that
produce, or are
capable of producing, a recombinant protein therein. Most preferred plant
tissues are
seeds that producem, or are capable of producing, a recombinant protein
therein.
Methods are provided for recovering recombinant proteins from plant tissue.
The methods comprise producing steep water by steeping plant tissue in a
steeping
solution. The plant tissue is from a plant that produces recombinant proteins
in such a
plant tissue. Such a plant is a transgenic plant that possesses a stably
integrated
nucleic acid construct, particularly a nucleic acid construct, within its
genome. The
nucleic acid construct comprises a nucleotide sequence encoding the
recombinant
protein operably linked to a promoter that drives expression in a plant cell.
However,
any plant tissue containing a recombinant protein can be utilized in the
methods of the
invention including, but not limited to, plant tissue from a stably
transformed plant
and plant tissue from a plant that produces recombinant proteins under the
direction of
recombinant DNA or RNA delivered to a plant by, for example, a virus or a
viral
vector.
By "steep water" is intended the solution that results from steeping plant
tissue
in a steeping solution. By "steeping" is intended bringing plant tissue into
contact
with a solution, herein referred to as a "steeping solution," or the act
thereof.
Generally, steeping is conducted over a period of time that is determined from
such
factors as, for example, the plant species, the plant tissue, the steeping
solution, the
environmental conditions of the steeping, the recombinant protein and the
like.
The steeping solution is comprised of water. Additionally, the steeping
solution can contain one or more other components including, but not limited
to:
sulfur dioxide; inorganic acids such as, for example, sulfurous acid, sulfuric
acid,
phosphoric acid, nitrous acid, nitric acid, hypochlorous acid, hydrochloric
acid,
carbonic, boric acid, and hydrofluoric acid; organic acids such as, for
example, lactic
acid, formic acid, succinic acid, malic acid, pyruvic acid, ascorbic acid,
malonic acid,
tartaric acid, oxalic acid, propionic acid, acetic acid, n-butyric acid,
isobutyric acid,
and citric acid; salts such as, for example, sodium acetate, calcium acetate,
potassium
acetate, ammonium acetate, magnesium acetate, sodium benzoate, sodium
chloride,
calcium chloride, potassium chloride, ammonium chloride, magnesium chloride,
sodium sulfate, calcium sulfate, potassium sulfate, ammonium sulfate,
magnesium
sulfate, sodium nitrate, calcium nitrate, potassium nitrate, ammonium nitrate,
4
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
magnesium nitrate, sodium nitrite, potassium nitrite, sodium carbonate,
calcium
carbonate, potassium carbonate, ammonium carbonate, magnesium carbonate,
sodium
phosphate, calcium phosphate, potassium phosphate, ammonium phosphate, and
magnesium phosphate; buffers; chelating agents, antimicrobial agents,
preservatives.
stabilizers, and the like.
Preferably, such components improve the recovery of the recombinant protein,
preserve the desired function or activity of the protein, or both. It is
recognized that
such a steeping solution can be comprised of process water that originates,
for
example, in downstream operations commonly used in the corn-refining industry.
By
"downstream operation" is intended any operation that follows the production
of steep
water.
Preferred embodiments of the invention make use of whole, unprocessed seeds
for producing steep water. However, the methods of the invention also
encompass the
use of seeds that have been previously processed by one or more methods
including,
but not limited to, grinding, milling, cracking, defatting, degerminating,
fermenting,
steaming, heating, cooling, freezing, thawing, pre-soaking in water or other
solvents,
and the like. Furthermore, the seeds of the invention can be washed or cleaned
in
some manner prior to steeping to remove or reduce the amount of undesired
materials
on the surface of the seeds. Such undesired materials include, but are not
limited to,
soil particles, insects, fungi, spores, and any undesired parts of a plant
that are
harvested with seeds such as, for example, husks, leaves, cobs, and any part
or
particles thereof. The seeds can be subjected to any one or more methods for
washing
or cleaning seeds. Such methods for washing or cleaning seeds can comprise the
use
of one or more components including, but not limited to, water, a solvent, and
a
pressured gas or mixture of gases, such as, for example, pressurized carbon
dioxide,
pressurized nitrogen, and pressurized air. While the washing and cleaning
procedures
described supra are directed toward seeds, those skilled in the art will
recognize that
other plant tissues of the invention can also be treated in a like manner
prior to
steeping.
Although the methods of the invention do not depend on a particular volume
of steeping solution per unit of plant tissue, those of ordinary skill in the
art
understand that altering the volume of steeping solution per unit of plant
tissue can
affect the speed of recovery of the recombinant protein, the total amount of
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
recombinant protein recovered or both. In addition, the cost of the steeping
solution,
including any costs of waste-water treatment or disposal, can also be used as
a
consideration in determining the appropriate volume of steeping solution to
use.
Thus, the volume of steeping solution depends on the desired outcome. In
certain
embodiments of the invention, the volume of the steeping solution per bushel
of seed
is preferably less than about 50 gallons, more preferably less than about 25
gallons,
most preferably less than about 10 gallons. Similarly volumes of steeping
solution
can also be utilized with other plant tissues.
The temperature of steeping can be controlled to improve recovery of the
recombinant protein in the steep water. Depending on the recombinant protein,
the
species of plant, the plant tissue, and the desired outcome, the temperature
selected is
generally a temperature that allows the maximum recovery of the recombinant
protein
in the desired form and in the shortest possible time. Typically, such a
desired form
of a recombinant protein is a form in which the protein is active or capable
of
1 S performing the intended function such as, for example, an enzymatic
activity.
Alternatively, a desired form of a recombinant protein can be a non-functional
or
denatured form. It is recognized that such a denatured form can be renatured
at a later
time by methods known to those of ordinary skill in the art. Generally, the
steeping
temperature is less than a temperature which is known to cause coagulation or
denaturation of the recombinant protein. Generally, the incubation temperature
is
greater than the freezing point but less than the boiling point of the
steeping solution.
Preferably, the incubation temperature is between about 20°C and about
70°C. More
preferably, the incubation temperature is between about 35°C and about
65°C. Most
preferably, the incubation temperature is between about 40°C and about
60°C.
While preferred methods of the invention employ atmospheric pressure,
embodiments of the invention can involve increasing or decreasing the pressure
during steeping and during the subsequent separation of the steep water from
the
steeped plant tissue. Decreasing the pressure during steeping, particularly in
the
initial phase, can facilitate the uptake of the steeping solution into the
plant tissue and
thus reduce the length of time of the incubation necessary to achieve the
desired
recovery of recombinant protein. Increasing the pressure, particularly at the
end of
steeping when the steep water is withdrawn from the plant tissue, can increase
the
volume of steep water recovered and thus increase the amount of recombinant
protein
6
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
recovered. By modifying pressure, increases in the speed of recovery of the
recombinant protein, the total amount of recombinant protein recovered or
both, can
be realized. The methods of the present invention do not depend on any
particular
method for altering pressure. Any method for altering pressure known to those
of
ordinary skill in the art can be employed.
The methods of the invention also encompass one or more additional measures
known to those of ordinary skill in the art which increase the speed of
recovery of the
recombinant protein, the total amount of recombinant protein recovered or
both.
During the steeping of plant tissues, the steeping solution can be, for
example, mixed,
stirred, agitated, shaken, re-circulated, aerated or de-aerated. The
particular additional
measures employed, if any, depend on factors such as, for example, the species
of
plant, the specific plant tissue, the specific recombinant protein and the
composition
of the steeping solution.
Depending on the desired use of the recombinant protein, further steps can be
employed, for example, to concentrate the recombinant protein, to remove
impurities
from the steeping solution, to separate the desired recombinant protein from
undesired
proteins and to obtain the recombinant protein in a dry form or in a form in
the
substantial absence of water. Methods for such steps are known to those of
ordinary
skill in the art. In addition, one or more components can be added to the
steeping
solution and/or steep water to preserve and/or stabilize the recombinant
protein.
The methods of the invention encompass the use of any protein purification
method known in the art. Such methods include, but are not limited to,
centrifugation,
ultrafiltration, salt precipitation, dialysis, gel-filtration chromatography,
ion-exchange
chromatography, affinity chromatography, immunoaffinity chromatography, high-
performance liquid chromatography (HPLC), reversed-phase high-performance
liquid
chromatography, ion-exchange high-performance liquid chromatography, size-
exclusion high-performance liquid chromatography, high-performance
chromatofocusing, hydrophobic interaction chromatography, one-dimensional gel
electrophoresis, two-dimensional gel electrophoresis and capillary
electrophoresis.
While a desired amount of recombinant protein can be recovered in the steep
water, one or more secondary extractions of the steeped plant tissue can be
employed
in the methods of the invention to increase the recovery of recombinant
protein from
the plant tissue. By "secondary extraction'' is intended any subsequent
extraction of a
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
plant tissue, or any part or parts thereof, that occurs after steeping. In
embodiments of
the invention involving seeds, preferred seed parts for a secondary extraction
include,
but are not limited to, an embryo (also referred to as a "germ"), an
endosperm, a
degerminated seed (i.e. a seed lacking a germ), a seed coat, a tip cap, and a
pericarp.
Any extraction methods known to those skilled in the art can be employed in
such a
secondary extraction including, but not limited to, incubating the steeped
plant tissue,
seed or seed parts in an extraction solution, grinding, and milling. The
extraction
solution is comprised of water and can additionally contain one or more other
components including, but not limited to, the components of a steeping
solution
described supra. Alternatively, the extraction solution is comprised of steep
water
and can additionally contain one or more other components including, but not
limited
to, the components of a steeping solution described supra. Typically, the
recombinant
protein is recovered from a secondary extraction in a solution and processed
further
by any one or more of the additional steps described supra for the steep
water. The
recovered solution can also be combined with the steep water before, during or
after
any such additional steps are employed. In preferred methods of the invention,
the
germs, degerminated seeds, or both are subjected to a secondary extraction
involving
combining the seed parts with an extraction solution comprising steep water.
The methods of the invention make use of any plant tissue that contains a
recombinant protein. In preferred embodiments of the invention, the plant
tissues
produce the recombinant protein under the direction of a nucleic acid
construct
optimized for recovery of a recombinant protein. Such a nucleic acid construct
comprises a nucleotide sequence encoding a recombinant protein operably linked
to a
promoter that drives expression in a plant cell. Nucleic acid constructs of
the
invention encompass both DNA constructs and RNA constructs. It is recognized
that
such DNA and RNA constructs can be either single stranded and double stranded.
Further, it is recognized that promoters of the invention also encompass
promoters
utilized for transcription by viral RNA polymerases.
By "optimized for recovery" is intended that the nucleotide sequence of the
nucleic acid construct has been manipulated by any means known to those of
ordinary
skill in the art wherein the recovery of a recombinant protein from plant
tissue is
improved.
8
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
By "manipulated" is intended modifying or altering the nucleotide sequence of
the nucleic acid construct in any way including, but not limited to,
nucleotide
substitutions, additions, deletions, inversions, rearrangements and selection
of the
promoter used to drive expression of the coding sequence of the recombinant
protein
of the invention.
By "recovery is improved" is intended at least one desired improvement in
recovery is achieved. Such a desired improvement in recovery can be, for
example,
an increase in the level of the recombinant protein in a plant tissue, an
increase in the
amount of the recombinant protein recovered in the steep water, an increase in
the
total amount of the recombinant protein recovered in steep water and from a
secondary extraction, and an increase in the amount recovered of a desired
form of the
recombinant protein. Alternatively, a desired improvement can be a reduction
in the
length or costs of extracting the recombinant protein from plant tissue. Thus,
optimizing a nucleic acid construct to improve recovery may or may not lead to
an
increased amount of a recombinant protein in the plant tissue or an increased
amount
of a recombinant protein recovered from such a plant tissue.
Most of the corn kernels produced in the United States are processed by the
corn-refining industry primarily to extract the starch present in the mature
corn
kernels. Some of the refined starch is sold as unmodified corn starch or
modified into
specialty starches prior to sale. However, the majority of the corn starch
produced by
the corn-refining industry is converted into ethanol, corn syrups, dextrose,
and
fructose.
The bulk of the corn starch produced in the United States is prepared by the
wet-milling process. The first step in the wet-milling process is to steep the
corn
kernels in an aqueous solution. Steeping the kernels serves two main purposes.
First
it softens the kernels for subsequent milling, and second, it allows undesired
soluble
proteins, peptides, minerals and other components to be extracted from the
kernels.
After steeping, the kernels are separated from the steep water and then wet
milled.
The steep water is typically concentrated by evaporation to yield a solution
referred to
as a corn steep liquor. Corn steep liquor typically contains about 3.~ pounds
dry
solids per bushel of corn kernels with a nitrogen content between 45-48%
(Blanchard
(1992) Technology of Corn Wet Milling and Associated Processes, Elsevier, New
York). Protein content in corn steep liquor has been estimated at about one
pound per
9
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
bushel of steeped corn which amounts to approximately 15-20% (w/w) of total
corn
kernel protein (Blanchard (1992) Technology of Corn Wet Milling and Associated
Processes, Elsevier, New York). Corn steep liquor is a low-value by-product
from
the corn wet-milling process and is currently sold as a feed additive or
fermentation
medium supplement at approximately $50 per ton dry solids.
In a first embodiment of the invention, methods are provided for recovering a
recombinant protein from corn kernels in steep water. The methods provide an
efficient and economical way to recover recombinant proteins from corn
kernels. The
cost of recovering recombinant proteins produced in corn kernels by the
methods of
the present invention is estimated to be approximately $0.50 per kg
recombinant
protein, assuming that approximately 10% of the dry solids in steep water is
recombinant protein. The major advantage of the methods of the invention over
existing methods for isolating recombinant proteins from corn kernels is the
integration of recombinant protein extraction in the corn wet-milling process.
In
preferred methods of the invention, kernels steeped by the methods of the
invention
are suitable for use in the milling or grinding step that occurs after
steeping in wet-
milling processes. Thus, the costs of retrofitting processing plants and/or
adjustments
to the corn-refining process are minimized. The methods of the invention also
find
use with any modified or improved version of the corn wet-milling process that
utilizes steeping or any aqueous treatment of corn for the purpose of
enhancing corn
starch and protein separation. Furthermore, the methods find use in increasing
the
economic value of corn steep liquor, thus providing the corn-refining industry
with a
potential new source of profits.
While the composition of the steeping solution, the temperature and duration
of steeping, will depend on the recombinant protein and its desired form for
recovery,
the methods of the present invention involve combining corn kernels with a
steeping
solution typically containing about 0.1 to about 0.2% sulfur dioxide.
Generally, the
corn kernels are steeped in such a steeping solution for about 12 to about 48
hours at a
temperature of about 50°C. The methods of invention do no depend on
steeping
kernels for a particular period of time. Typically, kernels are steeped for at
least
about 1 hour. Preferably, kernels are steeped for at least about 6 hours. More
preferably, kernels are steeped for at least about 12 hours. Most preferably,
kernels
are steeped for about 24 to about 48 hours.
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
Preferably, the pH of the steeping solution is in the range of about pH 3 to
about pH 4, and the volume of the steeping solution per bushel of corn kernels
is
between about 5 and about 15 gallons. At the end of the incubation, the steep
water is
withdrawn from the steeped corn kernels. In preferred embodiments of the
invention,
a desired amount of recombinant protein, preferably in a desired form, is
recovered in
the steep water, and no further extraction of the steeped kernels is
conducted.
However, in other embodiments, one or more secondary extractions can be
additionally employed to increase the total amount of protein recovered from
the
kernels.
While sulfur dioxide is routinely employed in existing methods of corn wet
milling, the methods of the present invention do not depend on the presence of
sulfur
dioxide in the steeping solution. In fact, the presence of sulfur dioxide in a
steeping
solution may be detrimental to recovering certain recombinant proteins in a
desired
form, particularly those desired forms that depend on one or more disulfide
bonds,
sulfhydryl groups or both. Such disulfide bonds and sulfhydryl groups may be
important for the structure and/or function of a recombinant protein of the
invention.
The disulfide bonds and sulfhydryl groups of proteins are known to those of
ordinary
skill in the art and may be involved in functions of a recombinant protein
such as, for
example, enzyme or catalytic activity, binding activity and channel activity.
If
desired, the steep solution can contain any one or more of the sulfhydryl
reagents
typically employed in protein purificaiton methods such as, for example, (3-
mercaptoethanol, dithiothreitol, and dithioerythritol.
While typical corn wet-milling processes employ a steeping that ranges from
12 to 48 hours, other wet-milling processes such as, for example, those known
as the
dry-grind process and the intermittent-milling-and-dynamic-steeping process
involve
an initial steeping of shorter duration and can additional involve steeping at
a higher
temperature. Typically, the dry-grind and intermittent-milling-and-dynamic-
steeping
processes involve a steeping of whole kernels for about 12 hours or less at
temperatures of about 60°C. The main objective of a such a short
initial steeping is to
hydrate the embryo or germ. Breaking open the kernel after such a short
initial
steeping reduces the damage to the germ as compared to dry milling. The
hydrated
germ can then be recovered by methods typically utilized in the wet-milling
process.
The degerminated kernel fraction can then be subjected to a second steeping
with
11
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
additional grinding or milling to facilitate removal of soluble material from
the kernel
particles. See, Singh and Eckhoff (1996) Cereal Chem. 73:716-720 and Lopes-
Filho
et al. (1997) Cereal Chem. 74:633-638; herein incorporated by reference.
In a second embodiment of the invention, methods are provided for recovering
a recombinant protein from corn kernels comprising producing steep water which
comprises steeping kernels for a period of time of less than about 12 hours,
preferably
less than about 6 hours, more preferably less than about 3 hours, most
preferably
between about 1 hour and 3 hours. The steeping solution is comprised of water.
The
steeping solution can additionally contain any one or more of the other
components of
a steeping solution described supra. The recombinant protein of the invention
can be
recovered in the steep water. Additionally or alternatively, the recovered
germ and/or
degerminated kernel fraction can be subjected to at least one secondary
extraction to
recover the recombinant protein. Such a secondary extraction involves
combining the
germ, degerminated kernel fraction, or both, with an extraction solution,
preferably an
extraction solution comprising steep water, and incubating the combination.
In preferred methods of the invention, the recombinant protein, particularly a
recombinant protein that is expressed in the embryo or endosperm, is recovered
from
steep water. Such preferred methods can additionally involve a secondary
extraction
of the recovered germ or degerminated kernel to increase the recovery of the
recombinant protein. In such preferred methods, the particle size of the
recovered
germ or degerminated kernel can be reduced to facilitate recovery of the
recombinant
protein resulting in, for example, an increased recovery of the recombinant
protein
from the secondary extraction or a reduction in the duration of the secondary
extraction.
In preferred methods of the invention, the corn kernels are from a transgenic
corn plant that has a stably integrated nucleic acid construct that has been
optimized
for recovery of a recombinant protein from kernels. Preferably, such a nucleic
acid
construct possesses a promoter that directs expression to parts of the kernel
that are
favored for recovery of the recombinant protein in steep water including, but
not
limited, to the embryo, endosperm, seed coat, tip cap, and pericarp. The
optimized
nucleic acid construct can also possess a nucleotide sequence encoding a
signal
peptide for cell secretion operably linked to the coding sequence of the
recombinant
protein.
12
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
Methods are provided for optimizing a nucleic acid construct for the recovery
of a recombinant protein. The methods involve manipulating the nucleotide
sequence
of a nucleic acid construct to improve recovery of a recombinant protein from
plant
tissue. The nucleotide sequence of the nucleic acid construct can be
manipulated by
any means known in the art. The particular manipulations that can be employed
depend on factors such as, for example, the particular recombinant protein,
the plant
species, the plant tissue and the particular process by which the recombinant
protein is
recovered from the seed. Such manipulations include, but are not limited to,
operably
linking a promoter that directs expression of the recombinant protein to a
desired
plant tissue, operably linking a nucleotide sequence that encodes a signal
peptide and
modifying the nucleotide sequence that encodes the recombinant protein.
Promoters of interest are tissue-preferred, inducible, chemical-regulated, and
constitutive promoters. Such tissue-preferred promoters are known in the art
and
include, but are not limited to, seed-preferred, root-preferred, tuber-
preferred, and leaf
preferred promoters. Preferred promoters of the invention are those that
preferentially
direct expression of the recombinant protein to a plant tissue that provides a
desired
improvement in recovery of the recombinant protein. In methods involving the
optimization of a nucleic acid construct for the recovery of a recombinant
protein
from seeds, preferred promoters are seed-preferred promoters including, but
not
limited to, promoters that preferentially direct expression to the embryo, the
endosperm, the pericarp, the tip cap, the seed coat or combinations thereof.
If desired, the nucleic acid construct can be manipulated in such a manner
that
the encoded recombinant protein contains the necessary signal for secretion
from a
plant cell. Typically, a nucleotide sequence encoding a signal peptide is
operably
linked to the coding sequence of the recombinant protein. Thus, the encoded
recombinant protein will contain a signal peptide domain within its
polypeptide chain.
Such a signal peptide domain directs the secretion of the recombinant protein
from a
cell and can be removed from or retained in the secreted protein. Within a
plant, such
a secreted protein is typically found in the cell wall regions or
intercellular spaces.
Additionally, the nucleic acid construct can be manipulated to change the
nucleotide sequence encoding the recombinant protein. Such changes may or may
not
alter the amino acid sequence of the recombinant protein. Changes that do not
affect
the amino acid sequence of the recombinant protein include, for example, codon
13
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
optimization. Such codon optimization is known to those skilled in the art and
involves changing codons to those preferred for translation by the organism of
interest. Preferred codons of an organism are determined by analyzing codon
usage
frequencies for each amino acid using the coding sequences of cloned genes.
Preferred codons for an amino acid are those that are used with the highest
frequency
in the coding sequences of an organism. See, for example, U.S. Patent Nos.
5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-
498,
herein incorporated by reference. Generally, codon optimization involves
replacing a
non-preferred codon that specifies a particular amino acid with a preferred
codon that
specifies the same amino acid. Thus, changing such a non-preferred codon to
such a
preferred codon increases translation in the plant tissue of interest and can
increase
the amount of recombinant protein in the plant tissue.
The methods of the invention also encompass changes in the nucleotide
sequence encoding the recombinant protein. Generally, such changes do not
substantially alter the intended function of the protein. Such changes can,
however,
alter the amino acid sequence of the recombinant protein and include both
conservative and non-conservative amino acid substitutions as well as
additions and
deletions of one or more amino acids. It is recognized that any one or more
characteristics or activities of the recombinant protein can be modified
including, but
not limited to, disulfide bonds, glycosylation sites, myristylation sites,
phosphorylation sites, quaternary structure, endoplasmic reticulum retention
signals,
and catalytic properties such as, for example, substrate specificity, product
specificity,
K~ac, Km and Vm~. Furthermore, in the case of recombinant proteins that
possess two
or more distinct functional domains, one or more of such domains having an
undesired function can be removed, or otherwise rendered non-functional, by
manipulating the nucleotide sequence encoding the recombinant protein.
Domains can be added to the protein to improve protein recovery. Such a
domain can, for example, help stabilize the protein during isolation.
Alternatively,
such a domain can aid in isolating the protein once it is liberated from plant
tissue by
protein isolation techniques such as, for example, affinity or immunoaffinity
chromatography, and other affinity-based and immunological methods. Such
domains include, but are not limited to, a poly-histidine-tag and a domain
that
interacts with a specific antibody.
14
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
To determine if the desired optimization for recovery has been achieved, the
nucleic acid construct can, for example, be used to transform a plant. Plant
tissue
from such a transformed plant or from transformed progeny thereof, is utilized
in at
least one of the methods of the invention for recovering a recombinant protein
from
plant tissue. With such an approach, one or more of the various manipulations
of the
nucleic acid construct described supra can be tested singly or in combination
for their
effect on recovery of a recombinant protein. Those of ordinary skill in the
art will
recognize that such an approach can be used to select both nucleic acid
constructs and
plants, optimized for recovery of any recombinant protein.
In a third embodiment of the invention, methods are provided for optimizing a
nucleic acid construct for the recovery of a recombinant protein from a grain
seed,
particularly a corn kernel. The nucleic acid construct is optimized by
operably linking
the nucleotide sequence encoding the recombinant protein to a promoter that is
capable of preferentially directing the expression of the recombinant protein
to
preferred portions of the corn kernel for improving recovery in steep water.
Such a
promoter is capable of driving expression in a corn kernel, preferably in the
endosperm, embryo, pericarp, tip cap or seed coat of such a corn kernel, more
preferably in the embryo, pericarp, tip cap or seed coat, most preferably in
the
embryo. In preferred methods of the present invention, the nucleic acid
construct is
further manipulated to operably link a nucleotide sequence encoding a signal
peptide
for secretion from a plant cell.
A major source of protein in steep water, that is produced by the typical
methods utilized in the corn-refining industry for steeping corn kernels
before wet
milling, is the corn embryo. Additionally, proteins from the embryo are known
to
appear in the steep water at a relatively faster rate than proteins from other
parts of the
corn kernel, such as, for example, the endosperm. Thus, preferred methods of
the
present invention involve a nucleic acid construct comprising a promoter that
drives
expression preferentially in an embryo.
Methods are provided for optimizing a plant for recovery of a recombinant
protein from tissues of the plant. The methods involve stably integrating into
the
genome of a plant a nucleic acid construct optimized for the recovery of a
recombinant protein as described supra. The methods find use in providing a
plant
that is genetically engineered for optimal recovery of a recombinant protein
from its
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
tissues. Such a plant is capable of producing, for example, seeds that contain
a
recombinant protein, in a desired cellular or subcellular location, in a
desired form, or
both, for optimal recovery of the recombinant protein from the seed.
In a fourth embodiment of the invention, methods are provided for optimizing
a corn plant for recovery of a recombinant protein from kernels comprising
stably
integrating a nucleic acid construct that is optimized for recovery of a
recombinant
protein from corn kernels. Preferably, such a nucleic acid construct is
optimized for
recovery of a recombinant protein from corn kernels essentially as described
for the
third embodiment supra.
Methods for producing a recombinant protein are provided. The methods
involve providing a plant with at least one nucleic acid construct comprising
a
nucleotide sequence encoding a recombinant protein operably linked to a
promoter
that drives expression in a plant. Such a nucleic acid construct is capable of
directing
the expression of a recombinant protein within the plant. The methods
additionally
involve synthesizing the recombinant protein in the plant, harvesting the
plant tissue,
using the plant tissue to produce steep water by steeping the plant tissue
with a
steeping solution and recovering the recombinant protein from the steep water.
In a fifth embodiment of the invention, methods are provided for producing a
recombinant protein involving stably integrating a nucleic acid construct into
the
genome of a crop plant, preferably a grain or oilseed plant, more preferably a
corn
plant. The nucleic acid construct comprises a nucleotide sequence that encodes
the
recombinant protein operably linked to a promoter that drives expression in a
plant
cell, particularly a cell in a seed. The methods additionally involve growing
the plant,
harvesting seeds of the plant and producing steep water by steeping the seeds
in a
steeping solution. In preferred embodiments of the invention, a desired
portion of the
total recombinant protein in the seed is recovered in the steep water.
Alternatively, if
desired, secondary extractions of the steeped seeds can be employed to recover
additional recombinant protein. Exemplary embodiments of methods for producing
a
recombinant protein make use of nucleic acid constructs optimized for recovery
of a
recombinant protein. Such nucleic acid constructs of the invention are
prepared as
described supra.
Plants transformed with the nucleic acid constructs optimized for recovery of
a
recombinant protein and seeds thereof, are provided. Transformed plant cells
and
16
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
tissues are also provided. Such plants and seeds find use in methods for
producing,
isolating or recovering recombinant proteins in plants and are particularly
directed to
the optimal recovery of a recombinant proteins from seeds.
The recombinant proteins of the invention comprise any recombinant protein
that can be produced in a plant. Recombinant proteins of interest include, but
are not
limited to, brazzein, avidin, streptavidin, aprotinin, (3-glucuronidase,
alkaline
phosphatase, insulin, bovine somatotropin, human growth hormone, fibrinogen,
thrombin, factor IX, factor XIII, serum albumin, plasma proteins, protein C,
invertase,
superoxide dismutase, catalase, urease, lysozyme, lactase, glucose isomerase,
a-
amylase, glucoamylase, pullulanase, isoamylase, (3-glucanase, xylanase,
papain,
trypsin, chymotrypsin, pepsin, proteases, protease inhibitors, esterases,
peroxidases,
hydrolases, phosphatases, kinases, ribonucleases, deoxyribonucleases,
antibodies,
phytases, lipases, phospholipases, cellulases, hemicellulases, pectinase,
peptide
hormones, pesticidal proteins, enzymes, and fusion proteins. Of particular
interest are
soluble, recombinant proteins having commercial value.
Preferably, the recombinant proteins of the invention are selected from
industrial enzymes, antibodies, insecticidal proteins, therapeutic proteins,
and proteins
that are nutritional supplements, nutraceuticals or food additives. More
preferably,
the recombinant protein is selected from the group consisting of avidin,
aprotinin, (3-
glucuronidase, and brazzein. Most preferably, the recombinant protein is the
sweetener protein, brazzein. See, U.S. Patent Nos. 5,326,580; 5,346,998;
5,527,555;
and 5, 741,537; herein incorporated by reference.
The recombinant proteins of the invention can be altered in various ways to
optimize recovery from plant tissue including, but not limited to, amino acid
substitutions, deletions, and insertions. Methods for such manipulations are
generally
known in the art. For example, amino acid sequence variants of the recombinant
proteins can be prepared by mutations in the DNA. Methods for mutagenesis and
nucleotide sequence alterations are well known in the art. See, for example,
Kunkel
(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in
Enzymol. 154:367-382; US Patent No. 4,873,192; Walker and Gaastra, eds. (1983)
Techniques in Molecular Biology (MacMillan Publishing Company, New York) and
the references cited therein. Guidance as to appropriate amino acid
substitutions that
do not affect biological activity of the protein of interest can be found in
the model of
17
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
Dayhoff et al. ( 1978) Atlas of Protein Sequence and Structure (Natl. Biomed.
Res.
Found., Washington, D.C.), herein incorporated by reference.
In addition, mutagenic and recombinogenic strategies for such as, for example,
DNA shuffling can be employed in altering the recombinant proteins of the
invention.
See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;
Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.
1~:436-
438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc.
Natl.
Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and
U.S.
Patent Nos. 5,605,793 and 5,837,458.
The deletions, insertions, and substitutions of the recombinant protein
sequences encompassed herein are not expected to produce radical changes in
desired
characteristics or activities of the protein. However, when it is difficult to
predict the
exact effect of the substitution, deletion, or insertion in advance of doing
so, one
skilled in the art will appreciate that the effect will be evaluated by
routine screening
assays to ensure the continued presence of the desired characteristics or
activities.
The use of the term "nucleic acid constructs" herein is not intended to limit
the
present invention to nucleic acid constructs comprising DNA. Those of ordinary
skill
in the art will recognize that nucleic acid constructs, particularly
polynucleotides and
oligonucleotides, comprised of ribonucleotides and combinations of
ribonucleotides
and deoxyribonucleotides may also be employed in the methods disclosed herein.
Thus, the nucleic acid constructs of the present invention encompass all
nucleic acid
constructs that can be employed in the methods of the present invention for
transforming plants including, but not limited to, those comprised of
deoxyribonucleotides, ribonucleotides, and combinations thereof. Such
deoxyribonucleotides and ribonucleotides include both naturally occurring
molecules
and synthetic analogues. The nucleic acid constructs of the invention also
encompass
all forms of nucleic acid constructs including, but not limited to, single-
stranded
forms, double-stranded forms, hairpins, stem-and-loop structures, and the
like.
The nucleic acid constructs of the invention encompass expression cassettes
for expression in the plant of interest. The cassette will include 5' and 3'
regulatory
sequences operably linked to a nucleotide sequence encoding a recombinant
protein
of the invention. By "operably linked" is intended a functional linkage
between a
promoter and a second sequence, wherein the promoter sequence initiates and
18
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
mediates transcription of the nucleotide sequence corresponding to the second
sequence. Generally, operably linked means that the nucleic acid sequences
being
linked are contiguous and, where necessary to join two protein coding regions,
contiguous and in the same reading frame. The nucleic acid construct can
additionally contain at least one additional gene, such as for example, a
selectable
marker gene.
Such an expression cassette is provided with a plurality of restriction sites
for
insertion of the coding sequence for the recombinant protein of the invention
to be
under the transcriptional regulation of the regulatory regions.
The expression cassette will include in the 5'-3' direction of transcription,
a
transcriptional and translational initiation region, a coding sequence for a
recombinant
protein of the invention, and a transcriptional and translational termination
region
functional in plants. The transcriptional initiation region, the promoter, can
be native
or analogous or foreign or heterologous to the plant host. Additionally, the
promoter
can be the natural sequence or alternatively a synthetic sequence. By
"foreign" is
intended that the transcriptional initiation region is not found in the native
plant into
which the transcriptional initiation region is introduced.
In additional to a promoter, the expression cassette can include one or more
enhancers. By "enhancer" is intended a cis-acting sequence that increases the
utilization of a promoter. Such enhancers can be native to a gene or from a
heterologous gene. Further, it is recognized that some promoters can contain
one or
more native, enhancers or enhancer-like elements.
The termination region can be native with the transcriptional initiation
region,
can be native with the operably linked DNA sequence of interest, or can be
derived
from another source. Convenient termination regions are available from the Ti-
plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase
termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-
144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;
Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-
158;
Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987)
Nucleic
Acid Res. 1:9627-9639.
Additional sequence modifications are known to enhance gene expression in a
plant. These include elimination of sequences encoding spurious
polyadenylation
19
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
signals, exon-intron splice site signals, transposon-like repeats, and other
such well-
characterized sequences that may be deleterious to gene expression. The G-C
content
of the sequence may be adjusted to levels average for a given cellular host,
as
calculated by reference to known genes expressed in the host cell. When
possible, the
sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes can additionally contain 5'-leader sequences in the
expression cassette construct. Such leader sequences can act to enhance
translation.
Translation leaders are known in the art and include: picornavirus leaders,
for
example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et
al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic
Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding
protein
(BiP), (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the
coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature
32:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in
Molecular
Biology of RNA, ed. Czech (Liss, New York), pp. 237-256); and maize chlorotic
mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See
also,
Delta-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods known to
enhance translation can also be utilized, for example, introns, and the like.
In preparing the nucleic acid construct, the various DNA fragments can be
manipulated, so as to provide for the DNA sequences in the proper orientation
and, as
appropriate, in the proper reading frame. Toward this end, adapters or linkers
can be
employed to join the DNA fragments or other manipulations may be involved to
provide for convenient restriction sites, removal of superfluous DNA, removal
of
restriction sites, or the like. For this purpose, in vitro mutagenesis, primer
repair,
restriction, annealing, resubstitutions, e.g., transitions and transversions,
may be
involved.
In the methods of the invention, a number of promoters that direct expression
of a gene in a plant can be employed. Such promoters can be selected from
constitutive, chemical-regulated, inducible, tissue-specific, and seed-
preferred
promoters. Constitutive promoters include, for example, the core CaMV 35S
promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al.
(1990)
Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.
12:619-
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et
al.
(1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBOJ 3:2723-
2730), and the like. Other constitutive promoters include, for example, U.S.
Patent
Nos: 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463;
and 5,608,142.
Chemical-regulated promoters can be used to modulate the expression of a
gene in a plant through the application of an exogenous chemical regulator.
Chemical-inducible promoters are known in the art and include, but are not
limited to,
the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide
safeners, the maize GST promoter, which is activated by hydrophobic
electrophilic
compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a
promoter, which is activated by salicylic acid. Other chemical-regulated
promoters of
interest include steroid-responsive promoters (see, for example, the
glucocorticoid-
inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-
10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-
inducible
and tetracycline-repressible promoters (see, for example, Gatz et al. ( 1991 )
Mol. Gen.
Genet. 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156), herein
incorporated by reference.
Inducible promoters can be employed in the methods of the invention such as,
for example, chemical-inducible promoters described supra, wound-inducible
promoters, pathogen-inducible promoters and plant-growth-regulator-inducible
promoters. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol.
89:245-254;
Uknes et al. (1992) Plant Cell 4:645-656; Van Loon (1985) Plant Mol. Virol.
4:111-
116; Ryan (1990) Ann. Rev. Phytopath. 28:425-449; U.S. Patent No. 5,428,148,
Rohmeier et al. ( 1993) Plant Mol. Biol. 22:783-792; Abe et al. ( 1997) Plant
Cell
9:1859-1868; Sakai et al. (1996) Plant Cell Physiol. 37:906-13; Ono et al.
(1996)
Plant Physiol. 112:483-91; and Wang and Kutler (1995) Plant Mol. Biol. 28:619-
34;
all of which are herein incorporated by reference.
The preferred promoters of the invention are seed-preferred promoters that are
active during seed development. For dicots, seed-preferred promoters include,
but are
not limited to, bean (3-phaseolin, napin, (3-conglycinin, soybean lectin,
cruciferin, and
the like. For monocots, seed-preferred promoters include, but are not limited
to,
maize 15 kDa zero, 22 kDa zero, 27 kDa zero, y-zero, waxy, shrunken l,
shrunken 2,
21
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
globulin 1, etc. Seed-preferred promoters of particular interest are those
promoters
that direct gene expression predominantly to specific tissues within the seed
such as,
for example, the endosperm-preferred promoter of y-zero, and the embryo-
preferred
promoter of Glob-1.
The methods of the invention involve providing a plant with a nucleic acid
construct comprising a nucleotide sequence encoding a recombinant protein. By
''providing'' is intended presenting to the plant the nucleic acid construct
in such a
manner that the construct gains access to the interior of the cell. The
methods of the
invention further involve the production of the recombinant protein in the
plant tissue
as a result of the presence of the nucleic acid construct within cells of the
plant tissue.
The methods of the invention do not depend on a particular method for
providing the
cells of a plant tissue with such a nucleic acid construct, only that the
production of
the recombinant protein therein depends on the nucleic acid construct. Methods
for
providing plants and cells thereof with a nucleic acid construct are known in
the art
including, but not limited to stable transformation methods, transient
transformation
methods and viral methods.
By ''stable transformation" is intended that the nucleic acid introduced into
a
plant integrates into the genome of the plant is capable of being inherited by
progeny
thereof. By "transient transformation" is intended that a nucleic acid
introduced into a
plant does not integrate into the genome of the plant.
The nucleic acids of the invention can be provided to the plant by contacting
the plant with a virus or viral nucleic acids. Generally, such methods involve
incorporating the nucleic acid construct of interest within a viral DNA or RNA
molecule. It is recognized that the recombinant protein of the invention can
be
initially synthesized as part of a viral polyprotein which later can be
processed by
proteolysis in vivo or in vitro to produce the desired recombinant protein.
Methods
for providing plants with nucleic acid constructs and producing the encoded
recombinant proteins in the plants, which involve viral DNA or RNA molecules
are
known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190,
5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.
Preferred methods of the invention for providing a plant with a nucleic acid
construct involve transforming a plant to stably integrate a nucleic acid
construct into
the genome of the plant. Transformation protocols as well as protocols for
22
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
introducing nucleotide sequences into plants can vary depending on the plant
or plant
cell targeted for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent stable integration into the plant
genome
include microinjection (Crossway et al. (1986) Biotechniques =1:320-334),
electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,
Agrobacterium-mediated transformation (Townsend et al., U.S. Pat No.
5,563,055),
direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and
ballistic
particle acceleration (see, for example, Sanford et al., U.S. Patent No.
4,945,050;
Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via
Microprojectile
Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,
ed.
Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)
Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet.
22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37
(onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et
al.
( 1988) BiolTechnology 6:923-926 (soybean); Finer and McMullen ( 1991 ) In
Vitro
Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.
96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice);
Klein et al.
(1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)
Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et
al.,
U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA
Transfer
into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell,
Tissue, and
Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)
(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al.
(1990)
Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature
(London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA
84:5345-
5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of
Ovule
Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen);
Kaeppler et
al. ( 1990) Plant Cell Reports 9:415-418 and Kaeppler et al. ( 1992) Theor.
Appl.
Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)
Plant
Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-
255
and Christou and Ford (1995) Annals of Botany 7:407-413 (rice); Osjoda et al.
( 1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens);
all
of which are herein incorporated by reference.
23
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
Generally. the nucleic acid construct will additionally comprise a selectable
marker gene for the selection of transformed cells. Selectable marker genes
are utilized
for the selection of transformed cells or tissues. Such selectable marker
genes and
methods of their use in selecting for transformed cells and/or plant tissues
are known in
the art. Selectable marker genes include, but are not limited to, genes
encoding
antibiotic resistance, such as those encoding neomycin phosphotransferase II
(NEO) and
hygromycin phosphotransferase (HPT), as well as genes conferring resistance to
herbicidal compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and
2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr.
Opin.
Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad Sci. USA
89:6314-
6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-
2422;
Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell ;8:555-
566;
Brown et al. (1987) Cell 19:603-612; Figge et al. (1988) Cell 52:713-722;
Deuschle et
al. (1989) Proc. Natl. Acad Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.
Natl.
Acad Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen
(1993) Ph.D. Thesis, University of Heidelberg; Refines et al (1993) Proc.
Natl. Acad
Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et
al. (1992) Proc. Natl. Acad Sci. USA 89:3952-3956; Baim et al. (1991) Proc.
Natl.
Acad Sci. USA 88:5072-5076; Wyborski et al. ( 1991 ) Nucleic Acids Res.
19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al.
(1991)
Antimicrob. Agents Chemother. 3:1591-1595; Kleinschnidt et al. (1988)
Biochemistry
27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et
al.
(1992) Proc. Natl. Acad Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.
Agents
Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental
Pharmacology,
Vol. 78 ( Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724.
Such
disclosures are herein incorporated by reference.
The above list of selectable marker genes is not meant to be limiting.
Any selectable marker gene can be used in the present invention.
Transformed plant cells and tissues can be regenerated into plants by standard
methods. See, for example, McCormick et al. (1986) Plant Cell Reports x:81-84.
These plants may then be grown, and either pollinated with the same
transformed
strain or different strains, and the resulting plants producing the desired
recombinant
protein of the invention. Two or more generations may be grown to ensure that
24
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
production of the desired recombinant protein is stably maintained and
inherited and
then seeds harvested and tested to ensure they possess the desired recombinant
protein.
The methods of the invention find use with any plant species capable of
producing a recombinant protein. Plants of the invention include, but are not
limited
to, corn (Zea mays or maize), sorghum (Sorghum bicolor, S. vulgare), wheat
(Triticum
aestivum), rice (Oryza saliva), rye (Secale cereale), soybean (Glycine max),
oats
(Avena saliva), barley (Hordeum vulgare), sunflower (Helianthus annuus),
safflower
(Carthamus tinctorius), canola (Brassica napus, B. rapa, B. juncea), oilseed
rape
(Brassica spp.), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum, G.
barbadense), flax (Linum usitatissimum), peas (Pisum sativum, Lathyrus spp.),
tobacco (Nicotiana tabacum), beans (Phaseolus spp.), fava bean (Vicia faba),
mung
bean (Vigna radiata), chickpea (Cicer arientinum), cowpea (Vigna sinensis, V
unguiculata), lentil (Lens culinaris), lupines (Lupinus spp.), alfalfa
(Medicago saliva),
potato (Solanum tuberosum), tomato (Lycopersicon esculentum), peppers
(Capsicum
annuum), sugar beet (Beta vulgaris), cassava (Manihot esculenta), cocoa
(Theobroma
cacao), carrot (Daucus carota), cabbage (Brassica oleracea var. capitata),
broccoli
(Brassica oleracea var. botrytis), cauliflower (Brassica oleracea var.
botrytis), lettuce
(Lactuca saliva), sweet potato (Ipomoea batatus), melons (Cucumis spp.),
watermelon
(Citrullus lanatus), squashes (Curcurbita spp.), cucumber (Cucumis. sativus),
apple
(Malus domestica), citrus trees (Citrus spp.), almond (Prunus amygdalus),
olive (Olea
europaea), avocado (Persea americana), mango (Mangifera indica), papaya
(Carica
papaya), cashew (Anacardium occidentale), coffee (Coffea spp.), guava (Psidium
guajava), grapes (Vitus spp.), millet (Pennisetum glaucum, Panicum miliaceum,
Setaria
italica), Eleusine coracana), palms (Phoenix dactylifera, Elaeis oleifera, E.
guineensis), coconut (Cocos nucifera), banana (Musa spp.), duckweed (Lemna
spp.),
onion (Album cepa), garlic (Allium sativum), and sugarcane (Saccharum spp.).
Preferably, the plant species are crop plant species. More preferably, the
plant
species are selected from the grain and oilseed plants including, but not
limited to,
corn, sorghum, wheat, millet, rice, rye, soybean, oats, barley, sunflower,
safflower,
canola, oilseed rape, peanuts, palm, coconut, cotton, and flax. Most
preferably, the
plant species are corn, wheat, rice, barley, sorghum, canola, cotton, and
soybeans.
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
The following examples are offered by way of illustration and not by way of
limitation.
EXPERIMENTAL
EXAMPLE 1
Transgenic Corn Plants that Accumulate Aprotinin in Kernels
Aprotinin, the active ingredient in the therapeutic agent, TRASYLOL, is a
serine protease inhibitor also referred to as bovine pancreatic trypsin
inhibitor.
TRASYLOL is indicated for prophylactic use to reduce perioperative blood loss
and
the need for blood transfusion in patients undergoing cardiopulmonary bypass
in the
course of coronary artery bypass graft surgery. Currently, commercial
preparations of
aprotinin are purified from bovine pancreas and lung. However, there is a
growing
concern that the bovine tissues used to prepare aprotinin may harbor prions
that may
be pathogenic to humans (Jefferey et al. (1995) Micron 26:277-298; Smith and
Copings (1995) Essays Biochem. 29:157-174). Thus, alternative sources of
aprotinin
are desired.
The production of aprotinin in plants can provide an alternative source of
aprotinin for therapeutic preparations such as TRASYLOL. Thus, corn plants
were
genetically engineered to produce aprotinin in their kernels. An optimized DNA
sequence for the aprotinin gene with preferred maize codons was prepared from
the
known amino acid sequence of the bovine protein. (Anderson and Kingston (1983)
Proc. Natl. Acad. Sci. USA 80:6838-42). The DNA sequence was optimized for
expression in corn by reverse translating the amino acid sequence of the
bovine
protein using preferred corn codons, and operably linked to a nucleotide
sequence
encoding a barley a-amylase signal peptide. Such a signal peptide is known to
direct
the secretion of operably linked proteins from plant cells. Additionally, the
maize
ubiquitin promoter and the potato pinll transcriptional terminator were
operably
linked to the 5' and 3' ends, respectively, of the signal peptide/aprotinin
nucleic acid
construct. Using this nucleic acid construct, transgenic corn plants were
produced
that accumulate aprotinin in kernels.
26
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
EXAMPLE 2
Recovery of Aprotinin from Kernels of Transgenic Corn Plants by Steeping
Because aprotinin was successfully produced in the kernels of transgenic corn
plants, experiments were initiated to develop efficient methods for recovering
the
recombinant protein from kernels. Preferably, such methods can be integrated
into
existing corn-refining systems such as those involving, for example, wet-
milling
processes, dry-grind processes and intermittent-milling-and-dynamic-steeping
processes.
Fifty grams of kernels from an aprotinin-expressing, transgenic corn plant
described in Example 1 were combined with a steeping solution of either 100 mL
water or 100 mL of an aqueous solution of 0.5% lactic acid and 0.2% SOZ (LA-
SOZ).
Lactic acid was added to the steeping solution to better simulate the
conditions of
industrial steeping processes which permit the growth of Lactobacillus sp.
(Singh and
Eckhoff (1996) Cereal Chem. 73:716-720; Lopes-Filho et al. (1997) Cereal Chem.
74:633-638). The kernels were steeped at 52°C with constant agitation
at 200 rpm.
At 6, 12, 24, and 48 hours of incubation, duplicate flasks were removed and
the steep
water was drained from each flask and assayed for total protein by the
Bradford
method and for aprotinin by an ELISA (enzyme-linked immunosorbent assay). The
steeped kernels were air dried and ground, and 2 g of the ground material was
extracted with 20 mL of PBS-T (phosphate-buffered saline + Tween 20) to
determine
the residual levels of total protein (Bradford) and aprotinin (ELISA).
After six hours of steeping, approximately 16 times more aprotinin was
recovered in the steep water from the LA-SOZ treatment than in steep water
from the
water treatment (Figure 1 and Table 1 ). By steeping kernels in the LA-SOZ
solution
for 24 hours, about 1 ~g of aprotinin was recovered in steep water per g of
moisture-
and oil-free kernel compared to 0.3 ~g when water was used. At the end of 24
hours
of steeping, the concentration of aprotinin in steep water from the LA-SO~ and
water
treatments was 0.1 % and 0.05% (w/w) of total soluble protein , respectively.
As
expected more corn protein was extracted during the first 24 hours by steeping
with
the LA-SOZ treatment than with the water treatment. However, the 48-hour
steeping
with water yielded a higher level of corn protein, but not aprotinin, than did
the LA-
27
CA 02384828 2002-03-12
WO 01/21270 PCT/CTS00/26005
SOZ steeping. While higher levels of both corn protein and aprotinin were
measured
in steep water from the water treatment after 48 hours of steeping than after
24 hours,
the opposite was true for the LA-SO~ samples. After 48 hours of steeping, the
levels
of both corn protein and aprotinin in steep water from the LA-SO~ treatment
were
lower than those measured after 24 hours of steeping.
The residual, extractable levels of both aprotinin and corn protein remaining
in
the kernels after steeping were determined (Figure 1 and Table 2). The
residual levels
of aprotinin after steeping with water were 10-20% more than after steeping
with LA-
S02. After six hours of steeping, substantially more corn protein remained in
the
water-steeped kernels than in the LA-SOZ-steeped kernels. However, when
steeping
was conducted for more than six hours, the differences in the levels of
residual corn
protein in between water-steeped kernels and LA-S02-steeped kernels were less
than
10%.
28
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
Table 1
Recovery of Aprotinin by Steeping
Kernels Steep
before Water
extraction
SteepingTime Weight Oil MoistureVolume AprotininProtein
solution(h) (g) contentcontent (ml) (~g/g (mg/g
(%) (%) dry dry
kernel) kernel)
Water 6 50 3.4 16 74 0.016 0.09
12 50 3.4 16 73 0.141 0.43
24 50 3.4 16 71 0.310 0.65
48 50 3.4 16 72 0.414 1.05
0.5% 6 50 3.4 16 74 0.262 0.31
lactic 12 50 3.4 16 73 0.547 0.55
acid 24 50 3.4 16 71 1.047 0.99
+
0.2% 48 50 3.4 16 71 ~ 0.965 0.71
S02 I
*All values are the average of two replicates.
Table 2
Residual Aprotinin and Corn Protein in the Kernels after Steeping
Kernels Residual
after in kernels
extraction
Steeping Time Weight Oil Moisture AprotininProtein
solution (h) (g) contentcontent (~g/g (mg/g dry
(%) (%) dry kernel)
kernel)
Water 0 2.0 3.4 16 4.483 4.31
6 2.0 2.7 13 4.406 5.60
12 2.0 2.0 13 2.557 4.21
24 2.0 2.0 13 1.616 3.34
48 2.0 1.8 13 1.545 2.71
0.5% lactic0 2.0 3.4 16 4.483 4.31
acid +0.2%6 2.0 2.2 12 3.542 4.72
SOZ 12 2.0 2.2 12 2.055 3.88
24 2.0 1.7 12 1.247 3.65
48 2.0 1.6 12 1.070 2.64
*All values are the average of two replicates.
29
CA 02384828 2002-03-12
WO 01/21270 PCT/US00/26005
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention
pertains. All
publications and patent applications are herein incorporated by reference to
the same
extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
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
appended claims.
