Note: Descriptions are shown in the official language in which they were submitted.
CA 02270289 1999-04-30
.o~ '~icant Ref. No.: 05718-PC'f.app
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PROTEINS WITH ENHANCED LEVELS
OF ESSENTIAL ArvIINO ACIDS
Field of the Invention
$ The present invention relates to the field of protein engineering wherein
changing
amino acid compositions effects improvements in the nutrition content of feed.
Specifically, the present invention relates to methods of enhancing the
nutritional content
of animal feed by expressing derivatives of a protease inhibitor to provide
higher
percentages of essential amino acids in plants.
Background of the Invention
Feed formula ns are required to provide animals essential nutrients critical
to
growth. However, crop plants are generally rendered food sources of poor
nutritional
quality because they contain low proportions of several amino acids which are
essential
I 5 for, but cannot be synthesized by, monogastric ;animals.
For many years researchers have attempted to improve the balance of essential
amino acids in the seed proteins of important crops through breeding programs.
As more
becomes known about seed storage proteins and the expression of the genes
which encode
these proteins, and as transformation systems are developed for a greater
variety of plants,
molecular approaches for improving the nutritional quality of seed proteins
can provide
alternatives to the more conventional approaches. Thus, specific amino acid
levels can be
enhanced in a given crop via biotechnology.
One_alternative method is to express .a heterologous protein of favorable
amino
acid composition at levels sufficient to obviate feed supplementation. For
example, a
number of seed proteins rich in sulfur amino acids have been identified. A key
to good
expression of such proteins involves efficient: expression cassettes with
tissue-preferred
promoters. Not only must the gene-controlling regions direct the synthesis of
high levels
of mRNA, the mRNA must be translated into a stable protein and over expression
of this
protein must not be detrimental to plant or animal health.
Among the essential amino acids needed for animal nutrition, often limiting in
crop plants, are methionine, threonine, lysine, isoleucine, leucine, valine,
tryptophan,
phenylalanine, and histidine. Attempts to increase the levels of these free
amino acids by
1 AMENDED SHEET
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breeding, mutant selection and/or changing the composition of the storage
prc~a~;ins
accumulated in crop plants has met with limited success.
A transgenic example is the phaseolin-promoted Brazil nut 2S expression
cassette.
However, even though Brazil nut protein increases the amount of total
methionine and
bound methionine, thereby improving nutritional value, there appears to be a
threshold
limitation as to the total amount of methionine that is accumulated in the
seeds. The
seeds remain insufficient as sources of methionine and methionine
supplementation is
required in diets utilizing the above soybeans.
An alternative to the enhancement of specific amino acid levels by altering
the
levels of proteins containing the desired amino acid is modification of amino
acid
biosynthesis. Recombinant DNA and gene transfer technologies have been applied
to
alter enzyme activity catalyzing key steps in the amino acid biosynthetic
pathway. See
Glassman, U.S. Patent No. 5,258,300; Galili, et al., European Patent
Application No.
485970; (1992). However, modification of the amino
acid levels in seeds is not always correlated with changes in the level of
proteins that
incorporate those amino acids. See Burrow, et al., Mol. Gen. Genet.; Vol. 241;
pp. 431-
439; (1993). Increases in free lysine
levels in leaves and seeds have been obtained by selection for DHDPS mutants
or by
expressing the E. coli DHDPS in plants. However, since the level of free amino
acids in
seeds, in general, is only a minor fraction of the total amino acid content,
these increases
have been insufficient to significantly increase the total amino acid content
of seed.
The IysC gene is a mutant bacterial aspartate kinase which is desensitized to
feedback inhibition by lysine and threonine. Expression of this gene results
in an increase
in the level of lysine and threonine biosynthesis. However, expression of this
gene with
seed-specific expression cassettes has resulted in only a 6-7% increase in the
level of Total
threonine - -or lysine in the seed. See Karchi, et al., The Plant J.; Vol. 3;
pp. 721-7; (1993.
Thus, there is minimal impact on the
nutritional value of seeds, and supplementation with essential amino acids is
still
required.
In another study (Falco et al., Biotechnology 13:577-582, 1995), manipulateon
of
bacterial DHDPs and aspartate kinase did result in useful increases in free
lysine and total
2
CA 02270289 1999-04-30
AE-plicant Ref. No.: 05718-PCT.app
r t
seed lysine. However, abnormal accumulation of lysine catabolites was also
observed
suggesting that the free lysine ool was subject to catabolism.
Based on the foregoing, there exists a need for methods of increasing the
levels of
essential amino acids in seeds of plants. As can be seen from the prior art,
previous
S approaches have led to insufficient increases in the levels of both free and
bound amino
acids and insignificant enhancement of the nutriitional content of the feed.
Summary of thla Invention
It is one object of the present invention 1:o provide nucleic acids encoding
protease
inhibitors with modified levels of essential amino acids. It is an object to
reduce the
protease inhibitory activity in addition to modifying levels of essential
amino acids and
antigenic polypeptide fragments thereof. It is a further object of the present
invention to
provide transgenic plants comprising protease inhibitors with modified levels
of essential
amino acids. Additionally, it is an object of the present invention to provide
methods for
increasing the nutritional value of a plant and for providing an animal feed
composition
comprising the transgenic plants comprising protease inhibitors with modified
levels of
essential amino acids and reduced protease inhibitory activity. The protease
inhibitor CI-
2 has been modified to produce on 83 amino acid polypeptide and an amino-
terminal
truncated version of 65 amino acids residues.
Therefore, in one aspect, the present invention relates to a polypeptide
comprising
at least 10 contiguous amino acid residues frorrl a protein having Seq. ID No.
2, 4, 6, 8, 10
or 12,16,18,20,22,24; and wherein the polypeptide exhibits reduced protease
inhibitor
activity compared to a wild-type protein. In ont; embodiment, the present
invention relates
to the ahove-~entioned polypeptide comprisini; Seq. ID No. 2, 4, 6, 8, 10 or
12,
16,18,20,22,24 and the polypeptide wherein more than about 55%, but less than
about
95%, more than about 55%, but less than about. 90%, or more than about 55% but
less
than about 85%, of the amino acid residues are essential amino acids. In some
embodiments, the essential amino acid is lysine, tryptophan, methionine,
threonine or
mixtures thereof. In some embodiments, the present invention relates to the
nucleic acid
encoding the polypeptide referred to supra and in one embodiment, relates to
the nucleic
acid as DNA and in another embodiment to a second nucleic acid which is
complementary to the DNA. Another embodirrlent relates to the polypeptide
wherein
more than about 10% but less than about 40% .of the amino acid residues are
essential
3 ,",
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CA 02270289 2000-04-20
75529-49(S)
amino acids. Another embodiment relates to the transformed
plant containing the polypeptide supra. In some embodiments an
animal feed composition is provided.
In another embodiment, the polypeptide referred to
supra, comprises at least 20 contiguous amino acid residues.
In one aspect, the present invention relates to this
polypeptide which contains or is modified to contain essential
amino acids at positions 1, 8, 11, 17, 19, 34, 41, 56, 59, 62,
65, 67 or 73. In another aspect, the present invention relates
to polypeptide which contains or is modified to contain
essential amino acids at positions 1, 16, 23, 41, 44, 49 and
55. In other embodiments, the polypeptide comprises at least
30 contiguous amino acid residues.
In a further aspect, the present invention relates to
the modification of amino acid residues in the active site of
protease inhibitors. The above mentioned polypeptide contains,
or is modified to contain, non-wild type amino acid residues at
positions from about 53 to about 70. In some embodiments, the
non-wild type amino acid residues are located at positions 58-
60, 62, 65 or 67. In another embodiment, the polypeptide the
non-wild type amino acid residue is located at position 59. In
some embodiments, the present invention relates to the nucleic
acid encoding the polypeptide referred to supra.
In another aspect the polypeptide is about 7.3 Kda or
about 9.2 Kda and further comprises one or more additional
amino terminal amino acid residues, and in some embodiments,
the amino-terminal amino acid residue is methionine. The
number of additional amino terminal amino acid residues is
preferably less than 50. In another embodiment, the
polypeptide is a cleavage product and in yet another, the
polypeptide is recombinantly produced.
4
CA 02270289 2000-04-20
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In a further aspect, the present invention relates to
an expression cassette comprising the nucleic acids as
described supra, operably linked to a promoter providing for
protein expression. In some embodiments, the promoter provides
for protein expression in plants and in others the promoter
provides for protein expression in bacteria, yeast or virus.
In yet another aspect, the present invention is
directed to transformed plant cells containing the expression
cassette described supra.
4a
CA 02270289 1999-04-30
Ap,.~.licant Ref. No.: 057IR-PCT.app
In another aspect, the present invention is directed to transformed plants
containing at least one copy of the expression caasette described supra. In
some
embodiments, there is a seed of this transformed plant.
Another aspect of this invention provides a polypeptide produced by
substituting
an essential amino acid for at least one but less than 50 amino acid residues
in a protease
inhibitor for enhancing nutritional value of feed.
In another aspect, the present invention relates to polypeptides supra wherein
hydrogen bonding is disrupted in the active site loop of the inhibitor.
In yet another aspect, the present invention relates to the polypeptide sa~pra
which exhibits
decreased protease inhibitor activity as compared to the wild-type protein
which does not
have substituted amino acid residues. In some embodiments nucleic acid encodes
a
protease inhibitor protein with decreased inhibitory activity.
In another aspect, the present invention relates to the polypeptide supra
which
exhibits less than about 30% of the inhibitor activity compared to
corresponding wild-
type protein which does not have substituted amino acid residues.
In another aspect, the present invention relates to a nucleic acid comprising
the
sequence of SEQ ID No. 1,3,5,7,9,11,15,17,19,21, or 23 or a nucleic acid
having at least
70% identity thereto, wherein the nucleic acid encodes for a polypeptide which
exhibits
reduced protease inhibitor activity compared to a wild type protein. In one
embodiment,
the polypep~id_s exhibits 80% identity and in another embodiment, 90%.
In yet another aspect, the present invention relates to a nucleic acid
encoding a
protease inhibitor protein wherein nucleotides have been substituted to
increase the
number of essential amino acids in the encoded protein. In one embodiment, the
inhibitor
protein is derived from a plant. In another emb~~diment, the inhibitor protein
is a
chymotrypsin inhibitor- like protein.
In another aspect, the present invention relates to an expression cassette
comprising the nucleic acid encoding the polypeptide supra, operably linked to
a
promoter providing for protein expression. In some embodiments, the promoter
provides
5
e_v. ~ ..
CA 02270289 1999-04-30
AFplicant Ref. No.: 0571 R-PCT.app
r
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. . . < . < . .
< . ~ . .
for protein expression in plants. In some embodiments, the promoter provides
for protein
expression in bacteria, yeast or virus.
In yet another aspect, the transformed plant containing at least one copy of
the
expression casette supra. In some embodiments, the transformed plant is a
monocotyledonous plant and could be selected from the group consisting of
maize,
sorghum, wheat, rice and barley. In some embodiments, the transformed plant is
a
dicotyledonous plant and could be selected from the group consisting of
soybean, alfalfa,
canola, sunflower, tobacco, tomato and canola. :Preferably, the transformed
plant is maize
or soybeans. In some embodiments seed is produced by the transformed plant. In
some
embodiments an animal feed composition is provided, and in some, the animal
feed
composition is the seed.
In another aspect, the present invention relates to transformed plant cells
containing the expression cassette supra.
In another aspect, the present invention relates to a method for increasing
the
1 ~ nutritional value of a plant comprising introducing into the cells of the
plant the
expression cassette supra to yield transformed plant cells and regenerating a
transformed
plant from the transformed plant cells.
The present invention provides a method for genetically modifying protease
inhibitors to increase the level of at least, but not limited to one,
essential amino acid in a
plant so as to enhance the nutritional value of the plant. The methods
comprise the
introducW'on f an expression cassette into re;~enerable plant cells to yield
transformed
plant cells. The expression cassette comprises a nucleotide encoding a
protease inhibitor
operably linked to a promoter functional in plant cells.
A fertile transgenic plant is regenerated from the transformed cells, and
seeds are
isolated from the plant. The seeds comprise the polypeptide which is encoded
by the
DNA segment and which is produced in an amount sufficient to increase the
amount of
the essential amino acid in the seeds of the transformed plants, relative to
the amount of
the essential amino acid in the seeds of a corresponding untransformed plant,
e.g., the
seeds of a regenerated control plant that is not transformed or corresponding
untransformed seeds isolated from the transformed plant.
6
AMENDED S~tEET
CA 02270289 2000-12-08
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Preferably, the substantiated amino acid is an
essential amino acid. More preferably, tryptophan threonine,
methionine and lysine arE: the substituted essential amino acid.
Even more preferably, the additional essential amino acid is
lysine.
A preferred embodiment of the present invention is
the introduction of an Expression cassette into regenerable
plant cells. Also preferred is the introduction of an
expression cassette comprising a DNA segment encoding an
endogenous or modified polypeptide sequence.
The present invention also encompasses variations in
the sequences described above, wherein such variations are due
to site-directed mutagenesis, or other mechanisms known in the
art, to increase or decrease levels of selected amino acids of
interest. For example, :>ite-directed mutagenesis to increase
levels of essential amino acids is a preferred embodiment.
The present invention also provides a fertile
transgenic plant. The fertile transgenic plant contains an
isolated DNA segment comprising a promoter and encoding a
protein comprising a protease inhibitor, modified by increasing
the number of essential amino acids, under the control of the
promoter. The protease inhibitor is expressed as so that the
level of essential amino acids in the seeds of the transgenic
plant is increased above the level in the seeds of a plant
which only differ from t:he seeds of the transgenic plant in
that the DNA segment or t:he encoded seed protein is under the
control of a different promoter. The DNA segment is
transmitted through a cc>mplete normal sexual cycle of the
transgenic plant to the next generation. The present invention
provides nucleotide sequences encoding proteins containing
higher levels of essential amino acids by the substitution of
7
CA 02270289 2004-07-20
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one or more of the amino acid residues in the protease
inhibitor. Substitutions at one or more of, but not limited
to, positions 1, 8, 11, 17, 19, 34, 41, 56, 59, 62, 67 and
73 of the wild type protein are substituted with essential
amino acids. The present invention also involves the
expression of the present chymotrypsin inhibitor derivatives
or any derived protease inhibitor in plants to provide
higher percentages of essential amino acids in plants than
wild type plants.
In a preferred embodiment of the present
invention, the present derivatives also exhibit reduced
protease inhibitor activity. This is achieved by
substituting the amino acid residues from about amino acid
residue 53 to about amino acid residue 70 with residues
other than the wild type residues.
In one aspect, there is described an isolated
polypeptide comprising a modified variant of SEQ ID N0: 14,
or a modified variant of the sequence from position 19 to
position 83 of SEQ ID N0: 14, wherein the modified variant:
(a) contains a higher percentage of essential amino acids
than either SEQ ID N0: 14 or the sequence from position 19
to position 83 of SEQ ID NO: 14; (b) has greater than 600
amino acid similarity to SEQ ID N0: 14 or the sequence from
position 19 to position 83 of SEQ ID N0: 14, wherein the
percent sequence similarity is based on the entire sequence
and is determined by BLAST 2.0 using default parameters; and
(c) contains an essential amino acid at a position
corresponding to a position of SEQ ID N0: 14 selected from
the group consisting of 1, 8, 17, 19, 34, 41, and 67, or
contains a lysine at a position corresponding to a position
of SEQ ID N0: 14 selected from the group consisting of 56,
59, 62 and 73.
7a
CA 02270289 2004-07-20
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In another aspect, there is described an isolated
polypeptide comprising a modified variant of SEQ ID N0: 14,
or a modified variant of the sequence from position 19 to
position 83 of SEQ ID N0: 14, wherein the modified variant:
(a) contains a higher percentage of essential amino acids
than either SEQ ID N0: 14 or the sequence from position 19
to position 83 of SEQ ID N0: 14; (b) has greater than 600
amino acid similarity to SEQ ID NO: 14 or the sequence from
position 19 to position 83 of SEQ ID N0: 14, wherein the
percent sequence similarity is based on the entire sequence
and is determined by BLAST 2.0 using default parameters; and
(c) is modified at at least 11 positions of SEQ ID N0: 14 to
contain essential amino acids at said at least 11 positions.
In another aspect, there is described an isolated
polypeptide comprising a modified variant of SEQ ID N0: 14,
or a modified variant of the sequence from position 19 to
position 83 of SEQ ID N0: 14, wherein the modified variant:
(a) contains a higher percentage of essential amino acids
than either SEQ ID N0: 14 or the sequence from position 19
to position 83 of SEQ ID NO: 14; (b) has greater than 600
amino acid similarity to SEQ ID N0: 14 or the sequence from
position 19 to position 83 of SEQ ID N0: 14, wherein the
percent sequence similarity is based on the entire sequence
and is determined by BLAST 2.0 using default parameters; and
(c) contains a pair of cysteines at at least one pair of
positions corresponding to SEQ ID N0: 14 positions Glu-23
and Arg-81, Thr-22 and Val-82, or Val-53 and Val-70.
In another aspect, there is described an isolated
polypeptide comprising a modified variant of SEQ ID NO: 14,
or a modified variant of the sequence from position 19 to
position 83 of SEQ ID NO: 14, wherein the modified variant:
(a) contains at least 55~ essential amino acids; (b) has
greater than 60o amino acid similarity to SEQ ID N0: 14 or
7b
CA 02270289 2004-07-20
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the sequence from position 19 to position 83 of SEQ ID
N0: 14, wherein the percent sequence similarity is based on
the entire sequence and is determined by BLAST 2.0 using
default parameters; and (c) contains a pair of cysteines at
at least one pair of positions corresponding to SEQ ID
NO: 14 positions Glu-23 and Arg-81, Thr-22 and Val-82, or
Val-53 and Val-70.
In another aspect, there is described an isolated
nucleic acid encoding the polypeptide of the invention.
In another aspect, there is described a
recombinant expression cassette comprising the nucleic acid
of the invention operably linked to a promoter.
In another aspect, there is described a
transformed plant cell comprising the recombinant expression
cassette of the invention.
In another aspect, there is described an animal
feed composition comprising plant tissue, wherein the plant
tissue comprises the polypeptide of the invention.
In another aspect, there is described a method for
increasing the nutritional value of a plant comprising:
(a) introducing into cells of the plant a recombinant
expression cassette of the invention, wherein the promoter
provides for protein expression in plants, to yield
transformed plant cells, and (b) regenerating a transformed
plant from the transformed plant cells.
In another aspect, there is described use of at
least one recombinant expression cassette of the invention,
wherein the promoter provides for protein expression in
plants, in the preparation of a transformed plant.
7c
CA 02270289 2004-07-20
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In another aspect, there is described use of at
least one recombinant expression cassette of the invention,
wherein the promoter provides for protein expression in
plants, for the preparation of a seed of a transformed
plant.
In another aspect, there is described use of the
plant cell of the invention in the preparation of an animal
feed composition.
7d
CA 02270289 1999-04-30
AFplicant Ref. No.: 0571 R-PCT.app
Methods for expressing the modified protease inhibitors and for using plants
are
also provided to enhance the nutritional value ol~animal feed.
It is therefore an object of the present invention to provide methods for
increasing
the levels of the essential amino acids in the seeds of plants used for animal
feed.
It is a further object of the present invention to provide seeds for food
and/or feed
with higher levels of the essential amino acid, lysine, than wild type species
of the same
seeds.
It is a further object of the present invention to provide seeds for food
and/or feed
such that the level of the essential amino acids is increased such that the
need for feed
supplementation is greatly reduced or obviated.
It is one object of the present invention ro provide nucleic acids encoding
enzymes
involved in protease inhibition and antigenic polypeptide fragments thereof.
It is also an
object of the present invention to provide protease inhibitor polypeptides and
antigenic
fragments thereof. It is a further object of the present invention to provide
transgenic
1 S plants comprising protease inhibitor nucleic acids. Additionally, it is an
object of the
present invention to provide methods for modulating, in a transgenic plant,
the expression
of protease inhibitor polynucleotides of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic
acid
comprising a member selected from the group consisting of (a)a polynucleotide
having at
least 70% identity to a polynucleotide encoding a polypeptide selected from
the group
consisting of SEQ ID NOS: 2,4,6,8,10 and l2,116,18,20,22,24;and (b) a
polynucleotide
which is complementary to the polynucleotide of (a); and (c) a polynucleotide
comprising
at least 3~-~tiguous nucleotides from a polyrmcleotide of (a) or (b). In some
embodiments, the polynucleotide has a sequence selected from the group
consisting of
SEQ ID NOS: 1,3,5,7,9 and 11, 15,17,19,21, or 23 . The isolated nucleic acid
can be
DNA.
In another aspect, the present invention relates to recombinant expression
cassettes, comprising a nucleic acid as describf:d, supra, operably linked to
a promoter.
In some embodiments, the nucleic acid is operably linked in antisense
orientation to the
promoter.
In another aspect, the present invention is directed to a host cell
transfected with
the recombinant expression cassette as described, supra. In some embodiments,
the host
8
~1,~L'~J"r~': ~'i ~C.'
J _ .:, i:_~
Applicant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
cell is a maize, rye, barley, wheat, sorghum, oa~a, millet, rice, triticale,
sunflower, alfalfa,
rapeseed or soybean cell.
In a further aspect, the present invention relates to an isolated protein
comprising a
polypeptide of at least 10 contiguous amino acids encoded by the isolated
nucleic acid
referred to, supra. In some embodiments, the polypeptide has a sequence
selected from
the group consisting of SEQ ID NOS: 2,4,6,8,10 and 12,16,18,20,22,24.
In another aspect, the present invention relates to an isolated nucleic acid
comprising a polynucleotide of at least 30 nuclE;otides in length which
selectively
hybridizes under stringent conditions to a nucleic acid selected from the
group consisting
of SEQ ID NOS: 1,3,5,7,9 and 11, 15,17,19,21, 23 or a complement thereof. In
some
embodiments, the isolated nucleic acid is operably linked to a promoter.
In yet another aspect, the present invention relates to an isolated nucleic
acid
comprising a polynucleotide, the polynucleotide having at least 60% sequence
identity to
an identical length of a nucleic acid selected from the group consisting of
SEQ ID NOS:
1,3,5,7,9 and 1 l, 15,17,19,21, 23 or a complement thereof.
In another aspect, the present invention :relates to an isolated nucleic acid
comprising a polynucleotide having a sequence of a nucleic acid amplified from
a Zea
mays nucleic acid library using the primers selected from the group consisting
of: SEQ ID
NOS: 25 and 26 or complements thereof. In some embodiments, the nucleic acid
library
is a cDNA library.
In another aspect, the present invention :relates to a recombinant expression
cassette comprising a nucleic acid amplified from a library as referred to
szrpra, wherein
the nucleicd is operably linked to a promoter. In some embodiments, the
present
invention relates to a host cell transfected with l:his recombinant expression
cassette In
some embodiments, the present invention relates to a protease inhibitor
protein produced
from this host cell.
In a further aspect, the present invention relates to a heterologous promoter
operably linked to a non-isolated protease inhibitor polynucleotide encoding a
polypeptide, wherein the polypeptide is encoded by a nucleic acid amplified
from a
nucleic acid library as referred to, supra.
In yet another aspect, the present invention relates to a transgenic plant
comprising
a recombinant expression cassette comprising a plant promoter operably linked
to any of
9
: ' r,-
CA 02270289 2002-O1-28
75529-49(S)
the isolated nucleic acids referred to supra. In some
embodiments, the transgenic plant is Zea mays. The present
invention also provides transgenic seed from the transgenic
plant.
In a further aspect, the present invention relates
to a method of providing a modified protease inhibitor in a
plant, comprising the steps of (a) transforming a plant cell
with a recombinant expression casette comprising a protease
inhibitor polynucleotide operably linked to a promoter; (b)
growing the plant cell under plant growing conditions; and
(c) inducing expression of the polynucleotide.
Applicant Ref. No.: 05718-PCT.app
F~,ure IistinE
Figure 1 Protease Inhibition
Sequence identification
CA 02270289 1999-04-30
DETAILED DESCRIPTION
Barley High Lysine 1(BHL-1) is coded for by the polypeptides of SEQ ID
No. 2 which is encoded for by the nucleic acid of SEQ ID No. 1.
Barley High Lysine 2 (BHL-2) its coded for by the polypeptides of SEQ
ID No. 4 which is encoded for by the nucleic acid of SEQ ID No. 3.
Barley High Lysine 3 (BHL-3) i;s coded for by the polypeptides of SEQ ID
No. 6 which is encoded for by the nucleic acid of SEQ ID No. 5.
Barley High Lysine 3N (BHL-3N) is coded for by the polypeptides of SEQ
ID No. 8 which is encoded for by the nucleic acid of SEQ ID No. 7.
Barley High Lysine 1N (BHL-1N) is coded for by the polypeptides of SEQ
1 ~ ID No. 10 which is encoded for by the nucleic acid of SEQ ID No. 9.
Barley High Lysine 2N (BHL-2N) is coded for by the polypeptides of SEQ
ID No. 12 which is encoded for by the nucleic acid of SEQ ID No. 11.
Wild-type chymotrypsin inhibitor (WI-CI-2) is coded for by the
polypeptides of SEQ ID No. 14 which is encoded for by the nucleic acid of SEQ
ID No. 13.
Maize EST PI-1 is coded for by the polypeptides of SEQ ID No.l6 which
is encoded for by the nucleic acid of SEQ ID No. 15.
Maize EST PI-2 is coded for by the polypeptides of SEQ ID No.18 which
is encoded for by the nucleic acid of SEQ ID No. 17.
Maize EST PI-3 is coded for by the polypeptides of SEQ ID No.20 which
is encoded for by the nucleic acid of SEQ ID No. 19.
Maize EST PI-4 is coded for by the polypeptides of SEQ ID No.22 which
is encoded for by the nucleic acid of SEQ ID No. 21.
Maize EST PI-Sis coded for by t:he polypeptides of SEQ ID No. 24 which
is encoded for by the nucleic acid of SEQ ID No. 23.
The 5' and 3' PCR primer pairs A & B, are identified as SEQ ID Nos. 25
and 26, respectively.
11 AMENDED SHEET
Applicant Ref. No.: 05718-PCT.app
Definitions
CA 02270289 1999-04-30
Units, prefixes, and symbols may be denoted in their SI accepted form. Unless
otherwise indicated, nucleic acids are written left to right in 5' to 3'
orientation; amino
acid sequences are written left to right in amino to carboxy orientation,
respectively.
Numeric ranges are inclusive of the numbers defining the range. Amino acids
may be
referred to herein by either their commonly known three letter symbols or by
the one-
letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature
Commission. Nucleotides, likewise, may be referred to by their commonly
accepted
single-letter codes. The terms defined below are more fully defined by
reference to the
specification as a whole.
"Chymotrypsin inhibitor-like" protein is a protein with a sequence identity of
40%
or more to the CI-2 from barley.
"%" refers to molar % unless otherwise specified or implied.
"Essential amino acids" are amino acids that must be obtained from an external
source because they are not synthesized by the individual. They are comprised
of:
methionine, threonine, lysine, isoleucine, leucine, valine, tryptophan,
phenylalanine, and
histidine.
By "amplified" is meant the construction of multiple copies of a nucleic
acid sequence or multiple copies complementar)~ to the nucleic acid sequence
using at
least one of the nucleic acid sequences as a template. Amplification systems
include the
polymerase~ain reaction (PCR) system, ligase chain reaction (LCR) system,
nucleic
acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-
Beta
Replicase systems, transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., Dicxgnostic Molecular
Microbiology:
Principles and Applications, D. H. Persing et al., Ed., American Society for
Microbiology, Washington, D.C. (1993).
As used herein, "antisense orientation" includes reference to a duplex
polynucleotide sequence which is operably linked to a promoter in an
orientation where
12 l'e~~EPJCeB S;-~EE
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CA 02270289 1999-04-30
the antisense strand is transcribed. The antisense strand is sufficiently
complementary to
an endogenous transcription product such that translation of the endogenous
transcription
product is often inhibited.
As used herein, "chromosomal region" includes reference to a length of
chromosome which may be measured by reference to the linear segment of DNA
which it
comprises. The chromosomal region can be defined by reference to two unique
DNA
sequences, i.e., markers.
The term "conservatively modified variants" applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid sequences,
conservatively
modified variants refers to those nucleic acids which encode identical or
essentially
identical amino acid sequences, or where the nucleic acid does not encode an
amino acid
sequence, to essentially identical sequences. Because of the degeneracy of the
genetic
code, a large number of functionally identical nucleic acids encode any given
protein. For
instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon, the codon
can be altered
to any of the corresponding codons described without altering the encoded
polypeptide.
Such nucleic acid variations are "silent variations" and represent one species
of
conservatively modified variation. Every nucleic acid sequence herein which
encodes a
polypeptide also describes every possible silent variation of the nucleic
acid. One of
ordinary skill will recognize that each codon in a nucleic acid (except AUG,
which is
ordinarily the only codon for methionine, and TGG, which is ordinarily the
only codon
for tryptophan) can be modified to yield a functionally identical molecule.
Accordingly,
each sileiation of a nucleic acid which encodes a polypeptide of the present
invention is implicit in each described polypeptide sequence and incorporated
herein by
reference.
As to amino acid sequences, one of skill will recognize that individual
substitutions, deletions or additions to a nucleic acid, peptide, polypeptide,
or protein
sequence which alters, adds or deletes a single amino acid or a small
percentage of amino
acids in the encoded sequence is a "conservatimely modified variant" where the
alteration
results in the substitution of an amino acid with a chemically similar amino
acid. Thus,
any number of amino acid residues selected from the group of integers
consisting of from
1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10
alterations can be
13
:.-: ,~ ~ ~.y ~;
f. _ ~ . a
.:,U:..., ~.W :~ t:.
CA 02270289 1999-04-30
Applicant Ref. No.: 0571 R-PCT.app
made. Conservatively modified variants typically provide similar biological
activity as
the unmodified polypeptide sequence from which they are derived. For example,
substrate specificity, enzyme activity, or ligancUreceptor binding is
generally at least 30%,
40%, 50%, 60%, 70%, 80%, or 90% of the native protein for it's native
substrate.
Conservative substitution tables providing functionally similar amino acids
are well
known in the art.
The following six groups each contain s~mino acids that are conservative
substitutions for one another:
1 ) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton ( 1984) Proteins W.H. Freeman and Company.
By "encoding" or "encoded", with respect to a specified nucleic acid, is meant
comprising the information for translation into the specified protein. A
nucleic acid
encoding a protein may comprise non-translated sequence (e.g., introns) within
translated
regions of the nucleic acid, or may lack such intervening non-translated
sequences (e.g.,
as in cDNA). The information by which a protein is encoded is specified by the
use of
codons. Typically, the amino acid sequence is encoded by the nucleic acid
using the
"universal" genetic code. However, variants of the universal code, such as is
present in
some plant=aiiimal, and fungal mitochondria, the bacterium Mycoplasma
capricolz~m
(Proc. Natl. Acad. Sci. (USA), 82: 2306-2309 (1985)), or the ciliate
Macronucleus, may
be used when the nucleic acid is expressed using these organisms.
When the nucleic acid is prepared or altered synthetically, advantage can be
taken
of known codon preferences of the intended host where the nucleic acid is to
be
expressed. For example, although nucleic acid sequences of the present
invention may be
expressed in both monocotyledonous and dicot;yledonous plant species,
sequences can be
modified to account for the specific codon pref°rences and GC content
preferences of
monocotyledons or dicotyledons as these preferences have been shown to differ
(Murray
et al. Nucl. Acids Res. 17: 477-498 (1989)). Tllus, the maize preferred codon
for a
14 ' . _.' '-'; z~
~.. r
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CA 02270289 1999-04-30
particular amino acid may be derived from known gene sequences from maize.
Maize
codon usage for 28 genes from maize plants are listed in Table 4 of Murray et
al., szrpra.
As used herein "full-length sequence" includes reference to a protease
inhibitor
polynucleotide or the encoded protein having t:he entire amino acid sequence
of, a native
(non-synthetic), endogenous, catalytically active form of a protein involved
in protease
inhibition. A full-length sequence can be determined by size comparison
relative to a
control which is a native (non-synthetic) endogenous cellular protease
inhibitor nucleic
acid or protein. Methods to determine whether a sequence is full-length are
well known
in the art including such exemplary techniques as northern or western blots.
See, e.g.,
Plant Moleczrlar Biology: A Laboratory Manzrczl, Clark, Ed., Springer-Verlag,
Berlin
(1997). Comparison to known full-length homologous sequences can also be used
to
identify full-length sequences of the present invention. Additionally,
consensus
sequences typically present at the 5' and 3' unt:ranslated regions of mRNA aid
in the
identification of a polynucleotide as full-length. For example, the consensus
sequence
ANNNNAUGG, where the underlined codon represents the N-terminal methionine,
aids
in determining whether the polynucleotide has a complete 5' end. Consensus
sequences
at the 3' end, such as polyadenylation sequences, aid in determining whether
the
polynucleotide has a complete 3' end.
As used herein, "heterologous" in reference to a nucleic acid is a nucleic
acid that
originates from a foreign species, or, if from thc: same species, is
substantially modified
from its native form in composition and/or genomic locus. For example, a
promoter
operably linked to a heterologous structural gene is from a species different
from that
from whiE structural gene was derived, or, if from the same species, one or
both are
substantially modified from their original form. A heterologous protein may
originate
from a foreign species or, if from the same species, is substantially modified
from its
original form.
By "host cell" is meant a cell which contains a vector and supports the
replication
and/or expression of the expression vector. Host cells may be prokaryotic
cells such as E.
coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian
cells. Preferably,
host cells are monocotyledonous or dicotyledenous plant cells. A particularly
preferred
monocotyledonous host cell is a maize host cell.
Applicant Ref. No.: 0>71R-PCT.app
CA 02270289 1999-04-30
The term "hybridization complex" includes reference to a duplex nucleic acid
sequence formed by two single-stranded nucleic acid sequences which
selectively
hybridize with each other.
The terms "isolated" or "biologically pure" refer to material which is: (1)
substantially or essentially free from components which normally accompany or
interact
with it as found in its naturally occurring environment. The isolated material
optionally
comprises material not found with the material in its natural environment. (2)
If the
material is in its natural environment, the material has been synthetically
(non-naturally)
altered to a composition and/or placed at a loccls in the cell (e.g., genome)
not native to a
material found in that environment. The alteration to yield the synthetic
material can be
performed on the material within or removed from its natural state. For
example, a
naturally occurring nucleic acid becomes an isolated nucleic acid if it is
altered, or if it is
transcribed from DNA which is altered, by non-natural, synthetic (i.e., "man-
made")
methods performed within the cell from which it originates. See, e.g.,
Compounds and
Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Patent
No.
5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling
et al.,
PCT/LJS93/03868. Likewise, a naturally occurring nucleic acid (e.g., a
promoter) become
isolated if it is introduced by non-naturally occurring means to a locus of
the genome not
native to that nucleic acid.
The term "protease inhibitor nucleic acids" means an isolated nucleic acid
comprising a polynucleotide (a "protease inhibitor polynucleotide") encoding a
polypeptide involved in protease inhibition.
As-t»d herein, "localized within the chromosomal region defined by and
including" with respect to particular markers includes reference to a
contiguous length of
a chromosome delimited by and including the stated markers.
As used herein, "marker" includes referE:nce to a locus on a chromosome that
serves to identify a unique position on the chromosome. A "polymorphic marker"
includes reference to a marker which appears in multiple forms (alleles) such
that
different forms of the marker, when they are preaent in a homologous pair,
allow
transmission of each of the chromosomes in that pair to be followed. A
genotype may be
defined by use of a single or a plurality of markers.
16 ,~MFNDED ~:r'=1
CA 02270289 2004-07-20
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As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form, and unless
otherwise
limited, encompasses known analogues of natural nucleotides that hybridize to
single-
stranded nucleic acids in a manner similar to naturally occurring nucleotides
{e.g., peptide
nucleic acids).
By "nucleic acid library" is meant a collection of isolated DNA or RNA
molecules
which comprise and substantially represent the entire transcribed fraction of
a genome of
a specified organism. Construction of exemplary nucleic acid libraries, such
as genomic
and cDNA libraries, is taught in standard molecular biology references such as
Berger and
Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol.
152,
Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular
Cloning - A
Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in
Molecular
Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between
Greene
Publishing Associates, Inc, and John Wiley & Sons, Inc. (1994 Supplement).
I S As used herein "operably linked" includes reference to a functional
linkage
between a promoter and a second sequence, wherein the promoter sequence
initiates'and
mediates transcription of the DNA 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.
As used herein, the term "plant" includes reference to whole plants, plant
organs
(e.g., leaves, stems, roots, etc.), seeds and progeny of same. The class of
plants which can be
used in the methods of the invention is generally as broad as the class of
higher plants
amenable to transformation techniques, including both monocotyledonous and
dicotyledonous plants. Particularly preferred is Zea mays.
As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide,
ribopolynucleotide, or analogs thereof, that hybridize to nucleic acids in a
manner similar
to naturally occurring nucleotides. A polynucleotide can be full-length or a
sub-sequence
of a native or heterologous structural or regulatory gene. Unless otherwise
indicated, the
17
Applicant Ref. No.: 0571 R-PCT.app
CA 02270289 1999-04-30
term includes reference to the specified sequence as well as the complementary
sequence
thereof. Thus, DNAs or RNAs with backbones modified for stability or for other
reasons are
"polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs
comprising
unusual bases, such as inosine, or modified bases, such as tritylated bases,
to name just two
examples, are polynucleotidesas the term is used herein. It will be
appreciated that a great
variety of modifications have been made to DNE~ and RNA that serve many useful
purposes
known to those of skill in the art. The term polynucleotide as it is employed
herein
embraces such chemically, enzymaticallyor metabolicallymodified forms of
polynucleotides, as well as the chemical forms of DNA and RNA characteristic
of viruses
and cells, including inter alia, simple and complex cells.
The terms "polypeptide", "peptide" and "protein" are used interchangeably
herein
to refer to a polymer of amino acid residues. Tlhe terms apply to amino acid
polymers in
which one or more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to naturally
occurring amino
acid polymers. Among the known modifications which may be present in
polypeptides of
the present are, to name an illustrative few, acetylation, acylation, ADP-
ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a heme
moiety, covalent
attachment of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid
derivative, covalent attachment of phosphotidylinositol,cross-linking,
cyclization, disulfide
bond formation, demethylation, formation of covalent cross-links, formation of
cystine,
formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation,
GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic
processing,osphorylation,prenylation, racemization, selenoylation, sulfation,
transfer-
RNA mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
Such modifications are well known to those of skill and have been described in
great detail
in the scientific literature. Several particularly common modifications,
glycosylation, lipid
attachment, sulfation, gamma-carboxylationof g lutamic acid residues,
hydroxylation and
ADP-ribosylation, for instance, are described in most basic texts, such as,
for instance
Proteins - Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H.
Freeman
and Company, New York ( 1993). Many detailed reviews are available on this
subject, such
as, for example, those provided by Wold, F., PosttranslationalProtein
Modifications:
Perspectives and Prospects, pp. 1-12 in Posttranslational Covalent
Modification of Proteins,
18 ..,.'~~,;i;; :~-::~~;
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CA 02270289 1999-04-30
:.
.. . . . . . ~. . . , , ,
, . ,
. . ,' , . ,
,. ,. ' . ":. , ,.
B. C. Johnson, Ed., Academic Press, New York ( 1983); Seifter et al., Meth.
Enz-ymol. 182:
626-646 ( I 990) and Rattan et al., Protein Synthesis: Posttranslational
Modifications and
Aging, Ann. N.Y. Acad. Sci. 663: 48-62 ( 1992). It will be appreciated, as is
well known and
as noted above, that polypeptides are not always. entirely linear. For
instance, polypeptides
may be branched as a result of ubiquitination, arid they may be circular, with
or without
branching, generally as a result of posttranslation events, including natural
processing event
and events brought about by human manipulation which do not occur naturally.
Circular,
branched and branched circular polypeptides ma.y be synthesized by non-
translation natural
process and by entirely synthetic methods, as well. Modifications can occur
anywhere in a
polypeptide, including the peptide backbone, the: amino acid side-chains and
the amino or
carboxyl termini. In fact, blockage of the amino or carboxyl group in a
polypeptide, or both,
by a covalent modification, is common in naturally occurring and synthetic
polypeptides
and such modifications may be present in polypeptides of the present
invention, as well.
For instance, the amino terminal residue of polypeptides made in E. coli or
other cells, prior
to proteolytic processing, almost invariably will be N-formylmethionine.
During post-
translational modification of the peptide, a metlzionine residue at the NHZ-
terminus may
be deleted. Accordingly, this invention contemplates the use of both the
methionine-
containing and the methionineless amino terminal variants of the protein of
the invention.
In general, as used herein, the term polypeptide Encompasses all such
modifications,
particularly those that are present in polypeptides synthesized by expressing
a
polynucleotide in a host cell.
As used herein "promoter" includes reference to a region of DNA upstream from
the start o~a~scription and involved in recognition and binding of RNA
polymerase and
other proteins to initiate transcription. A "plans: promoter" is a promoter
capable of
initiating transcription in plant cells. Examples. of promoters under
developmental control
include promoters that preferentially initiate transcription in certain
tissues, such as
leaves, roots, seeds, fibers, xylem vessels, trach.eids, or sclerenchyma. Such
promoters
are referred to as "tissue preferred". Promoters which initiate transcription
only in certain
tissue are referred to as "tissue specific". A "cell type" specific promoter
is primarily
drives expression in certain cell types in one or more organs, for example,
vascular cells
in roots or leaves. An "inducible" promoter is a promoter which is under
environmental
control. Examples of environmental conditions. that may effect transcription
by inducible
19
Applicant Ref. No.: 0~71R-PCT. app
CA 02270289 1999-04-30
~: ..
. . . . . . r ~ :.
.
r
.
:.
promoters include anaerobic conditions or the presence of light. Tissue
specific, cell type
specific, and inducible promoters constitute th~~ class of "non-constitutive"
promoters. A
"constitutive" promoter is a promoter which is active under most environmental
conditions.
The terms "polypeptide involved in protease inhibition" or "protease inhibitor
polypeptide" refer to one or more proteins, in ~;lycosylated or non-
glycosylated form,
acting as a protease inhibitor. Examples are included as, but not limited to:
chymotrypsin
inhibitor, trypsin inhibitor, protease inhibitor, pre-pro-proteinase inhibitor
I, subtilisin-
chymotrypsin inhibitor, tumor-related protein, genetic tumor-related
proteinase inhibitor,
subtilisin inhibitor, endopeptidase inhibitor, serine protease inhibitor,
wound-inducible
proteinase inhibitor, and eglin c. The term is also inclusive of fragments,
variants,
homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof.
A "protease
inhibitor protein" comprises a protease inhibitor polypeptide.
As used herein "recombinant" includes reference to a cell, or nucleic acid, or
1 S vector, that has been modified by the introduction of a heterologous
nucleic acid or the
alteration or placement of a native nucleic acid to a form or to a locus not
native to that
cell, or that the cell is derived from a cell so modified. Thus, for example,
recombinant
cells express genes that are not found in identical form within the native
(non-
recombinant) form of the cell or express native genes that are otherwise
abnormally
expressed, under expressed or not expressed at all. The term "recombinant" as
used
herein does not encompass the alteration of the cell, nucleic acid or vector
by naturally
occurring events (e.g., spontaneous mutation, n~~tural
transformati~ftransduction/transposition) such as those occurring without
direct human
intervention.
As used herein, a "recombinant expression cassette" is a nucleic acid
construct,
generated recombinantly or synthetically, with a series of specified nucleic
acid elements
which permit transcription of a particular nucleic acid in a target cell. The
recombinant
expression cassette can be incorporated into a pllasmid, chromosome,
mitochondria) DNA,
plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant
expression
cassette portion of the expression vector includes, among other sequences, a
nucleic acid
to be transcribed, and a promoter.
APJIENDED SHEET
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PC'r.app
' . , _ . . . ~ . ~ . ' . . .
. . ' ~ .
., .. ." ."' ..
The term "residue" or "amino acid residue" or "amino acid" are used
interchangeably herein to refer to an amino acid that is incorporated into a
protein,
polypeptide, or peptide (collectively "protein"). The amino acid may be a
naturally
occurring amino acid and, unless otherwise limited, may encompass known
analogs of
natural amino acids that can function in a simil'.ar manner as naturally
occurring amino
acids.
The term "selectively hybridizes" includes reference to hybridization, under
stringent hybridization conditions, of a nucleic acid sequence to a specified
nucleic acid
target sequence to a detectably greater degree (e.g., at least 2-fold over
background) than
its hybridization to non-target nucleic acid sequences and to the substantial
exclusion of
non-target nucleic acids. Selectively hybridizing sequences typically have
about at least
80% sequence identity, preferably 90% sequence identity, and most preferably
100%
sequence identity (i.e., complementary) with each other.
The terms "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe will
hybridize to its
target sequence, to a detectably greater degree than other sequences (e.g., at
least 2-fold
over background). Stringent conditions are sequence-dependent and will be
different in
different circumstances. Longer sequences hybridize specifically at higher
temperatures.
Generally, stringent conditions are selected to be about 5 °C lower
than the thermal
melting point (Tm) for the specific sequence at a defined ionic strength and
pH. The Tm is
the temperature (under defined ionic strength and pH) at which 50% of a
complementary
target seque~e hybridizes to a perfectly matched probe. Typically, stringent
conditions
will be those in which the salt concentration is less than about 1.0 M Na ion,
typically
about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and
the
temperature is at least about 30°C for short probes (e.g., 10 to 50
nucleotides) and at least
about 60°C for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may
also be achieved with the addition of destabilizing agents such as formamide.
Exemplary
low stringency conditions include hybridization with a buffer solution of 30%
formamide,
1 M NaCI, 1% SDS at 37°C, and a wash in 2X SSC at 50°C.
Exemplary high stringency
conditions include hybridization in 50% formamide, 1 M NaCI, 1 % SDS at
37°C, and a
wash in O.1X SSC at 60°C.
21
ja A~ ~
r f ~Ff fJCLI Ci ~"~.~
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
., . " ,
. .. . . . . .~ . . . , .
. . ..
. . . . . . .
.. ~. ... .... ,.
Stringent hybridization conditions in the context of nucleic acid
hybridization
assay formats are sequence dependent, and are different under different
environmental
parameters. Longer sequences hybridize selectively at higher temperatures. An
extensive
guide to the hybridization of nucleic acids is found in Tijssen, Laboratory
Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes,
Part I,
Chapter 2 "Overview of principles of hybridization and the strategy of nucleic
acid probe
assays", Elsevier, New York (1993).
The terms "transfection" or "transformation" include reference to the
introduction
of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid
may be
incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid
or
mitochondria) DNA), converted into an autonomous replicon, or transiently
expressed
(e.g., transfected mRNA).
As used herein, "transgenic plant" includes reference to a plant which
comprises
within its genome a heterologous polynucleotide. Generally, the heterologous
polynucleotide is stably integrated within the genome such that the
polynucleotide is
passed on to successive generations. The heterologous polynucleotide may be
integrated
into the genome alone or as part of a recombinant expression cassette.
"Transgenic" is
used herein to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of
which has been altered by the presence of heterologous nucleic acid including
those
transgenics initially so altered as well as those created by sexual crosses or
asexual
propagation from the initial transgenic. The term "transgenic" as used herein
does not
encompass the
alteration~e genome (chromosomal or extra-chromosomal) by conventional plant
breeding methods or by naturally occurring events such as random cross-
fertilization,
non-recombinant viral infection, non-recombinant bacterial transformation, non
recombinant transposition, or spontaneous mutation.
As used herein, "vector" includes reference to a nucleic acid used in
transfection
of a host cell and into which can be inserted a polynucleotide. Vectors are
often
replicons. Expression vectors permit transcription of a nucleic acid inserted
therein.
The following terms are used to describe the sequence relationships between
two
or more nucleic acids or polynucleotides: (a) "reference sequence", (b)
"comparison
22
"'~ ~rG=_ , ,....,
CA 02270289 2000-04-20
75529-49(S)
window", (c) "sequence identity", (d) "percentage of sequence identity", and
(e)
"substantial identity".
(a) As used herein, "reference sequence" is a defined sequence used
as a basis for sequence comparison. A reference sequence may be a subset or
the entirety
of a specified sequence; for example, as a segment of a full-length cDNA or
gene
sequence, or the complete ~cDNA or gene sequence.
(b) As used herein, "comparison window" means includes
reference to a contiguous and specified segment of a polynucleotide sequence,
wherein
the polynucleotide sequence may be compared to a reference sequence and
wherein the
portion of the polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) compared to the reference sequence (which
does not
comprise additions or deletions) for optimal alignment of the two sequences.
Generally,
the comparison window is at least 20 contiguous nucleotides in length, and
optionally can
be 30, 40, 50, 100, or longer. Those of skill in the art understand that to
avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide sequence
a gap penalty is typically introduced and is subtracted from the number of
matches.
Methods of alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison may be conducted by the local
homology
algorithm of Smith and Waterman, Adv. Appl. Math 2: 482 (1981); by the
homology
alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by
the
search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:
2444
(1988); by computerized implementations of these algorithms, including, but
not limited
to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,
California,
GAP, BESTFIT, BLAST" FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin,
USA;
the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244
(1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic
Acids
Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the
Biosciences
8: 15~-65 (1992), and Pearson, et al., Methods in Molecular Biolosy 24: 307-
331 (1994);
preferred computer alignment methods also include the BLASTP, BLASTN, and
BLASTX algorithms. Altschul, et al., J. Mol. Biol. 215: 403-410 (1990).
Alignment is
also often performed by inspection and manual alignment.
Trade-mark 23
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Applicant Ref. No.: 05718-PCT.app
' . ,. . , . . ,~ , ~, , . .
. . , ". . . , , , ,
,. .. .., . "'
(c) As used herein, "sequence identity" or "identity" in the context
of two nucleic acid or polypeptide sequences includes reference to the
residues in the two
sequences which are the same when aligned fo:r maximum correspondence over a
specified comparison window. When percentage of sequence identity is used in
reference
to proteins it is recognized that residue positions which are not identical
often differ by
conservative amino acid substitutions, where amino acid residues are
substituted for other
amino acid residues with similar chemical properties (e.g. charge or
hydrophobicity) and
therefore do not change the functional propertifa of the molecule. Where
sequences differ
in conservative substitutions, the percent sequence identity may be adjusted
upwards to
correct for the conservative nature of the substitution. Sequences which
differ by such
conservative substitutions are said to have "sequence similarity" or
"similarity". Means
for making this adjustment are well-known to those of skill in the art.
Typically this
involves scoring a conservative substitution as a partial rather than a full
mismatch,
thereby increasing the percentage sequence identity. Thus, for example, where
an
identical amino acid is given a score of 1 and a non-conservative substitution
is given a
score of zero, a conservative substitution is given a score between zero and
1. The
scoring of conservative substitutions is calculated, e.g., according to the
algorithm of
Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., as
implemented in
the program PC/GENE (Intelligenetics, Mountain View, California, USA).
(d) As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over a
comparison
window, wherein the portion of the polynucleotide sequence in the comparison
window
may corrrpr~'s~ additions or deletions (i.e., gaps) as compared to the
reference sequence
(which does not comprise additions or deletion~~) for optimal alignment of the
two
sequences. The percentage is calculated by determining the number of positions
at which
the identical nucleic acid base or amino acid residue occurs in both sequences
to yield the
number of matched positions, dividing the number of matched positions by the
total
number of positions in the window of comparison and multiplying the result by
100 to
yield the percentage of sequence identity.
(e) (i) The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has at least 70%
sequence identity,
preferably at least 80%, more preferably at lease: 90% and most preferably at
least 95%,
24
A~,~ENDED SlIEET
Applicant Ref. No.: 0~71R-PCT.app
CA 02270289 1999-04-30
compared to a reference sequence using one of the alignment programs described
using
standard parameters. One of skill will recognize that these values can be
appropriately
adjusted to determine corresponding identity of proteins encoded by two
nucleotide
sequences by taking into account codon degeneracy, amino acid similarity,
reading frame
positioning and the like. Substantial identity of amino acid sequences for
these purposes
normally means sequence identity of at least 60'%, more preferably at least
70%, 80%,
90%, and most preferably at least 95%. Polypeptides which are "substantially
similar"
share sequences as noted above except that residue positions which are not
identical may
differ by conservative amino acid changes.
Another indication that nucleotide sequences are substantially identical is if
two
molecules hybridize to each other under stringent conditions. Generally,
stringent
conditions are selected to be about 5°C to about 20°C lower than
the thermal melting
point (Tm) for the specific sequence at a defined ionic strength and pH. The
T,n is the
temperature (under defined ionic strength and pH) at which 50% of the target
sequence
hybridizes to a perfectly matched probe. Typically, stringent wash conditions
are those in
which the salt concentration is about 0.02 molar at pH 7 and the temperature
is at least
about 50, 55, or 60°C. However, nucleic acids which do not hybridize to
each other under
stringent conditions are still substantially identical if the polypeptides
which they encode
are substantially identical. This may occur, e.g., when a copy of a nucleic
acid is created
using the maximum codon degeneracy permitted by the genetic code. One
indication that
two nucleic acid sequences are substantially identical is that the polypeptide
which the
first nucleic acid encodes is immunologically cross reactive with the
polypeptide encoded
by the sec~ucleic acid.
(e) (ii) The terms "substantial identity" in the context of a peptide
indicates
that a peptide comprises a sequence with at least 70% sequence identity to a
reference
sequence, preferably 80%, more preferably 85'%, most preferably at least 90%
or 95%
sequence identity to the reference sequence over a specified comparison
window.
Preferably, optimal alignment is conducted using the homology alignment
algorithm of
Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970). An indication that two
peptide
sequences are substantially identical is that one peptide is immunologically
reactive with
antibodies raised against the second peptide. Thus, a peptide is substantially
identical to a
r;'J!Er;r~~D SHEET
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
second peptide, for example, where the two peptides differ only by a
conservative
substitution.
S It has been unexpectedly discovered that a protease inhibitor can be
modified to
enhance its content of essential amino acids coupled with reduction in protese
inhibitor
activity. In a preferred embodiment of the present invention, derivatives of
the protease
inhibitor, CI-2, simultaneously exhibit both enhanced essential amino acid
content as well
as decreased protease inhibitor activity. The present compounds are thus
excellent
candidates for enhancing the nutritional value of feed.
The present invention provides, inter aha, compositions and methods for
modulating (i.e., increasing or decreasing) the total levels of essential
amino acids and/or
altering the ratios of essential amino acids in plants. Thus, the present
invention provides
utility in such exemplary applications as improving the nutritional properties
of fodder
crops, increasing the value of plant material for pulp and paper production,
altering the
protease inhibitory activity, as well as for improving the utility of plant
material where the
amount of essential amino acids or composition is important, such as the use
of plant as a
feed. In particular, protease inhibitor polypeptides may be expressed at times
or in
quantities which are not characteristic of natural plants.
The present invention also provides isolated nucleic acid comprising
polynucleotides of sufficient length and complementarity to a protease
inhibitor gene, to
use as probes or amplification primers in the detection, quantitation, or
isolation of gene
transcripts~r example, isolated nucleic acids of the present invention can be
used as
probes in detecting deficiencies in the level of mRNA in screenings for
desired transgenic
plants, for detecting mutations in the gene (e.g., substitutions, deletions,
or additions), for
monitoring upregulation of protease inhibition in screening assays for
compounds
affecting protease inhibition, or for use as molecular markers in plant
breeding programs.
The isolated nucleic acids of the present invention can also be used for
recombinant
expression of protease inhibitor polypeptides for use as immunogens in the
preparation
and/or screening of antibodies. The isolated nucleic acids of the present
invention can
also be employed for use in sense or antisense suppression of one or more
protease
inhibitor genes in a host cell, tissue, or plant. Further, using a primer
specific to an
26
. ,.,.- , ~t.~ -r
.. ~~ r
Applicant Ref. No.: 0571 R-PC1'.app
CA 02270289 1999-04-30
insertion sequence (e.g., transposon) and a primer which specifically
hybridizes to an
isolated nucleic acid of the present invention, one can use nucleic acid
amplification to
identity insertion sequence inactivated protease: inhibitor genes from a cDNA
library
prepared from insertion sequence mutagenized plants. Progeny seed from the
plants
S comprising the desired inactivated gene can be grown to a plant to study the
phenotypic
changes characteristic of that inactivation. See, Tools to Determine the
Function of Genes,
1995 Proceedings of the Fiftieth Annual Corn and Sorghum Industry Research
Conference, American Seed Trade Association., Washington, D.C., 1995.
The present invention also provides isolated proteins comprising polypeptides
having a minimal amino acid sequence from the polypeptides involved in
protease
inhibition as disclosed herein. The present invention also provides proteins
comprising at
least one epitope from a polypeptide involved in protease inhibition. The
proteins of the
present invention can be employed in assays fo:r enzyme agonists or
antagonists of
enzyme function, or for use as immunogens or antigens to obtain antibodies
specifically
immunoreactive with a protein of the present invention. Such antibodies can be
used in
assays for expression levels, for identifying andJor isolating nucleic acids
of the present
invention from expression libraries, or for purification of polypeptides
involved in
protease inhibition. In a preferred embodiment ~of the present invention, the
present
protein has both elevated essential amino acid content and reduced protease
inhibitor
activity.
The isolated nucleic acids of the present invention can be used over a broad
range
of plant types, including species from the genera Ctrcttrbita, Rosa, Vitis,
Juglans,
Fragariar.ls, Medicago, Onobrychis, Trifoli'um, Trigonella, Vigna, Citrus,
Linttm,
Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,
Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,
Digitalis,
Majorana, Ciahorium, Helianthus, Lactuca, Br~omus, Asparagus, Antirrhinum,
Heterocallis, Nemesis. Pelargonitrm, Panieum, Pennisetum, Ranunculus, Senecio,
Salpiglossis, Ctrcumis, Browaalia, Glycine, Pisium, Phaseolus, Lolium, Oryza,
Zea,
Avena, Hordeum, Secale, Triticum, Sorghum, Picea, and Populus.
27
. _ _- ~ --CL=y
CA 02270289 2000-12-08
75529-49(S)
The isolated nucleic acids of the present invention can be used
over a broad range of po--ypeptide types, including anti-
microbial peptides such as those described in Rao, G.,
Antimicrobial Peptides; Molecular Plant-Microbe Interactions
8:6-13 (1995).
Protease Inhibitor Nucleic Acids
The present invention provides, inter alia, isolated
and/or heterologous nucleic acids of RNA, DNA, and analogs
and/or chimeras thereof, comprising a protease inhibitor
polynucleotide encoding such proteins as: chymotrypsin
inhibitor, trypsin inhibitor, protease inhibitor, pre-pro-
proteinase inhibitor I, subtilisin-chymotrypsin inhibitor,
tumor-related protein, genetic tumor-related proteinase
inhibitor, subtilisin inhibitor, endopeptidase inhibitor,
serine protease inhibitor, wound-inducible proteinase
inhibitor, and eglin c. The protease inhibitor nucleic acids
of the present invention comprise protease inhibitor
polynucleotides which, are inclusive of:
(a) a polynucleotide encoding a protease inhibitor
polypeptide of SEQ ID NOS: 2,4,6,8,10, or 12,16,18,20,22,24 and
conservatively modified and polymorphic variants thereof,
including exemplary polynucleotides of SEQ ID NOS: 1,3,5,7,9
and 11,15,17,19,21,23 and. conservative changes
(b) a polynucleotide which is the product of
amplification from Zea mays nucleic acid library using primer
pairs from amongst. the consecutive pairs from SEQ ID NOS: 25
and 26, which amplify polynuc:leotides having substantial
identity to polynucleotides from amongst those having SEQ ID
NOS: 1,3,5,7,9 or 11,15,17,19,21,23
28
CA 02270289 2000-12-08
75529-49(S)
(c) a polynucl_eotide which selectively hybridizes
under stringent hybridization conditions consisting of washing
in a salt concentration of about 0.02 molar at pH 7 at 50°C, to
a polynucleotide of (a) or (b);
(d) a polynucl.eotide having at least 60o sequence
identity with Sequence I:D NOS: 1,3,5,7,9,11,15,17,19,21 or 23;
(e) a polynucl.eotide encoding a protein having a
specified number of contiguous amino acids from a prototype
polypeptide, wherein the protein is specifically recognized by
antisera elicited by prE:sentation of the protein and wherein
the protein does
28a
CA 02270289 1999-04-30
Applicant Rcf. No.: 057! R-PCT.app
' ~ - . .
. .~ . , ,
. . . . . . . , , ",
.. .. .,. . . .,
not detectably immunoreact to antisera which has been fully immunosorbed with
the
protein;
(f) complementary sequences oi'polynucleotides of (a), (b), (c), (d), or (e);
and
(g) a polynucleotide comprising; at least 20 contiguous nucleotides from a
polynucleotide of Sequence ID Nos. 1, 3, 5, 7, 9, 11, 15, 17, 19, 21 or 23.
A. Polynucleotides Encoding A Protease inhibitor Protein of SEQ ID NOS: 2, 4,
6, 8,10
and 12,16,18, 20, 22, 24 or Conservatively Mod ified or Polymorphic Variants
Thereof
As indicated in (a), sarpra, the present invention provides isolated and/or
heterologous nucleic acids comprising protease: inhibitor polynucleotides,
wherein the
polynucleotides encode the protease inhibitor polypeptides disclosed herein as
SEQ ID
NOS: 2,4,6,8,10 and 12,16,18,20,22,24 or conservatively modified or
polymorphic
variants thereof. Those of skill in the art will recognize that the degeneracy
of the genetic
1 ~ code allows for a plurality of polynucleotides to encode for the identical
amino acid
sequence. Thus, the present invention includes protease inhibitor
polynucleotides of SEQ
ID NOS: 1,3,5,7,9 and 11, 15,17,19,21, 23 and silent variations of
polynucleotides
encoding a protease inhibitor polypeptide of SF:Q ID NOS: 2,4,6,8,10 and
12,16,18,20,22,24. The present invention further provides isolated and/or
heterologous
nucleic acids comprising protease inhibitor pol;ynucleotides encoding
conservatively
modified variants of a protease inhibitor polypeptide of SEQ ID NOS:
2,4,6,8,10 and 12,
16,18,20,22,24. Additionally, the present invention further provides isolated
and/or
heterolog8a~iucleic acids comprising protease inhibitor polynucleotides
encoding one or
more polymorphic (allelic) variants of protease inhibitor
polypeptides/polynucleotides.
B. Polynucleotides Amplified from a Zea mays .Nucleic Acid Library
As indicated in (b), supra, the present invention provides isolated and/or
heterologous nucleic acids comprising protease inhibitor polynucleotides,
wherein the
polynucleotides are amplified from a Zea mays nucleic acid library. The
nucleic acid
library may be a cDNA library, a genomic library, or a library generally
constructed from
nuclear transcripts at any stage of intron processing. Nucleic acid libraries
from other
plants, both monocots and dicots could also be used in a similar fashion. The
29
~1~~PJDEt? c~.,~'T
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
polynucleotides of the present invention include those amplified using the
following
primer pairs:
SEQ ID NOS: 25 and 26 which yield an amplic,on comprising a sequence having
substantial identity to SEQ ID NOS: 7,9, and 11.
S Thus, the present invention provides protease inhibitor synthetic
polynucleotides
having the sequence of the gene, a nuclear transcript, a cDNA, or
complementary
sequences thereof. In preferred embodiments, l:he nucleic acid library is
constructed from
Zea mays, such as lines B73, PHRE1, A632, B1VIS-P2#10, and W23, each of which
are
known and publicly available. In particularly preferred embodiments, the
library is
constructed from tissue such as root, leaf, or tassel, or embryonic tissue.
The amplification products can be translated using expression systems well
known
to those of skill in the art and as discussed, infr~2. The resulting
translation products can
be confirmed as protease inhibitor polypeptides. of the present invention by,
for example,
assaying for the appropriate inhibition activity or verifying the presence of
a linear
epitope which is specific to a protease inhibitor polypeptide using standard
immunoassay
methods.
Those of ordinary skill will appreciate that primers which selectively
amplify,
under stringent conditions, the polynucleotides of the present invention (and
their
complements) can be constructed by reference 1:o the sequences provided herein
at SEQ
ID NOS: 1,3,5,7,9 and 11. In preferred embodiments, the primers will be
constructed to
anneal with the first three contiguous nucleotides at their 5' terminal end's
to the first
codon encoding the carboxy or amino terminal amino acid residue (or the
complements
thereof) opolynucleotides of the present invention. Typically, such primers
are at
least 15 nucleotides in length. The primer length in nucleotides is selected
from the group
of integers consisting of from at least 15 to 90. Thus, the primers can be at
least 15, 18,
20, 25, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length.
The amplification primers may optionally be elongated in the 3' direction with
contiguous nucleotide sequences from polynucleotide sequences of SEQ ID NOS:
1,3,5,7,9 and 11, 15,17,19,21, from which they are derived. The number of
nucleotides
by which the primers can be elongated is selected from the group of integers
consisting of
from at least 1 to 25. Thus, for example, the primers can be elongated with an
additional
l, 5, 10, or 1 S nucleotides. Those of skill will recognize that a lengthened
primer
30 '-'~w'~... -~_-
Elppli,:ant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
sequence can be employed to increase specificity of binding (i.e., annealing)
to a target
sequence.
C. Polynucleotides Which Selectively Hybridize to a Polynucleotide of (A) or
(B)
As indicated in (c), supra, the present invention provides isolated and/or
heterologous nucleic acids comprising protease inhibitor polynucleotides,
wherein the
polynucleotides selectively hybridize, under selective hybridization
conditions, to a
protease inhibitor polynucleotide of paragraphs (A) or (B) as discussed,
supra. Thus, the
polynucleotides of this embodiment can be used for isolating, detecting,
and/or
quantifying nucleic acids comprising the polynucleotides of (A) or (B). Low
stringency
hybridization conditions are typically, but not exclusively, employed with
sequences
having relatively small sequence identity. Moderate and high stringency
conditions can
optionally be employed for sequences of greater identity. Low stringency
conditions
allow selective hybridization of sequences having about 70% sequence identity.
D. Polynucleotides Having at Least 60% Seqzrence Identity with the
Polynzrcleotides of
(A), (B) or (C)
As indicated in (d), supra, the present invention provides isolated and/or
heterologous nucleic acids comprising protease inhibitor polynucleotides,
wherein the
polynucleotides have a specified identity at the nucleotide level to a
polynucleotide as
disclosed above in paragraphs (A), (B), (C), or (D). The percentage of
identity to a
reference sequence is at least 60% and, rounded upwards to the nearest
integer, can be
expressed.as:~ integer selected from the group of integers consisting of from
60 to 99.
Thus, for example, the percentage of identity to a reference sequence can be
at least 70%,
75%, 80%, 85%, 90%, or 95%.
The protease inhibitor polynucleotide optionally encodes a protein having a
molecular weight as the unglycosylated protein within 20% of the molecular
weight of the
truncated or full-length protease inhibitor polype:ptides as disclosed herein
(e.g., SEQ ID
NOS: 2,4,6,8,10 and 12). Preferably, the molecular weight is within 1 S% of a
full length
protease inhibitor polypeptide, more preferably within 10% or 5%, and most
preferably
31 . . .,.; '~:' '_'.' ._ ;~~ 7~
Applicant Ref. No.: 05718-PCT. app
CA 02270289 1999-04-30
within 3%, 2%, or 1% of a full length protease inhibitor polypeptide of the
present
invention.
Optionally, the protease inhibitor polynucleotides of this embodiment will
encode
a protein having an inhibitory activity less than or equal to 20%, 30%, 40%,
or 50% of the
native, endogenous (i.e., non-isolated), full-length protease inhibitor
polypeptide.
Determination of protein inhibition can be detemined by any number of means
well
known to those of skill in the art.
F. Polynucleotides Complementary to the Polyr~ucleotides of (A)-(E)
As indicated in (f), supra, the present invention provides isolated and/or
heterologous nucleic acids comprising protease inhibitor polynucleotides,
wherein the
polynucleotides are complementary to the polynucleotides of paragraphs A-E,
above. As
those of skill in the art will recognize, complementary sequences base-pair
throughout the
entirety of their length with the polynucleotides of (A)-(E) (i.e., have 100%
sequence
identity). Complementary bases associate through hydrogen bonding in double
stranded
nucleic acids. For example, the following base pairs are complementary:
guanine and
cytosine; adenine and thymine; and adenine and uracil.
G. Polynz~cleotides Which are Subseqzrences of ,the Polynucleotides of (A)-(F)
As indicated in (h), supra, the present invention provides isolated and/or
heterologous nucleic acids comprising protease inhibitor polynucleotides,
wherein the
polynucleotide comprises at least 15 contiguous bases from the polynucleotides
of (A)
through (F~.-discussed above. The length of tile polynucleotide is given as an
integer
selected from the group consisting of from at least 1 S to the length of the
nucleic acid
sequence from which the protease inhibitor polynucleotide is a subsequence of.
Thus, for
example, polynucleotides of the present invention are inclusive of
polynucleotides
comprising at least 15, 20, 25, 30, 40, 50, 60, 75, or 100 contiguous
nucleotides in length
from the polynucleotides of (A)-(F). Optionally, the number of such
subsequences
encoded by a polynucleotide of the instant embodiment can be any integer
selected from
the group consisting of from 1 to 20, such as 2, :3, 4, or 5.
Construction of Protease inhibitor Nucleic Acids
32
t-r
/cppli~ant Ref. No.: 0571 R-PCT.app
CA 02270289 1999-04-30
The isolated and/or heterologous protease inhibitor nucleic acids of the
present
invention can be made using (a) standard recombinant methods, (b) synthetic
techniques,
or combinations thereof. In some embodiments, the protease inhibitor
polynucleotides of
the present invention will be cloned, amplified, or otherwise constructed from
a plant.
The preferred plants are barley and Zea mays, such as inbred line B73 which is
publicly
known and available. Particularly preferred is the use of Zea mays tissue such
as roots,
leaves, tassels, seeds or embryonic tissue.
A. Recombinant Methods for Constructing Protease inhibitor Nucleic Acids
The isolated and/or heterologous nucleic; acid compositions of this invention,
such
as RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plant
biological
sources using any number of cloning methodologies known to those of skill in
the art.
The isolation of protease inhibitor polynucleotides may be accomplished by a
number of techniques. For instance, oligonucleotide probes based on the
sequences
disclosed here can be used to identify the desired gene in a cDNA or genomic
DNA
library. To construct genomic libraries, large segments of genomic DNA are
generated by
random fragmentation, e.g. using restriction ene!onucleases, and are ligated
with vector
DNA to form concatemers that can be packaged into the appropriate vector. To
prepare a
cDNA library, mRNA is isolated from the desired organ, such as sclerenchyma
and a
cDNA library which contains the gene encoding; for a protease inhibitor
protein (i.e., the
protease inhibitor gene) is prepared from the mF:NA. Alternatively, cDNA may
be
prepared from mRNA extracted from other tissues in which protease inhibitor
genes or
homologs.xpressed.
The DNA or genomic library can then bf: screened using a probe based upon the
sequence of a cloned protease inhibitor polynucl.eotide such as those
disclosed herein.
Probes may be used to hybridize with genomic I~NA or cDNA sequences to isolate
homologous genes in the same or different plant species. Those of skill in the
art will
appreciate that various degrees of stringency of hybridization can be employed
in the
assay; and either the hybridization or the wash medium can be stringent. As
the
conditions for hybridization become more stringent, there must be a greater
degree of
complementarity between the probe and the target for duplex formation to
occur. The
degree of stringency can be controlled by temperature, ionic strength, pH and
the presence
33
AMENDED SHEET
CA 02270289 1999-04-30
Applicant Ref. No.: 0~7tR-PCT.app
.. ..
. . , . . . .
. ~ . . . . . . ~..
.. .. .~. .... ..
of a partially denaturing solvent such as formamide. For example, the
stringency of
hybridization is conveniently varied by changing the polarity of the reactant
solution
through manipulation of the concentration of formamide within the range of 0%
to 50%.
Cloning methodologies to accomplish these ends, and sequencing methods to
S verify the sequence of nucleic acids are well known in the art. Examples of
appropriate
cloning and sequencing techniques, and instructions sufficient to direct
persons of skill
through many cloning exercises are found in S~~mbrook, et al., Molecular
Cloning. A
Laboratory Manual, 2nd Ed., Cold Spring Harlbor Laboratory Vols. 1-3 (1989),
Methods
in Enzymology, Vol. 152: Garide to Molecular Cloning Technigues, Berger and
Kimmel,
Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular
Biology,
Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (
1987); Plant
Molecular Biology: A Laboratory ManZral, Clark, Ed., Springer-Verlag, Berlin
(1997).
The nucleic acids of interest can also be; amplified from nucleic acid samples
using amplification techniques. For instance, polymerase chain reaction (PCR)
technology can be used to amplify the sequences of protease inhibitor
polynucleotides of
the present invention and related genes directly from genomic DNA or cDNA
libraries.
PCR and other in vitro amplification methods may also be useful, for example,
to clone
nucleic acid sequences that code for proteins to be expressed, to make nucleic
acids to use
as probes for detecting the presence of the desii:-ed mRNA in samples, for
nucleic acid
sequencing, or for other purposes.
The degree of complementarity (sequence identity) required for detectable
binding
will vary in accordance with the stringency of the hybridization medium and/or
wash
medium. -~ewdegree of complementarity will optimally be 100 percent; however,
it
should be understood that minor sequence variations in the probes and primers
may be
compensated for by reducing the stringency of the hybridization and/or wash
medium.
Examples of techniques sufficient to direct persons of skill through in vitro
amplification methods are found in Berger, Sarnbrook, and Ausubel, as well as
Mullis et
al., U.S. Patent No. 4,683,202 (1987); PCR Protocols A Guide to Methods and
Applications, Innis et al., Eds., Academic Press; Inc., San Diego, CA (1990);
Arnheim &
Levinson, C&EN pp. 36-47 (October 1, 1990).
B. Synthetic Methods for Constructing Protease inhibitor Nucleic Acids
34
~r:~~~~~r:~ s~L~r
Hppl~cant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
' ~ ~ ~ ~ ,
.. ~. . . .. . ..
The isolated nucleic acids of the present invention can also be prepared by
direct
chemical synthesis by methods such as the phosphotr~iester method of Narang et
al., Meth.
Enzvmol. 68: 90-99 ( 1979)and the phosphodiester method of Brown et al., Meth.
Enzymol. 68: 109-151 (1979). The isolated nucleic acids of the present
invention
can also be modified through methods such as site directed mutogenesis, error
prone PCR
and known to one of skill.
Recombinant Expression Cassettes
The present invention further provides recombinant expression cassettes
comprising a protease inhibitor nucleic acid of the present invention. A
nucleic acid
sequence coding for the desired protease inhibitor polynucleotide, for example
a cDNA or
a genomic sequence encoding a full length protease inhibitor protein, can be
used to
construct a recombinant expression cassette wr~ich can be introduced into the
desired host
cell. A recombinant expression cassette will typically comprise a protease
inhibitor
polynucleotide operably linked to transcriptional initiation regulatory
sequences which
will direct the transcription of the protease inhibitor polynucleotide in the
intended host
cell, such as tissues of a transformed plant.
For example, plant expression vectors may include (1) a cloned plant gene
under the transcriptional control of 5' and 3' regulatory sequences and (2) a
dominant selectable marker. Such plant expression vectors may also contain, if
desired, a promoter regulatory region (e.g., one conferring inducible or
constitutivvironmentally- or developmentally-regulated, or cell- or
tissue-specific/selective expression), a transcription initiation start site,
a ribosome
binding site, an RNA processing signal, a transcription termination site,
and/or
a polyadenylation signal. Highly preferred plant expression cassettes will be
designed to
include one or more selectable marker genes, .such as kanamycin resistance or
herbicide
tolerance genes.
A plant promoter fragment may be employed which will direct expression of the
protease inhibitor polynucleotide in all tissues of a regenerated plant. Such
promoters are
referred to herein as "constitutive" promoters arid are active under most
environmental
~,t,p~r,~~cr c~..c~-.
~.~ ....,:... i
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
. , . "' , , ~~,
, , ,
,. .. , . ,.
conditions and states of development or cell differentiation. Examples of
constitutive
promoters include the cauliflower mosaic virus (CaMV) 35S transcription
initiation
region, the 1'- or 2'- promoter derived from T-L>NA ofAgrobacterium
tumefat;iens, the
ubiquitin 1 promoter, the Smas promoter, the ci:nnamyl alcohol dehydrogenase
promoter
(U.S. Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco
promoter,
the GRP1-8 promoter, and other transcription initiation regions from various
plant genes
known to those of skill. In a preferred embodirrlent, the gamma zero promoter
of maize
would be used.
Alternatively, the plant promoter may direct expression of the protease
inhibitor
polynucleotide in a specific tissue or may be otherwise under more precise
environmental
or developmental control Examples of promoters under developmental control
include promoters that initiate transcription only, or preferentially, in
certain tissues, such
as leaves, roots, fruit, seeds, or flowers. The operation of a promoter may
also vary
depending on its location in the genome. Thus, an inducible promoter may
become fully
or partially constitutive in certain locations.
Both heterologous and non-heterologous (i.e., endogenous) promoters can be
employed to direct expression of the protease inhibitor nucleic acids of the
present
invention. These promoters can also be used, fir example, in recombinant
expression
cassettes to drive expression of antisense nucleic acids to reduce, increase,
or alter
protease inhibitor content and/or composition in a desired tissue
Methods for identifying promoters with a particular expression pattern, in
terms of, e.g., tissue type, cell type, stage of development, andJor
environmental
conditiorrs~=a~e well known in the art. See, e.g., The Maize Handbook,
Chapters 114-115,
Freeling and Walbot, Eds., Springer, New York: (1994); Corn and Corn
Improvement, 3'a
edition, Chapter 6, Sprague and Dudley, Eds., American Society of Agronomy,
Madison,
Wisconsin (1988). A typical step in promoter isolation methods is
identification of gene
products that are expressed with some degree of specificity in the target
tissue. Amongst
the range of methodologies are: differential hybridization to cDNA libraries;
subtractive
hybridization; differential display; differential 2 -D gel electrophoresis;
DNA probe arrays;
and isolation of proteins known to be expressed. with some specificity in the
target tissue.
Such methods are well known to those of skill in the art. Commercially
available
36
_ _., c_'_:
CA 02270289 2000-04-20
75529-49(S)
products for identifying promoters are known in the art such as CloneTech's
(Palo Alto,
CA) PROMOTERFINDER DNA Walking Kit
Once promoter and/or gene sequences are known, a region of suitable size is
selected from the genomic DNA that is 5' to the transcriptional start, or the
translational
start site, and such sequences are then linked to a coding sequence. If the
transcriptional
start site is used as the point of fusion, any of a number of possible 5'
untranslated regions
can be used in between the transcriptional start site and the partial coding
sequence. If the
translational start site at the 3' end of the specific promoter is used, then
it is linked
directly to the methionine start codon of a coding sequence.
If polypeptide expression is desired, it is generally desirable to include a
polyadenylation region at the 3'-end of the protease inhibitor polynucleotide
coding
region. An intron sequence can be added to the ~' untranslated region or the
coding
sequence of the partial coding sequence to increase the amount of the mature
message that
accumulates in the cytosol Use of maize introns Adhl-S intron 1, 2, and 6, the
Bronze-I
intron are known in the art. See generally, The Maize Handbook, Chapter 116,
Freeling
and Walbot, Eds., Springer, New York ( 1994).
The vector comprising the sequences from a protease inhibitor nucleic acid
will
typically comprise a marker gene which confers a selectable phenotype on plant
cells.
Usually, the selectable marker gene will encode antibiotic resistance, with
suitable genes
including genes coding for resistance to the antibiotic spectinomycin (e.g.,
the aada gene),
the streptomycin phosphotransferase (SPT) gene coding for streptomycin
resistance, the
neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin
resistance,
the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance,
genes
coding for resistance to herbicides which act to inhibit the action of
acetolactate synthase
(ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate
synthase
(ALS) gene containing mutations leading to such resistance in particular the
S4 and/or
Hra mutations), genes coding for resistance to herbicides which act to inhibit
action of
glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or
other such
genes known in the art. The bar gene encodes resistance to the herbicide
basta, the nptll
gene encodes resistance to the antibiotics kanamycin and geneticin, and the
ALS gene
encodes resistance to the herbicide chlorsulfuron.
Trade-mark
37
CA 02270289 1999-04-30
Applicant Ref. No.: 071 R-PCT.app
' . . .. ,.
. . , " . . , ,
. , . , . . . ,.
,. ,. ". .,.. ,
Typical vectors useful for expression of genes in higher plants are well known
in
the art and include vectors derived from the tumor-inducing (Ti) plasmid of
Agrobacterium tumefaciens described by Rogers et al., Meth. In Enzymol.,
153:253-277
(1987). These vectors are plant integrating vect~~rs in that on
transformation, the vectors
integrate a portion of vector DNA into the genome of the host plant. Exemplary
A.
tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et
al.,
Gene, 61:1-11 (1987) and Berger et al., Proc. Natl. Acad. Sci. U.S.A., 86:8402-
8406
(1989). Another useful vector herein is plasmid pBI101.2 that is available
from Clontech
Laboratories, Inc. (Palo
Alto, CA).
The protease inhibitor polynucleotide of the present invention can be
expressed in
either sense or anti-sense orientation as desired.
Protease inhibitor Proteins
The isolated protease inhibitor proteins of the present invention comprise a
protease inhibitor polypeptide having at least 10 amino acids encoded by any
one of the
protease inhibitor polynucleotides as discussed more fully, sarpra, or
polypeptides which
are conservatively modified variants thereof. Exemplary protease inhibitor
polypeptide
sequences are provided in SEQ ID NOS: 2,4,6,8,10 and 12. The protease
inhibitor
proteins of the present invention or variants thereof can comprise any number
of
contiguous amino acid residues from a protease; inhibitor protein, wherein
that number is
selected from the group of integers consisting of from 10 to the number of
residues in a
full-length protease inhibitor polypeptide. Optionally, this subsequence of
contiguous
amino acids at least 15, 20, 25, 30, 35, or 40 amino acids in length, often at
least 50, 60,
70, 80, or 90 amino acids in length. Further, the number of such subsequences
can be any
integer selected from the group consisting of from 1 to 20, such as 2, 3, 4,
or 5.
As those of skill will appreciate, the preaent invention includes protease
inhibitor
polypeptides with less inhibitory activity. Less inhibitory protease inhibitor
polypeptides
have an inhibitory activity at least 20%, 30%, or 40%, and preferably at least
50% or
60%, below that of the native (non-synthetic), endogenous protease inhibitor
polypeptide.
A preferred immunoassay is a competitive immunoassay as discussed, infra.
Thus, the protease inhibitor proteins can be employed as immunogens for
constructing
38
ftpplicant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
1 1 1 1
1 1 1 ! a 1 1 1 . 1 1
n I ~ I . 1 1 a 1 . 1
n n 1 v 1 n ! 1
1 ~ n . 1 a 1 1 1 o i
,
antibodies immunoreactive to a protease inhibitor protein for such exemplary
utilities as
immunoassays or protein purification techniquca.
Expression of Proteins in Host Cells
Using the nucleic acids of the present invention, one may express a protease
inhibitor protein in a recombinantly engineered cell such as bacteria, yeast,
insect,
mammalian, or preferably plant cells. The cells produce the protein in a non-
natural
condition (e.g., in quantity, composition, location, and/or time), because
they have been
genetically altered through human intervention to do so.
It is expected that those of skill in the act are knowledgeable in the
numerous
expression systems available for expression of nucleic acids encoding protease
inhibitor
proteins. No attempt to describe in detail the various methods known for the
expression
of proteins in prokaryotes or eukaryotes will be made.
IS
B. Expression in Eukaryotes
A variety of eukaryotic expression systems such as yeast, insect cell lines,
plant
and mammalian cells, are known to those of skill in the art. As explained
briefly below,
protease inhibitor proteins of the present invention may be expressed in these
eukaryotic
systems. In some embodiments, transformed/transfected plant cells, as
discussed infra,
are employed as expression systems for production of the proteins of the
instant
invention.
Transfection/Transformation of Cells
The method of transformation/transfection is not critical to the instant
invention;
various methods of transformation or transfection are currently available. As
newer
methods are available to transform crops or othE:r host cells they may be
directly applied.
Accordingly, a wide variety of methods have been developed to insert a DNA
sequence
into the genome of a host cell to obtain the transcription and/or translation
of the sequence
to effect phenotypic changes in the organism. Thus, any method which provides
for
efficient transformation/transfection may be employed.
39
AMENDED SN~ET
CA 02270289 1999-04-30
Applicant Ref. No.: 0571 R-PCT.app
A. Plant Transformation
A DNA sequence coding for the desired protease inhibitor polynucleotide, for
example a cDNA or a genomic sequence encoding a full length protein, will be
used to
construct a recombinant expression cassette which can be introduced into the
desired
plant.
Isolated nucleic acids of the present invention can be introduced into plants
according to techniques known in the art. Generally, recombinant expression
cassettes as
described above and suitable for transformation of plant cells are prepared.
Techniques
for transforming a wide variety of higher plant species are well known and
described in
the technical, scientific, and patent literature. Se:e, for example, Weising
et al., Ann. Rev.
Genet. 22: 421-477 (1988). For example, the DIVA construct may be introduced
directly
into the genomic DNA of the plant cell using techniques such as
electroporation, PEG
poration, particle bombardment, silicon fiber dellivery, or microinjection of
plant cell
protoplasts or embryogenic callus. Alternatively, the DNA constructs may be
combined
with suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium
tumefaciens host vector. The virulence functions of the Agrobacterium
tumefaciens host
will direct the insertion of the construct and adjacent marker into the plant
cell DNA
when the cell is infected by the bacteria.
The introduction of DNA constructs using polyethylene glycol precipitation is
described in Paszkowski et al., Embo J. 3: 2717-2722 (1984). Electroporation
techniques
are described in Fromm et al., Proc. Natl. Acad. Sci. 82: 5824 (1985).
Ballistic
transforms r ii techniques are described in Klein et al., Nature 327: 70-73
(1987).
Agrobacterium tumefaciens-meditated transformation techniques are well
described in the
scientific literature. See, for example Horsch et al., Science 233: 496-498
(1984), and
Fraley et al., Proc. Natl. Acad. Sci. 80: 4803 (1583). Although Agrobacterium
is useful
primarily in dicots, certain monocots can be transformed by Agrobacterium. For
instance,
Agrobacterium transformation of maize is described in U.S. Patent No.
5,550,318.
Other methods of transfection or transformation include (1) Agrobacteria~m
rhizogenes-mediated transformation (see, e.g., L,ichtenstein and Fuller In:
Genetic
Engineering, vol. 6, PWJ Rigby, Ed., London, Academic Press, 1987; and
Lichtenstein,
C. P., and Draper, J,. In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford,
IRI Press,
~;"J~~;DEI~ ~~-tEEi
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1985),Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)
describes
the use of A.rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens
vectors
pARC8 or pARC 16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al.,
Plant
Cell Physiol. 25: 1353, 1984), (3) the vortexing method (see, e.g., Kindle,
Proc. Natl.
Acad. Sci., USA 87: 1228, (1990).
DNA can also be introduced into plants by direct DNA transfer into pollen as
described by Zhou et al., Methods in Enzymology, 101:433 (1983); D. Hess,
Intern
Rev. Cytol., 107:367 (I987); Luo et al., Plane Mol. Biol. Reporter, 6:165
(1988). Expression ofpolypeptide coding genes can be obtained by injection of
the DNA into reproductive organs of a plant as described by Pena et al.,
Nature,
325.:274 (1987). DNA can also be injected directly into the cells of immature
embryos and the rehydration of desiccated embryos as described by Neuhaus et
al., Theor. Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings
Bio
Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 ( 1986). A variety of plant
viruses
that can be employed as vectors are known in the art and include cauliflower
mosaic virus
(CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
Synthesis of Proteins
Protease inhibitor proteins of the present invention can be constructed using
non-
20. cellular synthetic methods. Solid phase synthesis of protease inhibitor
proteins of less
than about 50 amino acids in length may be accomplished by attaching the C-
terminal
amino acid of the sequence to an insoluble support followed by sequential
addition of the
remaining amino acids in the sequence. Techniques for solid phase synthesis
are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284
in The
Peptides: Analysis, Synthesis, Biology. Yol. 2: Special Methods in Peptide
Synthesis,
Part A.; Merrifield, et al., J. Am. Chem. Soc. 85: 2149-2156 (1963), and
Stewart et al.,
Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill.
(1984). Also,
the compounds can be synthesized on an applied Biosystems model 431 a peptide
synthesizer using fastmocTM chemistry involving hbtu [2-(lh-benzotriazol-1-yl)-
1,1,3,3-
tetramethyluronium hexafluorophosphate, as published by Rao, et al., Int. J.
Pecr. Prot.
Res.; Vol. 40; pp. 508-515; (1992),
Peptides can be cleaved following standard protocols and purified by reverse
phase
41
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chromatography using standard methods. The amino acid sequence of each peptide
can
be confirmed by automated edman degradation on an applied biosystems 477a
protein
sequencer/120a pth analyzer. Protease inhibitor proteins of greater length may
be
synthesized by condensation of the amino and carboxy termini of shorter
fragments.
Methods of forming peptide bonds by activation. of a carboxy terminal end
(e.g., by the
use of the coupling reagent N,N'-dicycylohexylcarbodiimide)) is known to those
of skill.
Purification of Proteins
The protease inhibitor proteins of the present invention may be purified by
standard techniques well known to those of skill in the art. Recombinantly
produced
protease inhibitor proteins can be directly expressed or expressed as a fusion
protein. The
recombinant protease inhibitor protein is purified by a combination of cell
lysis (e.g.,
sonication, French press) and affinity chromatography. For fusion products,
subsequent
digestion of the fusion protein with an appropriate proteolytic enzyme
releases the desired
1 S recombinant protease inhibitor protein.
The protease inhibitor proteins of this invention, recombinant or synthetic,
may be
purified to substantial purity by standard techniques well known in the art,
including
selective precipitation with such substances as ammonium sulfate, column
chromatography, immunopurification methods, and others. See, for instance, R.
Scopes,
Protein Purification: Principles and Practice, Springer-Verlag: New York
(1982);
Deutscher, Guide to Protein Purification, Acadf:mic Press (1990). For example,
antibodies may be raised to the protease inhibitor proteins as described
herein.
Purificatie~om E. coli can be achieved following procedures described in U.S.
Patent
No. 4,511,503. The protein may then be isolated from cells expressing the
protease
inhibitor protein and further purified by standard protein chemistry
techniques as
described herein. Detection of the expressed protein is achieved by methods
known in the
art and include, for example, radioimmunoassays, Western blotting techniques,
protease
inhibition assays, or immunoprecipitation.
Trans>=enic Plant Re ~neration
Transformed plant cells which are derivE:d by any of the above transformation
techniques can be cultured to regenerate a whole plant which possesses the
transformed
42
. ~.,~:r~'. ~-..,-,__.
. . ..' ~. s . ~ .. .....
Applicant Ref. No.: Oi7lR-PCT.app
CA 02270289 1999-04-30
genotype and thus the desired protease inhibitor content and/or composition
phenotype.
Such regeneration techniques often rely on manipulation of certain
phytohormones in a
tissue culture growth medium, typically relying on a biocide and/or herbicide
marker
which has been introduced together with the protease inhibitor polynucleotide.
Plants cells transformed with a plant expression vector can be regenerated,
e.g., from single cells, callus tissue or leaf discs according to standard
plant
tissue culture techniques. It is well known in th.e art that various cells,
tissues, and organs from almost any plant can b~e successfully cultured to
regenerate an entire plant. Plant regeneration from cultured protoplasts is
described in
Evans et al., Protoplasts Isolation and Culture, Handbook ofPlant Cell
Carlture,
Macmillilan Publishing Company, New York, pp. 124-176 (1983); and Binding,
Regeneration ofPlants, Plant Protoplasts, CRC', Press, Boca Raton, pp. 21-73
(1985).
The regeneration of plants containing the foreign gene introduced by
Agrobacterium from leaf explants can be achieved as described by Horsch et
al.,
Science, 227:1229-1231 (1985
Regeneration can also be obtained from plant callus, explants, organs, or
parts
thereof. Such regeneration techniques are described generally in Klee et al.,
Ann. Rev. of
Plant Phys. 38: 467-486 (1987For maize cell culture and regeneration see
generally, The
Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and
Corn
Improvement, 3'd edition, Sprague and Dudley E,ds., American Society of
Agronomy,
Madison, Wisconsin (1988).
One of skill will recognize that after the recombinant expression cassette is
stably
incorporate~.n transgenic plants and confirmed to be operable, it can be
introduced into
other plants by sexual crossing. Any of a number of standard breeding
techniques can be
used, depending upon the species to be crossed.
In vegetatively propagated crops, mature transgenic plants can be propagated
by
the taking of cuttings or by tissue culture techniques to produce multiple
identical plants.
Selection of desirable transgenics is made and new varieties are obtained and
propagated
vegetatively for commercial use. In seed propagated crops, mature transgenic
plants can
be self crossed to produce a homozygous inbred plant. The inbred plant
produces seed
containing the newly introduced heterologous nucleic acid. These seeds can be
grown to
43 ~ - _ ;, : ,
CA 02270289 1999-04-30
Applicant Ref. No.: 057IR-PCT.app
~ ~ . , ,.
.. . . . . .. . , . .
. ~ . ... . . . .:. .
produce plants that would produce the selected phenotype, (e.g., altered
protease inhibitor
content or composition).
Parts obtained from the regenerated plant, such as flowers, seeds, leaves,
branches, fruit, and the like are included in the invention, provided that
these
parts comprise cells comprising the isolated nucleic acid of the present
invention. Progeny
and variants, and mutants of the regenerated plants are also included within
the
scope of the invention, provided that these parts comprise the introduced
nucleic acid sequences.
Transgenic plants expressing the selectable marker can be screened for
transmission of the protease inhibitor nucleic acrid of the present invention
by, for
example, standard immunoblot and DNA detection techniques. Transgenic lines
are also
typically evaluated on levels of expression of the heterologous nucleic acid.
Expression at
the RNA level can be determined initially to identify and quantitate
expression-positive
plants. Standard techniques for RNA analysis can be employed and include PCR
amplification assays using oligonucleotide primers designed to amplify only
the
heterologous RNA templates and solution hybridization assays using
heterologous nucleic
acid-specific probes. The RNA-positive plants can then analyzed for protein
expression
by Western immunoblot analysis using the protease inhibitor specific
antibodies of the
present invention. In addition, in situ hybridization and immunocytochemistry
according
to standard protocols can be done using heterologous nucleic acid specific
polynucleotide
probes and antibodies, respectively, to localize sites of expression within
transgenic
tissue. Generally, a number of transgenic lines are usually screened for the
incorporated
nucleic aci~"o identify and select plants with the most appropriate expression
profiles.
A preferred embodiment is a transgenic plant that is homozygous for the added
heterologous nucleic acid; i.e., a transgenic plant that contains two added
nucleic acid
sequences, one gene at the same locus on each chromosome of a chromosome pair.
A
homozygous transgenic plant can be obtained by sexually mating (selfing) a
heterozygous
transgenic plant that contains a single added heterologous nucleic acid,
germinating some
of the seed produced and analyzing the resulting plants produced for altered
activity
relative to a control plant (i.e., native, non-transgenic). Back-crossing to a
parental plant and out-crossing with a non- transgenic plant are also
contemplated.
~,rn'r~aF~ s~;~~r
44
CA 02270289 2000-04-20
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Protein structure and amino acid substitution
It can be difficult to predict the ultimate effect of substitution on the
tertiary
structure and folding of the protein. Both tertiary structure and folding are
critical to the
stability and adequate expression of the protein in vivo. It is critical to
undertake analysis
and functional modeling of the wild type compound to determine whether
substitutions
can be made without disrupting biological activity.
The biological activity of a protein is dictated by its three dimensional
structure
which is intrinsically related to the folding of the protein. The folding of a
protein into its
functional domains is a direct consequence of the primary amino acid sequence.
While it
is true that many proteins tolerate amino acid changes without affecting the
folding or
function of the protein, there is no a rp iori method of predicting which
amino acid may be
substituted or deleted without affecting the folding pathway. Each protein is
unique and
the folding process is necessarily an experimental determination. As has been
concluded
by Zabin et al., ("Approaches to Predicting Effects of Single Amino Acid
Substitutions on
the Function of a Protein"; Biochemistry; Vol. 30; pp. 6230-6240; 1991),
neither the
frequency of exchange of amino acids between homologous proteins nor any other
measure of the properties of the amino acids are particularly useful by
themselves in
predicting whether a protein with an amino acid substitution will be
functional. The
scientific literature is replete with examples where seemingly conservative
substitutions
have resulted in major perturbations of structure and activity and vice versa,
see e.g.;
Summers, et al., "A Conservative Amino Acid Substitution, Arginine for Lysine,
Abolishes Export of a Hybrid Protein in E. Coli." J. Biol. Chem., Vol. 264,
pp. 20082-
20088, (1989); Ringe, D., "The Sheep in Wolfs Clothing" Nature, Vol. 339, pp.
6S8-659,
(1989); Hirabayashi et al., "Effect of Amino Acid Substitution by Site-
directed
Mutagenesis on the Carbohydrate Recognition and Stability of Human 14-kDa (3-
galactoside-binding Lectin," J. Biol. Chem., Vol. 266, pp. 23648-23653,
(1991); and van
Eijsden, et al., "Mutational Analysis of Pea Lectin: Substitution of Asn125
for Asp in the
Monosachharide-binding Site Eliminates Mannose/Glucose -binding Activity,"
Plant
Mol. Biol., Vol. 20, pp. 1049-1058 (1992).
The 3D structure of many proteins, including enzymes and protein inhibitors
such
as the barley chymotrypsin inhibitor has been solved. The three dimensional
structure of a
CA 02270289 2000-04-20
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truncated fragment of CI-2 (with 65 residues) that is missing the N-terminal
I8 residues
has been determined by x-ray crystallography as well as by NMR spectroscopy
(McPhalen, et al., Biochemistry; Vol. 26; pp. 261-269; (1987); and Clore, et
al., Protein
Ene.; Vol. 1, pp. 3I3-318; (1987)). In the wild type CI-2 the first 18
residues do not
assume any ordered conformation and also do not contribute to the structural
integrity of
the molecule (see e.g. Kjaer, et al., Carlsberg Res. Commun.; Vol. 53; pp. 327-
354;
(1987?. This polypeptide is found in the
endosperm of grain and is isolated as an 83 residue protein with no disulfide
bridges. See
e.g. Jonassen, L, Carlsbere Res. Commun.; Vol. 45; pp. 47-48; (1980); and
Svendsen, L,
et al., Carlsberg Res. Commun.; Vol. 45; pp. 79-85; (1980). The 3D structure
of CI-2 has
been determined. See McPhalen, et al., 1987.
CI-2 is predominantly a (3-sheet protein, devoid of disulfide bonds and
containing a wide loop of approximately 18 residues (residue 53-70 in the CI-2
molecule)
in the extended conformation. This is the reactive site loop that contains a
methionine
I5 residue at position 59 which confers the property of chymotrypsin
inhibition. A
constrained peptide containing these residues has been synthesized and shown
to retain
full chymotrypsin inhibitory activity. See Leatherbarrow, et al., Biochem.,
Vol. 30, pp.
10717-10721 (1991). In the absence of any disulfide bonds, the integrity of
the reactive
site loop is maintained by strong hydrogen bond interactions between G1u60 -~
Arg65
and Thr58 -~ Arg67. Mutants of CI-2 in which Thr58 and G1u60 have been
replaced with
Ala are not only less stable proteins but also have little or no protease
inhibitory activity.
See Jackson, et al., Biochem., Vol. 33, pp. 13880-13887 (1994); and Jandu, et
al.,
Biochem., Vol. 33, pp. 6264-6269 (1990). These studies have demonstrated that
the
reactive site loop is a key structural feature essential for the function of
protease
inhibition.
Molecular Markers
The present invention provides a method of genotyping a plant comprising a
protease inhibitor polynucleotide. Preferably, the plant is a monocot, such as
maize or
sorghum. Genotyping provides a means of distinguishing homologs of a
chromosome
pair and can be used to differentiate segregants in a plant population.
46
r
Appli.;ant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
. . . , " ,
.. ., . . . . , . , .
. . . ,.. . , . ,
. . . . ,
. ,. ., , , ,
Molecular marker methods can be used for phylogenetic studies, characterizing
genetic
relationships among crop varieties, identifying crosses or somatic hybrids,
localizing
chromosomal segments affecting monogenic traits, map based cloning, and the
study of
quantitative inheritance. See, e.g., Plant Molecular Biology: A Laboratory
Manual,
Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1.997). For molecular marker
methods,
see generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in:
Genome
Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R. G. Landis
Company,
Austin, Texas, pp.7-21.
Detection of Protease Inhibitor Nucleic Acids
The present invention further provides methods for detecting protease
inhibitor polynucleotides of the present invention in a nucleic acid sample
suspected of
comprising a protease inhibitor polynucleotide, such as a plant cell lysate,
particularly a
lysate of corn. In some embodiments, a proteasE; inhibitor gene or portion
thereof can be
amplified prior to the step of contacting the nucleic acid sample with a
protease inhibitor
polynucleotide. The nucleic acid sample is contacted with the protease
inhibitor
polynucleotide to form a hybridization complex. The protease inhibitor
polynucleotide
hybridizes under stringent conditions to a gene encoding a protease inhibitor
polypeptide.
Formation of the hybridization complex is used to detect a gene encoding a
protease
inhibitor polypeptide in the nucleic acid sample. Those of skill will
appreciate that an
isolated nucleic acid comprising a protease inhil:>itor polynucleotide should
lack cross-
hybridizing sequences with non-protease inhibitor genes that would yield a
false positive
result.
Detection of the hybridization complex can be achieved using any number
of well known methods. For example, the nucleic acid sample, or a portion
thereof, may
be assayed by hybridization formats including but not limited to, solution
phase, solid
phase, mixed phase, or in situ hybridization assays.
Protease Inhibitor Protein Immunoassays
47 . , .~,~-.,
t~pplicant Ref. No.: 0571 R-PCT.app
CA 02270289 1999-04-30
.. , ,
.. . ,
. . . ... . . . ..
. . . . . .
.. .. ... .... ,.
Means of detecting the protease inhibitor proteins of the present invention
are not critical aspects of the present invention. In a preferred embodiment,
the protease
inhibitor proteins are detected and/or quantified using any of a number of
well recognized
immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110;
4,517,288;
and 4,837,168). For a review of the general immunoassays, see also Methods in
Cell
Biology, Vol. 37: Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc.
New York
(1993); Basic and Clinical Immunology 7th Edition, Stites & Ten, Eds. (1991).
D. Other Assay Formats
In a particularly preferred embodiment, Western blot (immunoblot)
analysis is used to detect and quantify the presence of protease inhibitor
protein in the
sample. The technique generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the separated
proteins to a
suitable solid support, (such as a nitrocellulose i:llter, a nylon filter, or
derivatized nylon
I 5 filter), and incubating the sample with the antibodies that specifically
bind protease
inhibitor protein. The anti-protease inhibitor protein antibodies specifically
bind to
protease inhibitor protein on the solid support. 'These antibodies may be
directly labeled
or alternatively may be subsequently detected using labeled antibodies (e.g.,
labeled sheep
anti-mouse antibodies) that specifically bind to ~,he anti-protease inhibitor
protein.
E. Quantifrcation of Protease inhibitor Proteins.
Protease inhibitor proteins may b~e detected and quantified by any of a
number of~ans well known to those of skill in the art. These include analytic
biochemical methods such as electrophoresis, capillary electrophoresis, high
performance
liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion
chromatography, and the like, and various immunological methods such as fluid
or gel
precipitin reactions, immunodiffusion (single or double),
immunoelectrophoresis,
radioimmunoassays (RIAs), enzyme-linked imnlunosorbent assays (ELISAs),
immunofluorescent assays, and the like.
48 ,
7559-49 (S)
CA 02270289 2000-04-20
Example 1: Isolation of DNA Coding for Protease inhibitor Protein from Zea
mays
or other plant library
The polynucleotides having DNA sequences given in SEQ ID Nos: 15, 17, 19, 21,
and 23
were obtained from the sequencing of cDNA clones prepared from maize.
SEQ ID NO 15 is a contig comprised of 28 cDNA clones. 20 of the cDNA clones
were
from libraries prepared from leaves treated with jasmonic acid. One was from a
root
library. Four were from libraries prepared from corn rootworm-infested roots.
One was
from a tassel library. One was from a library prepared from seedlings
recovering from
heat shock. One was from a shoot culture library.
SEQ ID NO 17 is a contig comprised of two cDNA clones. One was from a jasmonic
acid
treated leaf library. The other was from an induced resistance leaf library.
SEQ ID NO 19 is a contig comprised of two cDNA clones. One was from a
germinating
maize seedling library. The other was from jasmonic acid treated leaf library.
SEQ ID NO 21 is a contig comprised of 4 cDNA clones. All four were from
libraries
prepared from jasmonic acid treated leaves.
SEQ ID NO 23 is a contig comprised of two cDNA clones. One was from a library
prepared from silks, 24 hours post pollination. The other was from a library
prepared
from root tips less than 5 mm in length.
One skilled in the art could apply these same methods to other plant
nucleotide containing
libraries.
Example 2: Engineering BHL for nutritional enhancement
Wild type CI-2 (from barley) contains 49.4% essential amino acids (41/83) and
9.6% lysine (8/83). Using the strategies outlined below, six different BHL
variants with
increasing amounts of lysine have been proposed. The lysine percentages are
21.5%,
24.1%, 23.1%,and 25.3%, for BHL-1, BHL-1N, BHL-2, BHL-2N, BHL-3, and BHL-3N,
respectively. Construct BHL-1N contains the same eight substitutions as BHL-1,
plus
lysine substitutions in the 18 additional amino acid residues in the amino
terminal
region. BHL-2 is the same as BHL-1 but with changes of amino acid residues 40
and 42
49
Applicant Ref. No.: 057IR-PCT.app
CA 02270289 1999-04-30
. . ~~ .. . .,
. . .. . . . . .
. . . .~.
. . . .
.. .. .., .~,. .. .
to Ala and amino acid residue 47 to lysine. Construct BHL-2N contains the same
11
substitutions as BHL-2, plus four lysine substitutions in the 18 additional
amino acid
residues in the amino terminal region. BHL-3 is the same as BHL-2 except that
residues
40 and 42 are changed to Gly and His, respectively. Construct BHL-3N contains
the
same 11 substitutions as BHL-3, plus the four lysine substitutions in 18
additional amino
acid residues in the amino terminal region. Onc: skilled in the art will
realize that essential
and non-wild-type amino acid residue substitutions will be tolerated at both
the same
positions substituted with lysine, and at other positions.
The active site loop region encompasses an extended loop region from about
amino acid residue 53 to about amino acid residue 70. Destabilization of the
reactive loop
was achieved by substituting the non-wild type amino acids residues at about
positions 53
to about 70. Amino acid residues were changed by primer mutagenesis.
Preferably, the
following mutations are made: Arg62 ~ Lys62, Arg65 -~ Lys65, Arg67 -~ Lys67,
Thr58
-~ A1a58 or G1y58, Met59 --~ Lys59, and GIuE~O ~ A1a60 or His60. However, it
will be
readily apparent to one skilled in the art that functionally equivalent
substitutions to those
described above will also be effective in the present invention.
In a preferred embodiment of the prese;nt invention, the present protein has
both
elevated essential amino acid content and reduced protease inhibitor activity.
Modification in the area by amino acid substitution or other means, destroys
the
hydrogen bonding and changes or reduces the protease inhibitor activity of
BHL.
Substitution of amino acid residues threonine, at position 58, and glutamic
acid, at
position 60, with glycine and histidine, respecaively, resulted in a protein
with lowered
protease iitor activity. Residue 59 is a critical residue in modifying
protease inhibitor
activity and changing specificity. When this residue was changed to a lysine,
the protease
inhibition specificity was changed from a chymotrypin inhibitor to a trypsin
inhibitor.
The present invention provides for the creation of a nutritionally enhanced
feed
from WT CI-2 through at least one lysine substitution of residues
1,18,11,17,19,34,41,56,59,62,67 and 73 (long versions BHL-1N, 2N, 3N) plus
residue 67
in BH2-2N and BH2-3N. Lysine substitutions in BHL-1,2 and 3 are at amino acid
residues 1,16,23,41,44,49 and 55, plus residue 47 in BHL-2 and BHL-3.
Example 3- Construction of Expression Cassettes
50 t~,%~ :rJV!~ SNP
h
Applicant Rcf. No.: 0571 R-PCT.app
CA 02270289 1999-04-30
., ..
,.
. . , , . , . .
..
. ~ , . , . . . ,.
. . . .
..
. . ., .. ... .. . ..
Vector construction was based upon the; published WT CI-2A sequence
information Williamson et al, Eur. J. Biochem 165: 99-106 (1987) and SEQ ID NO
13.
Methods for obtaining full length or truncated wild-type CI-2 DNA include, but
are not
limited to PCR amplification, from a barley (or other plant ) endosperm cDNA
library
using oligonucleotides derived from Seq. ID no 13 or from the published
sequence supra,
using probes derived from the same on a barley (or other plant ) endosperm
cDNA
library, or using a set of overlapping oligonuclc:otides that encompass the
gene.
BHL-1
The BHL-1 insert corresponds to SEQ ID NO '.l, plus start and stop codons.
Oligonucleotide pairs, N4394/N4395, and N4396/N4397, were annealed and ligated
together to make a 202 base pair double strandf:d DNA molecule with overhangs
compatible with Rca I and Nhe I restriction sites. PCR was performed on the
annealed
molecule using primers N5045 and N5046 to add a 5' Spe I site and 3' Hind III
site. The
PCR product was then restriction digested at those sites and ligated into
pBluescript II
KS+ at Spe I and Hind III sites. The insert was then removed by restriction
digestion with
Rca I and Hind III and was ligated into the Nco I and Hind III sites of pET28a
(Novagen)
to form the BHL-1 construct.
Oligonucleotide and primer sequences (5' to 3'):
N4394
1 CATGAAGCTG AAGACAGAGT GGCCGGAGTT GGTGGGGAAA
TCGGTGGAGA
51 AAGCCAAGAA GGTGATCCTG AAGGACAAGC CAGAGGCGCA
AATCATAGTT
101 CTGC
N4395
1 CAACCGGCAG AACTATGATT TGCGCCTCTG GCTTGTCCTT
CAGGATCACC
S 1 TTCTTGGCTT TCTCCACCGA TTTC:CCCACC AACTCCGGCC
ACTCTGTCTT
101 CAGCTT
51 _
r~ ~ .err.
..;';L':; ~:i_cj
Applicant Ref. No.: 05718-PCT.app
N4396
. ,. . ~ .. ,
.. ., . , , . ~ , . ,
..
. . . . . .., . ~ . . .
. . . , . .
. .. ., . >.
1 CGGTTGGTAC AAAGGTGACG AAGGAATATA AGATCGACCG
CGTCAAGCTC
51 TTTGTGGATA AAAAGGACAA CA7'CGCGCAG GTCCCCAGGG TCGG
N4397
1 CTAGCCGACC CTGGGGACCT GCGCGATGTT GTCCTTTTTA
TCCACAAAGA
51 GCTTGACGCG GTCGATCTTA TAT"TCCTTCG TCACCTTTGT AC
N5045
1 GTACTAGTCA TGAAGCTGAA GACAGA
N5046
1 GAGAAGCTTG CTAGCCGACC CTGGGGAC
b. BHL-2: The BHL-2 construct insert corresponds to SEQ ID NO 3, plus start
and stop codons. An overlap PCR strategy was used to make the BHL-2 construct.
PWO
polymerase from Boehringer-Mannheim was used for all PCR reactions.The primers
were
chosen to change 3 amino acids in the BHL-1 active site loop region, and to
create unique
AgeI and Hind III restriction sites flanking the active site loop, to
facilitate loop
replacement iri future constructs. A unique Rca I site (compatible with Nco I)
was
included at the S' end, and a unique Xho I site was included at the 3' end.
The overlap
PCR was done as follows: PCR was done with primers N13561 and N13564, using
the
BHL-1 construct as template. A separate PCR was done with primers N13563 and
N13562, again using the BHL-1 construct as template. The products from both
reactions
were gel purified and combined. Primer N13565, which overlapped regions on
both of
the PCR products, was then added and another l?CR was done to generate the
full-length
insert. The resulting product was amplified by ~~nother PCR with primers
N13561 and
N13562. It was subsequently suspected that a deletion was present in N13562
that caused
a frameshift near the 3' end of the PCR product. To avoid this frameshift
problem, a final
CA 02270289 1999-04-30
52
.. _. .~- , , -j~ ~ .
Applicant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
., ,. . ., ,. .
. . . ,. .
. . , ~ . . , . .., .
. . . . . . . . .
.. ,. m. ",. ~. .
PCR reaction was done with primers N13562 and N13905. The final PCR product
was
digested with Rca I and Xho I, and then ligated into the Nco I and Xho I sites
of pET 28b.
Note: Some primers had 6-oligonucleotide extensions to improve restriction
digestion
efficiency.
Primer sequences (5' TO 3'):
N13561
1 TTTTTTTCATGAAGCTGAAGACA
N13562 (as ordered)
1 TTTTTTCTCGAGGCTAGCCGACCCTGGGGA
N13563
1 ATCGACAAGGTCAAGCTTTTTC~TGGATAAAAAGGA
N13564
1 CACCTTTGTACCAACCGGTAGAACTATGATTTGCGC
N13565
1 GTTGGTACAAAGGTGGCGAAG~GCCTATAAGATCGACAAGGTCAAG
N13905
1 TTTTTTCTCGAGGCTAGCCGACCCTGGGGACCTGCGCTA
c. BHL-3: The BHL-3 construct insert corresponds to SEQ ID NO 5, plus start
and
stop codons. The BHL-2 construct was digested with Age I and Hind III, and the
region
between these sites was removed by gel purification. Oligonucleotide pairs,
N14471 and
N 14472, were annealed to make a double stranded DNA molecule with overhangs
compatible with Age I and Hind III restriction sites. The annealed product was
ligated
into the Age I and Hind III sites of the digested BHL-2 construct to yield the
BHL-3
construct.
Oligonucleotide Primer sequences (5' to 3'):
N 14471
1 CCGGTTGGTACAAAGGTGGGTAAGCATTATAAGATCGACAAGGTCA
N 14472
1 AGCTTGACCTTGTCGATCTTATAATGCTTACCCACCTTTGTACCAA
d. BHL-I N, BHL-2N, and BHL-3N
The BHL-1N, BHL-2N, and BHL-3N construct inserts correspond to SEQ ID No 9,
SEQ
ID NO 11, and SEQ ID NO 7, respectively, plus start and stop codons. Three
separate
PCR reactions were done with either the BHL-1, BHL-2, or BHL-3 constructs as
template. The primers for these reactions were N13771 and N13905. The
resulting PCR
products were digested with Rca I and Xho I and ligated into the Nco I and Xho
I sites of
pET 28b to yield the BHL-1N, BHL-2N, and BI~IL-3N constructs.
Primer sequences (S' to 3'):
N13771
53
A~,~ENDED SHEET
75529-49(S)
CA 02270289 2000-04-20
1
TTTTTTTCATGAAGTCGGTGGAGAAGAAACCGAAGGGTGTGAAGACAGG
50 TGCGGGTGACAAGCATAAGCTGAAGACAGAGTG
N13905 (already provided in BHL-2 description)
BHL-1N is an 83 residue polypeptide in which residues 1,8,11, and 17 were also
replaced with lysine. The resulting compound has the protein sequence
indicated in
Sequence LD. No.lO.
BHL-2N is an 83 residue polypeptide in which residues 1,8,11, and 17 were also
replaced with lysine. The resulting compound has the protein sequence
indicated in
Sequence LD. No.l2.
BHL-3N is an 83 residue polypeptide in which residues 1,8,11, and 17 were also
replaced with lysine. The resulting compound has the protein sequence
indicated in
Sequence LD. No.B.
Example 3 - Expression of BHL-1 in E. colt
E.rpression in E. colt
BHL-I, BHL-2, BHL-3, BHL-3N, and the truncated wild-type CI-2 (residues 19
through
6~ of SEQ ID NO. 14) were expressed in E colt using materials and methods from
Novagen, Inc. The Novagen expression vector pET-28 was used (pET-28a for WT CI-
2
and BHL-1, and pET-28b for the other proteins). Ecoli strains BL21 (DE-3) or
BL21 (DE-
3)pLysS were used. Cultures were typically grown until an OD at 600 nm of 0.8
to 1.0,
and then induced with 1 mM IPTG and grown another 2.5 to 5 hours before
harvesting.
Induction at an OD as low as 0.4 was also done successfully. Growth
temperatures of 37
degrees centigrade and 30 degrees centigrade were both used successfully. The
media
used was 2xYT plus the appropriate antibiotic at the concentration recommended
in the
Novagen manual.
Purification
a. WT CI-2 (truncated)-- Lysis buffer was 50 mM Tris-HCI, pH 8.0, 1 mM EDTA,
150 mM NaCI. The protein was precipitated with 70% ammonium sulfate. The
pallet
was dissolved and dialyzed against 50 mM Tris-HCI, pH 8.6. The protein was
~.oa.d~d
onto a Hi-Trap Q column, and the unbound fraction was collected and
precipitated i~~ 70%
ammonium sulfate. The pellet was dissolved in 50 mM sodium phosphate, pH 7.0,
200
Trade-mark 54
CA 02270289 2000-04-20
75529-49(S)
mM NaCI, and fractionated on a Superdex-75 26/60 gel filtration column.
Fractions wire
pooled and concentrated.
b. BHL-I--Lysis buffer was 50 mM sodium phosphate, pH 7.0, 1 mM EDTA.
The protein was loaded onto an SP Sepharose FF 16/10 column, washed with 150
mM
NaCI in 50 mIVI sodium phosphate, pH 7.0, and then eluted with an NaCI
gradient in SO
mM sodium phosphate. BHL-1 eluted at approximately 200 mM NaCI. Fractions were
pooled and concentrated.
c. BHL-2, BHL-3, and BHL-3N--Lysis buffer was 50 mM Hepes, pH 8.0, 2mM
EDTA, 0.1% Triton X-100, and 0.5 mg/m1 Iysozyme. The protein was loaded onto
an
SP-Sepharose cation exchange column (typically a 5 to 10 ml size), washed with
150 mM
NaCI in 50 mM sodium phosphate, pH 7.0, and eluted with 500 mM NaCI in 50 mM
sodium phosphate, pH 7Ø The protein was concentrated and then subjected to
Superdex-
75 gel filtration chromatography twice.
d. BHL-1--Lysis buffer was SO mM sodium phosphate, pH 7.0, 1 mNl EDTA.
The protein was loaded onto an SP Sepharose FF 16/10 column, washed with 150
mM
NaC1 in 50 mM sodium phosphate, pH 7.0, and then eluted with an NaCI gradient
in 50
mM sodium phosphate. BHL-I eluted at approximately 200 mNI NaCI. Fractions
were
pooled and concentrated.
e. BHL-2, BHL-3, and BHL-3N--Lysis buffer was 50 mM Hepes, pH 8.0, 2miVI
EDTA, 0.1% Triton X-100, and 0.5 mg/ml lysozyme. The protein was loaded onto
an
SP-Sepharose cation exchange column (typically a 5 to 10 ml size), washed with
150 mM
NaCI in 50 mM sodium phosphate, pH 7.0, and eluted with 500 mM NaCI in 50 mM
sodium phosphate, pH 7Ø The protein was concentrated and then subjected to
Superdex-
75 gel filtration chromatography twice.
4. Storage
The purified proteins were stored long term by freezing in liquid nitrogen and
keeping frozen at -70 degrees centigrade.
5. Verification of recombinant protein identity.
a. DNA sequencing--
The insert region of these pET 28 constructs was co~rmed by DNA sequencing.
b. N-terminal protein sequencing --
Trade-mark 55
Appl'~.cant Ref. No.: 0571 R-PCT.app
CA 02270289 1999-04-30
..
.. . . .
~ ,j~
. . . . ..~ . ~ .
~ . ,. ..
. . .. ..~ ... ,~.. ,. ..
100 pg of purified BHL-3 were digested with 1 pg of chymotrypsin (Sigma
catalog # C-
4129) for 30 min at 37 degrees centigrade in 50 mM sodium phosphate, pH 7Ø
The
resulting chymotryptic fragments were purified by reversed phase
chromatography, using
an acetonitrile gradient for elution. Three pure peaks were observed and were
sent to the
University of Michigan Medical School Protein. Structure Facility for N-
terminal
sequencing (6 cycles). Peak 1 had an N-terminal sequence of val-asp-lys-lys-
asp-asn.
Peak 2 had an N-terminal sequence of lys-ile-as.p-lys-val-lys. Peak 3 had an N-
terminal
sequence of met-lys-leu-lys-thr-glu. These results demonstrate that
chymotrypsin cleaved
BHL-3 after tyr-61 and phe-69. The N-terminal sequences all match exactly the
BHL-3
expected sequence, assuming that the start methionine was largely retained in
the
recombinant protein. This experiment verifies that the protein we expressed in
and
purified from E. coli was BHL-3. Furthermore, SDS-PAGE analysis with 16.5%
Tris-
Tricine precast gels from Biorad showed a similar mobility of BHL-1 and BHL-2
with the
confirmed BHL-3 protein, as would be expected because BHL-1 and BHL-2 have
molecular masses very similar to that of BHL-3.
160 ~g of BHL-3N were digested with 1.6 ~g pepsin overnight, and the resulting
peptic fragments were purified by reversed phase chromatography. Five of the
resulting
peaks were sent to the Iowa State University Protein Facility for N-terminal
sequencing
through four cycles. The N-terminal sequences of the 5 peaks were: val-gly-lys-
ser, phe-
val-asp-lys, pro-val-gly-thr, met-lys-ser-val, and ile-ile-val-leu, all of
which exactly match
the expected BHL-3N sequence, assuming that 'the start methionine was largely
retained
in this recombinant protein. This experiment verifies that the protein we
expressed in and
purified fre~E. coli was BHL-3N.
c. Protease inhibition--
The obvious protease inhibitory activity observf:d for BHL-1 and for the wild-
type protein
are further evidence that we have purified the a};pected proteins from E coli.
The details
of these protease inhibition experiments are described next.
56
r ~.,._~ r;; .Tn c~~c-;~
CA 02270289 2000-04-20
75529-49 (S)
The following experiments utilized truncated wild type CI-2 as represented as
nt. SS-249
in Seq. ID NO. 13 with addition of start and stop codons.
E~camgle S - Protease Inhibition assays and Proteolitic Digests
S a. Chymotrypsin
Protease activity was measured by an increase in absorbance
at 40S nm.
Sigma Chymotrypsin type iI (Bovine pancreas) Cat. # C-4129.
Substrate - Sigma cat. # .~-7388. N-Succinyl-Ala-Ala-Pro-phe-p
vitro anilide
or BHL protein used, l nM chymotrypsin, 1mM substrate,
200 ul vol~ne
luM BSA included in control (no CI-2, no BHL).
Preincubated 30 min 37 C., then added substrate to start
and kept at 37 C.
Buffer 0.2M tris - HCl pH 8.0
Read Abs 40S nm - 30 min
Protease Activity - % of Control ABS. 40S nm
1S
Abs. At 405 nm
Rep. 1 Rep. 2 Mean (S.D.) Using % control data
Controll-value 0.350 0.299
control 100.0 100.0 100.0
WT CI-2-value .042 .018
control 12.0 6.0 9.0 (4.2)
BHL-1-value .289 .274
control 82.6 91.6 87.1 (6.4)
BHL-2-value .309 .318
control 88.3 106.4 97.4 (12.8)
BHL-3-value .346 .31 S
control 98.9 lOS.4 102.2 (4.6)
BHL-3N-value .318 .31 S
control 90.9 lOS.4 98.2 (10.3)
S7
CA 02270289 2000-04-20
75529-49 (S)
b. Subtilisin
Subtilisin Carlsberg lichenif'ormis (Sigma cat.
from Bacillus # P-5380)
Substrate and buffer
same as for chymotrypsin
exper. 200 ul reaction
volume
1 uM CI2 or BHL
1nM subtilisin
1mM Substrate
room temp (25 C)
30 min. preincubatedthen added
substrate
and read
absorbance
at 40~nm
30 min. data used
luM BSA used or BHL)
in control (no
CI2
Abs. At 405 nm
Rep. 1 Rep. 2 Mean (S.D.) Using % ontrol
c data
Controll-value 2.171 1.834
control 100.0 100.0 100.0
WT CI-2-value .014 .002
control 0.6 0 0.3 (0.4)
BHL-1-value .286 .295
control 13.2 16.1 14.7 {2.1)
BHL-2-value 1.692 1.569
control 77.9 85.6 81.8 (5.4)
BHL-3-value 7.056 1.960
control 94.7 106.9 100.8 (8.6)
BHL-3N-value 2.103 1.729
control 96.9 94.3 95.6 (1.8)
58
CA 02270289 2000-04-20
75529-49(S)
c. Trypsin
Bovine pancreas trypsin (Sigma cat #T-8919)
Substrate S-2222 (chromogenix): N-benzoyl-2-isolenuel-Lglutamyl-glycyl-L-
arginine-p-
nitroaniline
buffer: SOmMTris pH 7.5, 2mM NaCI, 2mM CaCl2, 0.005 % TritonX-100.
30 min. preincubation 25°, then added substrate and kept at 25°;
these are 30 minute
values.
1 mM substrate, SuM CI-2 or BHL, O.SnM trypsin, no BSA in control. 200 ul
reaction
volume .
Abs. At 405nm
Rep. 1 Rep. 2 Rep. 3 Rep. 4 Mean (S.D.) Using
Control Data
Control l- .505 .533 .473 .391
value
control 100.0 100.0 100.0 100.0 100.0
WT CI-2- .561 .533 .474 .420
value
control 111.1 100.0 100.2 107.4 104.7 (5.5)
BHL-1-value .072 .096 .041 .057
control 14.3 18.0 8.7 14.6 13.9 (3.9)
BHL-2-value .436 .481 .404 .405
control 86.3 90.2 85.4 103.5 91.4 (8.4)
BHL-3-value .536 .557 .456 .430
control 106.1 104.5 96.4 110.0 104.3 (5.7)
BHL-3N- .542 .583 .490 .437
value
control 107.3 109.4 103.6 111.8 108.0 (3.5)
Trade-mark
59
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
,. , . . .
.. .. . . . .. . . , , ,
. . , . . . , . , .
. , . . , ,
,. ., .,. .". ,. ..
d. Elastase
Porcine elastase IV (Sigma)
Type Cat#
E-0258
Substrate: Sigma
S-4760 N-succinyl-ala-ala-ala-p-nitroanile
buffer: 0.2M Tris
HC1 pH 8.0 200
ul reactive volume
50nM elastase,
2 uM CI-2 or BHL;
1mM substrate
luM BSA in control
min. preincub, , then ubstrate. Kept at 25; 30
added min. data
s
Abs. At 405 nm
10
Rep. 1 Rep. 2 Mean (sp) Using % control
data
Control 1-value 1.416 1.461
control 100.0 100.0 100.0
WT CI-2-value .030 .049
control 2.1 3.4 2.8 (0.9)
BHL-1-value 1.519 1.459
control 107.3 99.9 103.6 (5.2)
BHL-2-value 1.558 1.509
control 110.0 103.3 106.7 (4.7)
BHL-3-value 1.587 1.493
control 112.1 102.2 107.2 (7.0)
BHL-3N-value 1.527 1.481
control 107.8 101.4 104.6 (4.5)
r !
CA 02270289 2000-04-20
75529-49(S)
protease inhibition summary - % of control
Protein Chymotrypsin Trypsin Elastase Subtilisin
WT CI-2 9.0 104.7 2.8 0.3
BHL-1 87.1 13.9 103.6 14.7
BHL-2 97.4 91.4 106.7 81.8
BHL-3 102.2 104.3 107.2 100.8
BHL-3N 9$.2 108.0 104.6 95.6
These experiments show that BHL-2, BHL-3 and BHL-3N have reduced protease
inhibition activity compared to WT CI-2 .
Digestion by trypsin
The purified proteins were incubated at 37 degrees centigrade with a 100:1
(wt:wt)
ratio of BHL protein or wild-type CI-2 : trypsin for l5min, 30 min, 1 hr, 2
hr, or 4 hr.
Incubation buffer was 50 mM sodium phosphate, pH 7Ø Bovine pancreas trypsin
was
used (Sigma catalog # T-8918). Digestion was assessed by SDS-PAGE with 16.5%
Tris-
Tricine precast gels from Biorad. The BHL-2, BHL-3, and BHL-3N proteins were
digested by trypsin in 15 minutes. In contrast, the BHL-1 and wild-type
truncated CI-2
proteins were resistant to trypsin. This experiment confirmed that the BHL-2,
BHL-3,
and BHL-3N proteins are not effective inhibitors of trypsin.
Digestion by chymotrypsin.
The purified proteins were incubated at 37 degrees centigrade with a 100:1
(wt:wt)
ratio of BHL protein or wild-type CI-2 : chymotrypsin for 15min, 30 min, 1 hr,
2 hr, ox 4
hr. Incubation buffer was 50 mM sodium phosphate, pH 7Ø Bovine pancreas
chymotrypsin type II (Sigma catalog # S-7388 was used. Digestion was assessed
by
SDS-PAGE with 16.5% precast Tris-Tricine gels from Biorad. BHL-2, BHL-3, and
BHL-3N proteins were digested by chymotrypsin in 15 minutes. In contrast, BHL-
1 and
wild-type CI-2 proteins were resistant to chymotrypsin. This experiment
confirrraed that
BHL-2, BHL-3, and BHL-3N are not effective inhibitors of chymotrypsin.
Digestion in simulated gastric fluid.
61
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PC'T.app
.. , . .. . ,.
.. , . . .. . , , .
. . . ~ . ... . . . . ,., ,
. . . , .
. ,. ., ... ..,. .. .,
Simulated gastric fluid was prepared by dissolving 20 mg NaCI and 32 mg of
pepsin in 70 pl of HCl plus enough water to make 10 ml. Porcine stomach pepsin
(Sigma
cat # P-6887) was used. SO ~l of 1 mg/ml BHL-3N or wild-type CI-2 protein were
incubated with 250 pl simulated gastric fluid at :37 degrees centigrade. At 1
S sec, 30 sec,
1 min, 5 min, and 30 min, 40 ~1 aliquots were removed to a stop solution
consisting of 40
~l 2X Tris-Tricine SDS sample buffer (Biorad) that also contained 3 pl of 1 M
Tris-HC1,
pH 8.0 and 0.1 mg/ml pepstatin A (Boehringer-lvlannheim cat # 60010).
Digestion was
assessed by 16.5% Tris-Tricine SDS-PAGE (pre:cast gels from Biorad).
Both BHL-3N and wild-type CI-2 were digested in simulated gastric fluid in 15
seconds. This experiment suggests that our engineered proteins and even the
wild-type
protein would likely be digested into proteolytic; fragments in the stomach of
humans or
monogastric animals.
Digestion in simulated intestinal fluid.
Simulated intestinal fluid was prepared by dissolving 68 mg of monobasic
potassium phosphate in 2.5 ml of water, adding 1.9 ml of 0.2 N sodium
hydroxide and 4
ml of water. Then 2.0 g porcine pancreatin (Sigma catalog # P-7545) was added
and the
resulting solution was adjusted with 0.2N sodium hydroxide to a pH of 7.5.
Water was
added to make a final volume of 10 ml.
50 ~g of BHL-3N or wild-type CI-2 protein in 50 pl were incubated with 250 ~1
simulated-~estinal fluid at 37 degrees centigrade . At 15 sec, 30 sec, 1 min,
5 min, and
min, 40 pl aliquots were removed and added to 40 pl of a stop solution
consisting of
25 2X Tris-Tricine SDS sample buffer (Biorad) containing 2 mM EDTA and 2mM
phenylmethylsulfonyl fluoride (Sigma catalog 3# P-7626). Digestion was
assessed by 16.5
Tris-Tricine SDS-PAGE (precast gels form l3iorad).
BHL-3N was digested by simulated, intcatinal fluid in 15 seconds. In contrast,
30 wild-type CI-2 was resistant to digestion for 30' minutes. This experiment
shows that in
the intestine of humans or monogastric animal~~, our engineered protein would
likely be
more digestible than the wild-type protein would be. These results are
consistent with the
CA 02270289 1999-04-30
Appl~,cant Ref. No.: 0~71R-PCT.app
. ~. . . . ,.
. . . , . . . ,
..~ . . . ", ,.
. . , . .
. . " , .., .,.. ,.
protease inhibition assays showing that BHL-3N was not an effective protease
inhibitor.
The inventive protein was digested in less than i:me minutes, less than one
and less than
30 seconds.
Digestion in simulated gastric fluid
Simulated gastric fluid was prepared by dissolving 20 mg NaCI and 32 mg of
pepsin in 70 ~l of HCl plus enough water to make 10 ml. Porcine stomach pepsin
(Sigma
cat # P-6887) was used. 50 ~l of 1 mg/ml BHL-3N or wild-type CI-2 were
incubated
with 250 ~l simulated gastric fluid at 37 degrees centigrade. At 15 sec, 30
sec, 1 min, 5
min, and 30 min, 40 ~1 aliquots were removed to a stop solution consisting of
40 ul 2X
Tris-Tricine SDS sample buffer (Biorad) that al.>o contained 3 pl of 1 M Tris-
HCI, pH 8.0
and 0.1 mg/ml pepstatin A (Boehringer-Mannheim cat # 60010). Digestion was
assessed
by 16.5% Tris-Tricine SDS-PAGE (precast gels. from BioradTM).
Both BHL-3N and wild-type CI-2 were digested in simulated gastric fluid in 15
seconds. This experiment suggests that our engineered proteins and even the
wild-type
protein would likely be digested into proteolytic: fragments in the stomach of
humans or
monogastric animals.
Digestion in simulated intestinal fluid.
Simulated intestinal fluid was prepared by dissolving 68 mg of monobasic
potassium phosphate in 2.5 ml of water, adding 1.9 ml of 0.2 N sodium
hydroxide and 4
ml of water. Then 2.0 g porcine pancreatin (Sig:ma catalog # P-7545) was added
and the
resulting sel.~ion was adjusted with 0.2N sodium hydroxide to a pH of 7.5.
Water was
added to make a final volume of 10 ml.
50 ~1 of lmg/ml BHL-3N or wild-type CI-2 were incubated with 250 ~1 simulated
intestinal fluid at 37 degrees centigrade . At 15 sec, 30 sec, 1 min, 5 min,
and 30 min, 40
~l aliquots were removed and added to 40 ~1 of a stop solution consisting of
2X Tris-
Tricine SDS sample buffer (Biorad) containing 2 mM EDTA and 2mM
phenylmethylsulfonyl fluoride (Sigma catalog #~ P-7626). Digestion was
assessed by 16.5
Tris-Tricine SDS-PAGE (precast gels form Biorad).
63
A?~~ENDED SHEET
Applicant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
,.
. . . . ,. . , .
. . . ~ .., . . ~,
.
. . , .
.. .. ... .... " ..
BHL-3N was digested by simulated intestinal fluid in 15 seconds. In contrast,
wild-type
CI-2 was resistant to digestion for 30 minutes. ~Chis experiment shows that in
the
intestine of humans or monogastric animals, our engineered protein would
likely be more
digestible than the wild-type protein would be. 'These results are consistent
with the
protease inhibition assays showing that BHL-3I~1 was not an effective protease
inhibitor.
The inventive proteins were digested in less than five minutes, less than one
minute and
less than 30 seconds.
Example 6 - Protein Conformation
Wild type CI-2, BHL-I, BHL-2, BHL-3 and BHL-3N at proteins concentrations of
approximately 0.16mg/ml in IOmM sodium phosphate, pH = 7.0 were prepared and
sent
to the University of Michigan Medical Scho~~l Protein Structure Facility for
circular
dichroism analysis. Data indicates that the substituted proteins BHL-1, BHL-2
and BHL-
3 have very similar CD spectra confirming that the BHL proteins fold into a
structure
similar to the wild type CI-2.
Example 7 - Thermodynamic stability
Equilibrium denaturation experiments were done to assess the thermodynamic
stability of the engineered and wild-type proteins, following the method of
Pace et al.
(Meth. Enzym. 131:266-280). The engineered or wild-type proteins at a
concentration of
2 pM were incubated 18 hours at 25 degrees centigrade in 10 mM sodium
phosphate, pH
7.0, with various concentrations of guanidine-hydrochloride. Unfolding of the
proteins
was monite~l by measuring intrinsic fluorescence at 25 degrees centigrade,
using an
excitation wavelength of 280 nm and an emission wavelength of 356 nm. The
guanidine-
hydrochloride concentration sufficient for 50% unfolding was found to be 3.9M
for wild-
type, 2.4M for BHL-1, and 0.9M for BHL-2, BI:IL-3, and BHL-3N. These
experiments
showed that BHL-1 has a higher thermodynamic stability than do the other
engineered
proteins, but that all of the engineered proteins lhave a lower thermodynamic
stability than
does the wild-type protein.
Example 8 - Accessibility of the Tryptophan of BHL Proteins to Acrylamide
64
~It~Elw~~~~ Si'I~C
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
.. , ,
.. . . . . . .~ . . . . . .
. . . . .,. , . . . ". ,.
~ . . , . ~ , .
.. .. ... .." .. ..
Acrylamide effectively quenches the fluorescence of accessible tryptophan
residues in
proteins. We examined fluorescence quenching of the tryptophan residue of the
BHL
proteins and of the truncated WT CI-2, in the prcaence or absence of 6M
guanidine-
hydrochloride. An excitation wavelength of 295 nm was used. Emission
wavelengths of
337 nm and 356 nm were used for the samples v~ithout guanidine-HCl and with
guanidine-HCI, respectively. Protein concentrations of 20 pM or 2 pM were used
for the
samples without, and with guanidine-HCI, respectively. Samples were in 10 mM
sodium
phosphate, pH 7.0, and contained acrylamide at the following concentrations:
0, 0.0196M,
0.0385M, 0.0566M, 0.0741M, 0.0909M, 0.1071M, 0.01228M, or 0.1379M. The
equation
of Mclure and Edelman (Biochem 6: 559-566) vvas used to correct for self
absorption of
light by acrylamide. Fo/F was plotted against the molar acrylamide
concentration, where
Fo = fluorescence intensity without acrylamide, and F = fluorescence intensity
with
acrylamide. The slope of each line (known as the Stern-Volmer constant) was
determined. The mean of 2 experiments is presented below. Values in
parentheses are
standard deviations.
Protein 6M guanidine-HC1 Slope
BHL-1 - 3.5 (0.3)
BHL-1 + 16.9 (1.3)
BHL-2 - 4.6 (0.4)
BHL-2 + 19.0 (0.1)
BHL-3 - 2.4 (0.2)
BHL-3 + 17.5 (0.04)
BHL-3N - 5.8 (0.1)
BHL-3N + 16.6 (0.6)
WT CI-2 - 1.7 (0.1)
(truncated.
WT CI-2 + 15.7(2.1)
(truncated)
Example 9 - Stabilization ~ Disulfide Bonds,
An examination of the WI-CI 2 three dimensional structure has identified three
pairs of
residues (Glu-23 and Arg-81, Thr-22 and Val-82, and Val-53 and Val-70) with an
alpha
carbon distance appropriate for disulfide formation. Constructs designed to
substitute
these residues with cysteines will be prepared.
,,~. r.,.. ~.', i
CA 02270289 1999-08-OS
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: PIONEER HI-BRED INTERNATIONAL, INC.
(ii) TITLE OF INVENTION: PROTEINS WITH ENHANCED LEVELS
OF ESSENTIAL AMINO ACIDS
(iii) NUMBER OF SEQUENCES: 26
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
lO (C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
2 O (A) APPLICATION NUMBER: CA 2,270,289
(B) FILING DATE: 31-OCT-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/740,682
(B) FILING DATE: O1-NOV-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 75529-49
66
75529-49
, CA 02270289 1999-08-OS
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 195 base pairs
66a
75529-49
CA 02270289 1999-04-30
Applicant
Ref. " , , >. . , ,
No.: ~, > , >
05718-PCT.app ". .
' , ~ ,. .,. >".
(B) TYPE: nucleic >
acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY: linear
S (ii) MOLECULE
TYPE:
cDNA
(ix)
FEATURE:
(A) NAME/KEY: Coding quence:
Se
(B) LOCATION: 1...195
IO (D) OTHER INFORMATION:
(xi) SEQ7:DNO:1:
SEQUENCE
DESCRIPTION:
AAG CTG ACA GAG TGG CCG TTGG7.>GGGG AAATCGGTG GAGAAA 48
AAG GAG
1S Lys Leu Thr Glu Trp Pro LeuVa1Gly LysSerVal GluLys
Lys Glu
1 5 1C> 15
GCC AAG GTG ATC CTG AAG AAGCC:AGAG GCGCAAATC ATAGTT 96
AAG GAC
Ala Lys Val Ile Leu Lys LysProGlu AlaGlnIle IleVal
Lys Asp
20 20 25 30
CTG CCG GGT ACA AAG GTG AAGG~,ATAT AAGATCGAC CGCGTC 144
GTT ACG
Leu Pro Gly Thr Lys Val LysG7.uTyr LysIleAsp ArgVal
Val Thr
35 40 45
2S
AAG CTC GTG GAT AAA AAG AACA7.'CGCG CAGGTCCCC AGGGTC 192
TTT GAC
Lys Leu Val Asp Lys Lys AsnI7.eAla GlnValPro ArgVal
Phe Asp
50 55 60
30 GGC 195
Gly
65
3S
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
40 (B) TYPE: amino acid
---~: STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
4S (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: :CDN0:2:
SEQ
Lys Leu Lys Thr Glu Trp Pro ValGlyLys Ser Glu
Glu Leu Val Lys
SO i s l 15
Ala Lys Lys Val Ile Leu Lys ProGluAla Gln Ile
Asp Lys Ile Val
20 25 30
Leu Pro Val Gly Thr Lys Val G:LuTyrLys Ile Arg
Thr Lys Asp Val
35 40 45
SS Lys Leu Phe Val Asp Lys Lys I:LeAlaGln Val Arg
Asp Asn Pro Val
50 55 60
Gly
65
67
AMENDED ShiEET
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PC?.app
..
..
. . . . ., .~~. . . .
. . ~ . .., . . . ." ,
~ ~ . . . . . .
,. ., ... .". .. ,.
(2)
INFORMATION
FOR
SEQ
ID
N0:3:
S
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 195 base rs
pai
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
IS (A) NAME/KEY: Coding quence
Se
(B) LOCATION: 1...195
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION:SEQID N0:3:
AAG CTG AAG ACA GAG TGG CCG TTGGTGGGGAAA TCGGTGGAG AAA 48
GAG
Lys Leu Lys Thr Glu Trp Pro LeuValGlyLys SerValG1u Lys
Glu
1 5 10 15
2S GCC AAG AAG GTG ATC CTG AAG AAGCC'AGAGGCG CAAATCATA GTT 96
GAC
Ala Lys Lys Val Ile Leu Lys LysProGluAla GlnIleIle Val
Asp
20 25 30
CTA CCG GTT GGT ACA AAG GTG AAGGC'CTATAAG ATCGACAAG GTC 144
GCG
Leu Pro Val Gly Thr Lys Val LysAlaTyrLys IleAspLys Val
Ala
35 40 45
AAG CTT TTT GTG GAT AAA AAG AACATCGCGCAG GTCCCCAGG GTC 192
GAC
Lys Leu Phe Val Asp Lys Lys AsnIleAlaGln ValProArg Val
Asp
~S 50 55 60
GGC 195
Gly
40
(2) INFORMATION FOR SEQ ID NC1:4:
4S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
S0
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ 7:D N0:4:
SS
Lys Leu Lys Thr Glu Trp Pro Glu Leu Val Gly Lys Ser Val Glu Lys
1 5 10 15
Ala Lys Lys Val Ile Leu Lys Asp Lys Pro Glu Ala Gln Ile Ile Val
68
AMENDED SHEET
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
w '
.. , ~. . . , . . .~ , , .
.., . . . . ",
. . ~ . ~ ~ .
.. .. ... " .. .. ..
20 25 30
Leu Pro Val Gly Thr Lys Val Ala Lys Ala Tyr Lys Ile Asp Lys Val
35 40 45
Lys Leu Phe Val Asp Lys Lys Asp Asn Il.e Ala Gln Val Pro Arg Val
S 50 55 60
Gly
10
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 195 base rs
pai
IS (B)
TYPE:
nucleic
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
2O (ix) FEATURE:
(A) NAME/KEY: Coding quence.
Se
(B) LOCATION: 1...195
(D) OTHER INFORMATION:
2S
(xi) SEQUENCE DESCRIPTION:SEQ N0:5:
:CD
AAG CTG AAG ACA GAG TGG CCG TTGG'CGGGG AAATCGGTG GAGAAA 48
GAG
Lys Leu Lys Thr Glu Trp Pro LeuValGly LysSerVal GluLys
Glu
3O 1 5 17 15
GCC AAG AAG GTG ATC CTG AAG AAGCCAGAG GCGCAAATC ATAGTT 96
GAC
Ala Lys Lys Val Ile Leu Lys LysP:roGlu AlaGlnIle IleVal
Asp
20 25 30
3S
CTA CCG GTT GGT ACA AAG GTG AAGC..~TTAT AAGATCGAC AAGGTC 144
GGT
Leu Pro Val Gly Thr Lys Val LysHisTyr LysIleAsp LysVal
Gly
35 40 45
4O AAG CTT TTT GTG GAT AAA AAG AACATCGCG CAGGTCCCC AGGGTC 192
GAC
Lys Leu Phe Val Asp Lys Lys AsnIleAla GlnValPro ArgVal
Asp
50 55 60
GGC 195
4S G1y
SO (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
(B) TYPE: amino acid
SS (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
69
AMENDEt~ C'irrT
CA 02270289 1999-04-30
Applicant Ref. No.: 0571 R-PCT.app
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
~ ..
.. , ..
. , . . . . .. . . . . .
~ . . . ... . . . . ~.~ ..
. . . . .
.. ., .,. ..., .. .,
$ Lys Leu Lys Thr Glu Trp Pro Glu Leu V,~1 Gly Lys Ser Val Glu Lys
1 5 10 15
Ala Lys Lys Val Ile Leu Lys Asp Lys P:ro Glu Ala Gln Ile Ile Val
20 25 30
Leu Pro Val Gly Thr Lys Val Gly Lys His Tyr Lys Ile Asp Lys Val
35 40 45
Lys Leu Phe Val Asp Lys Lys Asp Asn ILe Ala Gln Val Pro Arg Val
50 55 60
Gly
1$
°,C~D H~Ei
,,,, r,<~
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
~ . .. ,
..
,. .. . . .~ . .. . . . . . .
. . . . . .,. . . . . .., ..
~ ~ . ,
.. .. . . ..,. .. ..
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 base pairs
S (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
lO (ix) FEATURE:
(A) NAME/KEY: Coding Sequence:
(B) LOCATION: 1...249
(D) OTHER INFORMATION:
1S
(xi) SEQUENCE DESCRIPTION: SEQ 7:D N0:7:
AAG TCG GTG GAG AAG AAA CCG AAG GGT G7.'G AAG ACA GGT GCG GGT GAC 48
Lys Ser Val Glu Lys Lys Pro Lys Gly Val Lys Thr Gly Ala Gly Asp
20 1 5 10 15
2S
AAG CAT AAG CTG AAG ACA GAG TGG CCG G71G TTG GTG GGG AAA TCG GTG 96
Lys His Lys Leu Lys Thr Glu Trp Pro G:_u Leu Val Gly Lys Ser Val
20 25 30
GAG AAA GCC AAG AAG GTG ATC CTG AAG G~~.C AAG CCA GAG GCG CAA ATC 144
Glu Lys Ala Lys Lys Val Ile Leu Lys Asp Lys Pro Glu Ala Gln Ile
35 40 45
3O ATA GTT CTA CCG GTT GGT ACA AAG GTG GGT AAG CAT TAT AAG ATC GAC 192
Ile Val Leu Pro Val Gly Thr Lys Val G:Ly Lys His Tyr Lys Ile Asp
50 55 60
AAG GTC AAG CTT TTT GTG GAT AAA AAG G~3C AAC ATC GCG CAG GTC CCC 240
3$ Lys Val Lys Leu Phe Val Asp Lys Lys Asp Asn Ile Ala Gln Val Pro
65 70 75 80
AGG GTC GGC 249
Arg Val Gly
(2) INFORMATION FOR SEQ ID Nc~:B:
4S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SO
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
SS
Lys Ser Val Glu Lys Lys Pro Lys Gly V,~1 Lys Thr Gly Ala Gly Asp
1 5 1D 15
Lys His Lys Leu Lys Thr Glu Trp Pro Glu Leu Val Gly Lys Ser Val
71
,4,';,-.n;r1'~; ~;:~~y_~:
CA 02270289 1999-04-30
Applicant Ref. No.: 0571 R-PCT.app
~. ., . .~ ,. "
. . . . .. . . , , , ,
. . . . . ." . . . . ,., ,
. . " ,
. . .. .. .~. .,.,
20 25 30
Glu Lys Ala Lys Lys Val Ile Leu Lys A;sp Lys Pro Glu Ala Gln Ile
35 40 45
Ile Val Leu Pro Val Gly Thr Lys Val G:Ly Lys His Tyr Lys Ile Asp
S 50 55 60
Lys Val Lys Leu Phe Val Asp Lys Lys As~~ Asn Ile Ala Gln Val Pro
65 70 75 80
Arg Val Gly
(2) INFORMATION FOR SEQ ID N0:9:
IS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
2S (B) LOCATION: 1...249
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
3O AAG TCG GTG GAG AAG AAA CCG AAG GGT G'TG AAG ACA GGT GCG GGT GAC 48
Lys Ser Val Glu Lys Lys Pro Lys Gly Val Lys Thr Gly Ala Gly Asp
1 5 10 15
AAG CAT AAG CTG AAG ACA GAG TGG CCG G.AG TTG GTG GGG AAA TCG GTG 96
3S Lys His Lys Leu Lys Thr Glu Trp Pro Glu Leu Val Gly Lys Ser Val
20 25 30
GAG AAA GCC AAG AAG GTG ATC CTG AAG G.AC AAG CCA GAG GCG CAA ATC 144
Glu Lys Ala Lys Lys Val Ile Leu Lys Asp Lys Pro Glu Ala Gln Ile
40 35 40 45
ATA GTT CTA CCG GTT GGT ACA AAG GTG ACG AAG GAA TAT AAG ATC GAC 192
Ile Val Leu Pro Val Gly Thr Lys Val Thr Lys Glu Tyr Lys Ile Asp
50 55 60
4S
CGC GTC AAG CTT TTT GTG GAT AAA AAG GAC AAC ATC GCG CAG GTC CCC 240
Arg Val Lys Leu Phe Val Asp Lys Lys Asp Asn Ile Ala Gln Val Pro
65 70 75 80
SO AGG GTC GGC 249
Arg Val Gly
SS (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids
72
;,i;: ~ ~~.: ~'-%
Applicant Rcf. No.: 05718-PCT.app
CA 02270289 1999-04-30
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
73
;; ~.;~ . ~:,=l-
CA 02270289 1999-04-30
Appi:cant Ref. No.: 05718-PCT.app
,. .. . .. ,.
.. , . . . . .. . . . , .
. . . , , .,. . . . . ."
. . . ,
" .. ... .... ,. .,
Lys Ser Val Glu Lys Lys Pro Lys Gly Val Lys Thr Gly Ala Gly Asp
1 5 10 15
Lys HisLys LeuLys ThrGluTrpPro G:LuLeuVa1 GlyLysSer Val
20 25 30
S Glu LysAla LysLys ValIleLeuLys AapLysPro GluAlaGln Ile
35 40 45
Ile ValLeu ProVal GlyThrLysVal TlzrLysGlu TyrLysIle Asp
50 55 60
Arg ValLys Phe LysLys Asp Ile Ala Val Pro
Leu Val Asn Gln
Asp
65 70 75 80
Arg ValGly
1S
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
ZS (ix) FEATURE:
(A) NAME/KEY: Coding Sequenc~_
(B) LOCATION: 1...249
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AAG TCG GTG GAG AAG AAA CCG AAG GGT G'TG AAG ACA GGT GCG GGT GAC 48
Lys Ser Val Glu Lys Lys Pro Lys Gly Val Lys Thr Gly Ala Gly Asp
3S 1 5 10 15
AAG CAT AAG CTG AAG ACA GAG TGG CCG G.AG TTG GTG GGG AAA TCG GTG 96
Lys His Lys Leu Lys Thr Glu Trp Pro Glu Leu Val Gly Lys Ser Val
20 25 30
GAG P.AA~C AAG AAG GTG ATC CTG AAG GAC AAG CCA GAG GCG CAA ATC 144
Glu Lys Ala Lys Lys Val Ile Leu Lys Asp Lys Pro Glu Ala Gln Ile
35 40 45
4S ATA GTT CTA CCG GTT GGT ACA AAG GTG GCG AAG GCC TAT AAG ATC GAC 192
Ile Val Leu Pro Val Gly Thr Lys Val Ala Lys Ala Tyr Lys Ile Asp
55 60
AAG GTC AAG CTT TTT GTG GAT AAA AAG GAC AAC ATC GCG CAG GTC CCC 240
SO Lys Val Lys Leu Phe Val Asp Lys Lys Asp Asn Ile Ala Gln Val Pro
65 70 75 80
SS
AGG GTC GGC 249
Arg Val Gly
(2) INFORMATION FOR SEQ TD N0:12:
74
AppIScant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
.. . .,
. . . . ,. . . , , . ,
. . . . , ... . . . ., ,
~ , . ,
~ . " " ." ,... " .,
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Lys Ser Val Glu Lys Lys Pro Lys Gly V;al Lys Thr Gly Ala Gly Asp
1 5 10 15
Lys His Lys Leu Lys Thr Glu Trp Pro G.Lu Leu Val Gly Lys Ser Val
25 30
Glu Lys Ala Lys Lys Val Ile Leu Lys A;sp Lys Pro Glu Ala Gln Ile
35 40 45
Ile Val Leu Pro Val Gly Thr Lys Val A:La Lys Ala Tyr Lys Ile Asp
20 50 55 60
Lys Val Lys Leu Phe Val Asp Lys Lys A;sp Asn Ile Ala Gln Val Pro
65 70 75 80
Arg Val Gly
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~S (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...249
4O (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ :ID N0:13:
AGT TCA GTG GAG AAG AAG CCG GAG GGA G'TG AAC ACC GGT GCT GGT GAC 48
4$ Ser Ser Val Glu Lys Lys Pro Glu Gly Val Asn Thr Gly Ala Gly Asp
1 5 10 15
CGT CAC AAC CTG AAG ACA GAG TGG CCA G:4G TTG GTG GGG AAA TCG GTG 96
Arg His Asn Leu Lys Thr Glu Trp Pro G:Lu Leu Val Gly Lys Ser Val
50 20 25 30
GAG GAG GCC AAG AAG GTG ATT CTG CAG GAC AAG CCA GAG GCG CAA ATC 144
Glu Glu Ala Lys Lys Val Ile Leu Gln A;sp Lys Pro Glu Ala Gln Ile
35 40 45
ATA GTT CTA CCG GTG GGG ACA ATT GTG ACC ATG GAA TAT CGG ATC GAC 192
Ile Val Leu Pro Val Gly Thr Ile Val Tlar Met Glu Tyr Arg Ile Asp
50 55 60
CA 02270289 1999-04-30
Appijcant Rcf. No.: 05718-PCT.app
. ~~ .. .
..
. . . ~. . . ~ ~ , .
. .. . . . . "
. ,
~. .~ . , .,» ,.
CGC GTC CGC CTC TTT GTC GAT AAA CTC GAC AAC ATT GCC CAG GTC CCC 240
Arg Val Arg Leu Phe Val Asp Lys Leu Asp Asn Ile Ala Gln Val Pro
65 70 75 80
S
AGG GTC GGC 249
Arg Val Gly
(2) INFORMATION FOR SEQ N0:14:
ID
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids
IS (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
ZO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: N0:14:
SEQ :LD
Ser Ser Val Glu Lys Lys Pro Va~lAsn ThrGly Gly
Glu Gly Ala Asp
ZS 1 5 10 15
Arg His Asn Leu Lys Thr Glu G:LuLeu ValGly Ser
Trp Pro Lys Val
20 25 30
Glu Glu Ala Lys Lys Val Ile AspLys ProGlu Gln
Leu Gln Ala Ile
35 40 45
30 Ile Val Leu Pro Val Gly Thr ThrMet GluTyr Ile
Ile Val Arg Asp
50 55 60
Arg Jal Arg Leu Phe Val Asp AspAsn IleAla Val
Lys Leu Gln Pro
65 70 75 80
Arg Val Gly
35
(2) INFORMATION FOR SEQ ID N0:15:
4O (i) SEQUENCE CHARACTERISTICS:
-~ LENGTH: 459 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
4S
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence:
SO (B) LOCATION: 1....288
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ).D N0:15:
SS GCA GTG CAA CAA GCA AGA TTT ACC TGC CC:A TCG ATC ATA TCG TCA ACT 48
Ala Val Gln Gln Ala Arg Phe Thr Cys Pro Ser Ile Ile Ser Ser Thr
1 5 1C1 15
76
Applicant Ref. No.: 0571 R-PCT.app
CA 02270289 1999-04-30
v ~ .r ,. , m ~ ,
~ ~ ~ ~ ~ , . , . . . , ~ , ,
. . . , , ,., . . , ,
' ~ ~ . , ,
~ r m , , , : , , , . , , ~
GGT CCG GCA GTT CGC GAC ACC ATG AGC TCC ACG GAG TGC GGC GGC GGC 96
Gly Pro Ala Val Arg Asp Thr Met Ser Ser Thr Glu Cys Gly Gly Gly
20 25 30
S GGC GGC GGC GCC AAG ACG TCG TGG CCT Gi~G GTG GTC GGG CTG AGC GTG 144
Gly Gly Gly Ala Lys Thr Ser Trp Pro G:Lu Val Val Gly Leu Ser Val
35 40 45
GAG GAC GCC AAG AAG GTG ATG GTC AAG Gl~C AAG CCG GAC GCC GAC ATC 192
Glu Asp Ala Lys Lys' Val Met Val Lys Asp Lys Pro Asp Ala Asp Ile
50 55 60
GTG GTG CTG CCC GTC GGC TCC GTG GTG ACC GCG GAT TAT CGC CCT AAC 240
Val Val Leu Pro Val Gly Ser Val Val Thr Ala Asp Tyr Arg Pro Asn
IS 65 70 75 80
CGT GTC CGC ATC TTC GTC GAC ATC GTC G(:C CAG ACG CCC CAC ATC GGC T 289
Arg Val Arg Ile Phe Val Asp Ile Val A7_a Gln Thr Pro His Ile Gly
85 90 95
GATAATATAT AAGCTAGCCG CTATTTCCTT TCCT7.'GCCCC AGAACTTGAA ATAAATATAT 349
ATACGATGAA ATAACGCGGG CATGCCGAAT ANATCdGANTG TGNNTGAATT CTCACTAATT 409
AAGTAATGNC ATAAATAAAC GTATTCAAAA AAAA7~.AAAAA P~~AAAAAA.AA 4 5 9
2S
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~S (ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ 7:D N0:16:
Ala Val Gln Gln Ala Arg Phe Thr Cys Pro Ser Ile Ile Ser Ser Thr
1 5 10 15
Gly Pro Ala Val Arg Asp Thr Met Ser Se:r Thr Glu Cys Gly Gly Gly
20 25 30
Gly Gly Gly Ala Lys Thr Ser Trp Pro Gl.u Val Val Gly Leu Ser Val
4S 35 40 45
Glu Asp Ala Lys Lys Val Met Val Lys A~,p Lys Pro Asp Ala Asp Ile
55 60
Val Val Leu Pro Val Gly Ser Val Val Thr Ala Asp Tyr Arg Pro Asn
65 70 75 80
SO Arg Val Arg Ile Phe Val Asp Ile Val Ala Gln Thr Pro His Ile Gly
85 90 95
SS !2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 428 base pairs
77
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
.. ., , .. ,
,. . . . . . ., . . . . . ,
", . . . ,
.. .. .n .... .. ..
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
S (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...303
IO (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ :CD N0:17:
CGA CCC ACG CGT CCG CCC ACG CGT CCG G(:A AGA TTT ACC TGC CCA TCG 48
IS Arg Pro Thr Arg Pro Pro Thr Arg Pro Al.a Arg Phe Thr Cys Pro Ser
1 5 10 15
ATC ATA TCG TCA ACT GGT CCG GCA GTT CGC GAC ACC ATG AGC TCC ACG 96
Ile Ile Ser Ser Thr Gly Pro Ala Val Arg Asp Thr Met Ser Ser Thr
20 20 25 30
GAG TGC GGC GGC GGC GGC GGC GGC GCC AF~G ACG TCG TGG CCT GAG GTG 144
Glu Cys Gly Gly Gly Gly Gly Gly Ala L~~s Thr Ser Trp Pro Glu Val
35 40 45
2S
GTC GGG CTG AGC GTG GAG GAC GCC AAG AF~G GTG ATC CTC AAG GAC AAG 192
Val Gly Leu Ser Val Glu Asp Ala Lys Lys Val Ile Leu Lys Asp Lys
50 55 60
3O CCG GAC GCC GAC ATC GTG GTG CTG CCC GT'C GGC TCC GTG GTG ACC GCG 240
Pro Asp Ala Asp Ile Val Val Leu Pro Va.l Gly Ser Val Val Thr Ala
65 70 75 80
GAT TAT CGC CCT AAC CGT GTC CGC ATC TT'C GTC GAC ATC GTC GCC CAG 288
~S Asp Tyr Arg Pro Asn Arg Val Arg Ile Phe Val Asp Ile Val Ala Gln
85 90 95
ACG CCC CAC ATC GGC TGATAATATA TAAGCTAGCC GCTATTTCCT TTCCTTGCCC C 344
Thr Pro His Ile Gly
4O 100
AGAACTTGAA ATAAATATAT ATACGATGAA ATAACGCGGG CATGCCGAAT AATGGATGTG 404
TGAAAAA.AAA F,~~P.AAP.AP.AA AAAA 4 2 8
4S
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
SO (A) LENGTH: 101 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SS (ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ I;,~ N0:18:
CA 02270289 1999-04-30
Applicant Ref. No.: 05718-PCT.app
r. .. . . ,.
.. . . . . , .: . . . . .
, . . . . .,. .
.
.. .. . . . .. .. .,
Arg Pro Thr Arg Pro Pro Thr Arg Pro A:La Arg Phe Thr Cys Pro Ser
1 5 10 15
Ile Ile SerSerThr Gly AlaVal A:rgAspThr MetSer SerThr
Pro
20 25 30
Glu Cys GlyGlyGly Gly GlyAla L!~sThrSer TrpPro GluVal
Gly
35 40 45
Val Gly LeuSerVal Glu AlaLys Ll~sValIle LeuLys AspLys
Asp
50 55 60
Pro Asp AlaAspIle Val LeuPro ValGlySer ValVal ThrAla
Val
65 70 75 80
Asp Tyr ArgProAsn Arg ArgIle PheValAsp IleVal AlaGln
Val
85 90 95
Thr Pro HisIleGly
loo
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence'
(B) LOCATION: 1...255
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ :CD N0:19:
3S
TTA ATT ATT GCC CTT TCA GTT NGC CAT CGG CAG CCG AGC ACC ATG AGC 48
Leu Ile Ile Ala Leu Ser Val Xaa His Arg Gln Pro Ser Thr Met Ser
1 5 10 15
4O TCC ACA GGC GGC GGC GAC GAT GGC GCC AAG AAG TCT TGG CCG GAA GTG 96
Ser Thr-'G1~ Gly Gly Asp Asp Gly ATa L;rs Lys Ser Trp Pro Glu Val
20 25 30
GTC GGG CTC AGC CTG GAA GAA GCC AAG AGG GTG ATC CTG TGC GAC AAG 144
45 Val Gly Leu Ser Leu Glu Glu Ala Lys Arg Val Ile Leu Cys Asp Lys
40 45
CCC GAC GCC GAC ATC GTC GTG CTG CCC G'.CC GGC ACG CCG GTG ACC ATG 192
Pro Asp Ala Asp Ile Val Val Leu Pro Val Gly Thr Pro Val Thr Met
50 50 55 60
$5
GAT TTC CGC CCC AAC CGC GTC CGC ATC T'.CC GTC GAC ACC GTC GCG GAG 240
Asp Phe Arg Pro Asn Arg Val Arg Ile Phe Val Asp Thr Val Ala Glu
65 70 75 80
GCA MCC CAC ATC GGC TGAGGTTAAA TCTACA)~AAT GAATGAYTCG GACATGCCAT G 296
Ala Xaa His Ile Gly
79
CA 02270289 1999-04-30
Applicant Rcf. No.: 05718-PCT.app
.. .. .
,. . . . . .~ . . . , , .
. . . . .., . . , . . ..
. ~ . , . .
.. .. ... .,.. .. .,
S
CGTACNTGTC CGTCGCCGAA TAATGGATGT GTGTGTGCTT CGATCGTTCC TAATAAGTTG 356
CTAGTNAAAA ATAATNGGCA TCGTCGTTAN TGCATGAATA AAAAGTATCA GAATAATGTT 416
CACCCTTTCN AAAAAAAAAA AAAAA 441
(2) INFORMATION FOR SEQ ID NC~:20:
lO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
1S
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ 7:D N0:20:
20
Leu IleIle AlaLeuSer ValXaaHis ArgGlnPro SerThrMet Ser
1 5 1 15
CI
Ser ThrGly GlyGlyAsp AspGlyAla L~~sLysSer TrpProGlu Val
20 25 30
2S Val GlyLeu SerLeuGlu GluAlaLys ArgValIle LeuCysAsp Lys
35 40 45
Pro AspAla AspIleVal ValLeuPro ValGlyThr ProValThr Met
50 55 60
Asp PheArg ProAsnArg ValArgIle PheValAsp ThrValAla Glu
30 65 70 75 80
Ala XaaHis IleGly
85
3S
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 382 base pairs
40 (B) TYPE: nucleic acid
~ STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
4S (ix) FEATURE:
(A) NAME/KEY: Coding Sequencf:
(B) LOCATION: 1...213
(D) OTHER INFORMATION:
SO
(xi) SEQUENCE DESCRIPTION: SEQ :CD N0:21:
GTG CGT CGT CGG CGA ACA GCC ACC GGC GGC AAG ACG TCG TGG CCG GAG 48
Val Arg Arg Arg Arg Thr Ala Thr Gly G:Ly Lys Thr Ser Trp Pro Glu
SS 1 5 10 15
GTG GTC GGG CTG AGC GTC GAG GAA GCC Ai4G AAG GTG ATT CTG GCG GAC 96
Val Val Gly Leu Ser Val Glu Glu Ala Lys Lys Val Ile Leu Ala Asp
8O
CA 02270289 1999-04-30
Applicant Ref. No.: 0571 R-PCT.app
.. ., . .. ..
.. . . . . . .. . . . . .
. . . .. , . ... .
. . .
.. .. ... .... .. ..
20 25 30
AAG CCG AAC GCC GAC ATC GTG GTG CTG CCC ACC ACC ACG CAG GCG GTG 144
Lys Pro Asn Ala Asp Ile Val Val Leu Pro Thr Thr Thr Gln Ala Val
S 35 40 45
ACC TCC GAC TTT GGG TTC GAC CGT GTC CGC GTC TTC GTC GGG ACC GTC 192
Thr Ser Asp Phe Gly Phe Asp Arg Val Arg Val Phe Val Gly Thr Val
50 55 60
GCC CAG ACG CCC CAT GTT GGC TAGGCTAGA3 CCTCAGCCTA GAGGTCGTCG GCAC 247
Ala Gln Thr Pro His Val Gly
65 70
IS CGCCGGCCAT GACCACCTGC TANTATGTCA CTNACTAGTA ATAAAGTATW AATAACAGGG 307
AGGATGCATG CTCATCNTTG GAATCTGTAC GCTTGTTGGA CTACTACTTG GCTACTTGAA 367
P~~;4AAAAAAA AAAAA 3 8 2
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 amino acids
2S (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Val Arg Arg Arg Arg Thr Ala Thr Gly Gly Lys Thr Ser Trp Pro Glu
~S 1 5 10 15
Val Val Gly Leu Ser Val Glu Glu Ala Lys Lys Val Ile Leu Ala Asp
20 25 30
Lys Pro Asn Ala Asp Ile Val Val Leu Pro Thr Thr Thr Gln Ala Val
40 45
Thr Ser Asp Phe Gly Phe Asp Arg Val Arg Val Phe Val Gly Thr Val
55 60
Ala Gln Thr Pro His Val Gly
65 70
4S
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
SO (A) LENGTH: 448 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SS (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
gl
Applicant Ref. No.: 05718-PCT.app
CA 02270289 1999-04-30
., . .. ,
.. .. .~ . . , . . . . .
.., . . .
, ,
.. .. .., . .. ..
(B) LOCATION: 1...240
(D) OTHER INFORMATION:
S
(xi) SEQUENCE DESCRIPTION: SEQ 7:D N0:23:
CGA TTT AGC TAT AGC AGG TCT CGA TCG GC:G GCC ATG AGC GGT AGC CGC 48
Arg Phe Ser Tyr Ser Arg Ser Arg Ser Al.a Ala Met Ser Gly Ser Arg
1 5 1 C1 15
IO AGC AAG AAG TCG TGG CCG GAG GTG GAG GCMG CTG CCG TCC GAG GTG GCC 96
Ser Lys Lys Ser Trp Pro Glu Val Glu Gl.y Leu Pro Ser Glu Val Ala
20 25 30
AAG CAG AAA ATT CTG GCC GAC CGC CCG GAC GTC CAG GTG GTC GTT CTG 144
1$ Lys Gln Lys Ile Leu Ala Asp Arg Pro A:>p Val Gln Val Val Val Leu
35 40 45
CCC GAC GGC TCC TTC GTC ACC ACT GAT TTC: AAC GAC AAG CGC GTC CGG 192
Pro Asp Gly Ser Phe Val Thr Thr Asp Phe Asn Asp Lys Arg Val Arg
20 50 55 60
30
GTC TTC GTC GAC AAC GCC GAC AAC GTC GC:C AAA GTC CCC AAG ATC GGC T 241
Val Phe Val Asp Asn Ala Asp Asn Val Al.a Lys Val Pro Lys Ile Gly
65 70 75 80
AGCTAGCTAG CTAGGCCCAA TCGTTCTAAT CAGC7.'AGTTT CTTTCTTTCA TAAATAAAAG 301
TCCTCTCTCG TACCCGGACT GTGATGTTTC CCTAGTTGTC TCGTACGTGT TGTTTTCTGT 361
CTTAATGGAT GCCATGGCGC CCGCGCGCGC CTYC.t~TCATG AAAAGCTACA TTTGAAACGA 421
TTTTNAGTAT TCTTTGCTGT TAAAAAA 448
(2) INFORMATION FOR SEQ ID N0:24:
3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(=~ MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ :CD N0:24:
Arg Phe SerTyrSer ArgSerArg SerA:LaAla MetSerGly SerArg
1 5 10 15
Ser Lys LysSerTrp ProGluVal GluG:lyLeu ProSerGlu ValAla
20 25 30
Lys Gln LysIleLeu AlaAspArg ProAspVal GlnValVal ValLeu
35 40 45
Pro Asp GlySerPhe ValThrThr AspPheAsn AspLysArg ValArg
50 55 60
Val Phe ValAspAsn AlaAspAsn ValA:laLys ValProLys IleGly
6s 70 75 so
82
-r..h ~.:
CA 02270289 1999-04-30
Applicant Ref. No.: 0571 R-PCT.app
.. , , ,.
.., . . . . , " , , . , ,
". . . . . ",
. , ,
.. " ." .,., .. ,.
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
S (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ I:D N0:25:
ATGAAGTCGG TGGAGAAG 18
1S
(2) INFORMATION FOR SEQ ID NC>:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
ZS (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ I:D N0:26:
GCCGACCCTG GGGACCTG 18
83
75529-49(S)
CA 02270289 2000-04-20
All publications and patent applications mentioned in this specification are
indicati~r~ of
the level of skill of those skilled in the art to which this invention
pertains.
Variations on the above embodiments are within the ability of one of ordzz~ar-
y skill
in the art, and such variations do not depart from the scope of the present
ira-v~;nti~~~ as
described in the following claims.
84
