Note: Descriptions are shown in the official language in which they were submitted.
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17 kDa FOAM PROTEIN
Background of the Invention
Field of the Invention
This invention relates to a novel gene sequence encoding a foam-
s related protein. More specifically, the invention relates to a nucleic acid
sequence
and the 17 kDa Foam Protein it encodes, which protein is useful to enhance the
production of foaming beverages, including beer.
Background of the Invention
For many types of products, the formation of a high and stable foam
is a desirable quality. For example, foam properties are an important
parameter in
high quality beverages. Formation of a stable head of foam on pouring a
beverage
such as beer is an important quality parameter considered by consumers as a
vital
characteristic. Much effort has been invested to identify and isolate factors
influencing foam quality, but the nature of the foam components is not yet
fully
elucidated. Lipids are generally considered destructive for foam whereas hop
bitter
components and proteins of malt origin are considered the most important foam
positive components.
Many studies have attempted to clarify which protein components of
beer are involved with foam stabilization, but a clear answer has not been
achieved.
Several molecular weight classes of proteins in beer have been suggested as
important to the foam: 90-100 kDa, 40 kDa, 15 kDa (Asano, et al., 1980, J.
Am.Soc. Brew. Chem., 38:129-137); 40 kDa (Yokoi, et al., 1989, Proc. Eur.
Brew.
Conv., 22nd Congress, pp. 593-600); l0 kDa (Sorensen, et al., 1993, MBAA
Techn.
Quart., 30:136-145), and 8 - 18 kDa (Douma, et al., 1997, Proc. Eur. Brew.
Conv.
26th Congress, pp. 671-679).
Beer proteins range in molecular weight from small polypeptides to
more than 150 kDa. Studies by Sharpe, et al. propose that the foam stability
of beer
is related to the ratio of high and low molecular weight polypeptides (Proc.
Eur.
Brew. Conv., 18th Congress, Copenhagen, 1981, pp.607-614). Yokoi, et al.,
(1989,
supra) disclose protein Z, a 40 kilodalton barley albumin, as playing the most
important role in foam stability. This conclusion is contrary to the results
of
Hollemans and Tonies, 1989 (Proc. Eur. Brew. Conv., 22nd Congress, Zurich,
1981,
pp.561-568), who showed only a limited effect on specific and complete removal
of
protein Z from beer.
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Thus, there remains a need for identifying and characterizing agents
which influence the quality of foam, and methods using such agents to enhance
the
quality of foam, including beer foam.
Summary of the Invention
The present invention describes the purification of a 17 kDa Foam
Protein from beer, from barley, from first wort and from rye; the
characterization of
the 17 kDa Foam Protein with respect to sequence and structure; the
establishment
of ELISA assays for quantification of the 17 kDa Foam Protein; and the use of
the
17 kDa Foam Protein to enhance foam production. The 17 kDa Foam Protein has
been demonstrated herein to have a positive effect on foam potential and on
foam
stability.
The instant invention includes a novel 17 kDa Foam Protein, nucleic
acid sequences encoding the protein, methods for enhancing foam quality, and
methods for producing enhanced amounts of the protein in host cells, including
yeast, plant cells and plants.
The invention further includes anti-17 kDa Foam Protein antibodies
useful in immunoassays for the analysis of 17 kDa Foam Protein content of a
sample, and immunoassay kits including the antibodies, and optionally 17 kDa
Foam
Protein standards.
Products of the invention include foaming products, such as
beverages which foam on pouring. Preferred products of the invention include
brewed and fermented products, in particular, beer, supplemented with enhanced
amounts of 17 kDa Foam Protein and having improved foam quality
characteristics
such as improved foam potential, foam stability, and foam half-life.
In the methods of the invention, foam quality is enhanced by
supplementation of 17 kDa Foam Protein. Although a product may include an
amount of 17 kDa Foam Protein naturally and under normal processing
conditions,
the method and products of the invention include an additional amount of 17
kDa
Foam Protein, supplementing the amount normally present, and resulting in an
enhanced amount of the 17 kDa Foam Protein in the product. Such
supplementation
is achieved by adding purified and isolated 17 kDa Foam Protein to the product
during its manufacturing steps, or by providing raw materials (for example,
barley or
wheat grain or yeast) that contain or produce enhanced amounts of 17 kDa Foam
Protein. In a preferred embodiment, a foaming product is manufactured from raw
materials such as barley or wheat grain or yeast, which raw materials are
transformed
with a supplemental nucleic acid sequence encoding 17 kDa Foam Protein. In a
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most preferred embodiment, foam quality is enhanced by supplementation with a
combination of 17 kDa Foam Protein and LTP-1 protein.
Brief Description of the Drawings_
Figure 1 is a diagrammatic representation of the structure of the 17
kDa Foam Protein polypeptide showing predicted disulfide bridges and protein
domains A, B, C, and D.
Figure 2A is a SDS-polyacrylamide gel showing 17 kDa Foam
Protein purified from beer (lane C).
Figure 2B is a Western blot showing selective reactivity of anti-I7
kDa Foam Protein antibody with the isolated and purified protein from beer
(lane C).
Figure 3 is a diagram showing the distribution of 17 kDa protein
between flotate and remanent during repeated flotations in a foam tower.
Figure 4 is a graph showing the effect of removing 17 kDa protein
from lager beer.
Figure 5 is a plasmid map of a self-replicating yeast expression
plasmid carrying the coding sequence of 17 kDa Foam Protein.
Figure 6 is a plasmid map of a yeast integration plasmid carrying the
coding sequence of 17 kDa Foam Protein.
Figures 7A-7C are graphs showing the relationship between foam
half-life and the total amount of 17 kDa Foam Protein and foam-type LTP 1 in
50
Danish lager beers at different carbonization levels (g C02/liter): 4.8-5.0
(A); S.1-
5.3 (B); 5.4-5.6 (C).
Figure 8 is a Western blot of barley, rye and wheat extracts probed
with anti-17 kDa Foam Protein (barley) antibody and showing antibody
recognition
of components in wheat and rye having approximately the same molecular mass
(17
kDa).
Figure 9 is a diagram showing a barley transformation cassette
carrying the coding sequence of the 17 kDa Foam Protein.
Detailed Description of the Invention
The present invention includes an isolated and purified 17 kDa Foam
Protein having foam-enhancing properties, and useful in the production of
foaming
products, including beer. The invention further includes nucleic acid
sequences
encoding 17 kDa Foam Protein, anti-17 kDa Foam Protein antibodies, and assays
for the detection and quantitation of 17 kDa Foam Protein in a sample. Methods
of
the invention include methods for the enhancement of foam quality in a foaming
product, and methods for the production of a foaming product, particularly
beer,
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having a content of 17 kDa Foam Protein that is enhanced over the amount of 17
kDa Foam Protein naturally present in the product. The content of 17 kDa Foam
Protein, and hence the foam quality of a product, is enhanced by adding
purified 17
kDa Foam Protein directly to the product during processing steps, and/or by
S providing raw materials genetically engineered to produce increased amounts
of 17
kDa Foam Protein. In a preferred embodiment, a combination of enhanced 17 kDa
Foam Protein and LTP-I is used to enhance foam quality.
Definitions
When used herein, the following terms have these defined meanings:
"17 kDa Foam Protein (polypeptide)": A novel protein isolated from
beer foam, first wort, barley or rye having a molecular weight of
approximately 17
kilodaltons (kDa) and having foam enhancing properties as described below.
Figure
1 shows a diagrammatic representation of the predicted 2-dimensional structure
of
the 17 kDa Foam Protein. While the amino acid sequence set forth in Tables 1
and 2
(SEQ ID NOS: 10 and 12) define one embodiment of the 17 kDa Foam Protein as
obtained from beer foam, first wort, and barley, it is anticipated that 17 kDa
Foam
Proteins (homologues) having similar foam enhancing properties and homologous
amino acid sequences will be similarly isolated from other grains by
conventional
methods. For example, a genomic DNA sequence encoding 17 kDa Foam Protein
isolated from wheat is shown in Table 4 (SEQ ID NOS: 20, 21). This wheat 17
kDa
Foam Protein was isolated using probes prepared from the barley 17 kDa Foam
Protein, as described below in Example 7. As another example, the amino acid
sequence of a 17 kDa Foam Protein obtained from rye is shown in Table I 0. For
purposes of the invention, "17 kDa Foam Protein" includes foam enhancing
proteins
derived from cereal grain and having the following characteristics of primary
structure useful for purposes of identification:
( 1 ) a molecular mass of about 17 kDa, that is, approximately 15
to 20 kilodaltons (kDa);
(2) a primary amino acid sequence which can be aligned with
distinct homology to the non-repetitive C-terminal domain of
the sulfur-rich prolamin storage proteins found in cereals.
More specifically, a primary sequence showing significant
homology (e.g., greater than 25%) to the C-terminal domain
of the monomeric y-type prolamins (such as y-gliadin in
wheat and y-hordein of barley). The alignment of the amino
acid sequence of the mature barley 17 kDa Foam Protein
(residues 1 to 130), with the C-terminal domain of y3 hordein
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(residues 119 to 276), shows a homology of 36%. The 17
kDa foam proteins are distinguished from these latter y-type
prolamins by the lack of an N-terminal proline- and
glutamine-rich domain composed of degenerate pentapeptide
repeats;
(3) a high content of cysteine residues (?8), that align with the
highly conserved cysteine residues found in members of the
sulfur-rich prolamins, in particular the y-type hordein
polypeptides shown in Table 3;
(4) a primary amino acid sequence which can be aligned with
high homology (>75%, preferably >85%) to other members of
this newly identified (~-type) class of prolamin. The primary
amino acid sequence homology between the 17 kDa Foam
Proteins found in wheat and barley is 86%, which is higher
I S than the homology found to members of other sulfur-rich
prolamin classes.
The 17 kDa foam proteins of the invention can also be identified by
its cross-reactivity with anti-17 kDa foam protein antibodies raised against
the
purified 17 kDa protein purified from barley. 17 kDa foam protein can be
purified
from cereal grain with the use of 17 kDa specific antibodies for
identification
purposes.
The 17 kDa foam proteins are members of a newly identified class of
storage polypeptides (e-type) present in cereal grain, belonging to the
prolamin
storage protein family. As such, they are found in the endosperm tissue of
mature
cereal grain and are synthesized during grain development.
"Foam Enhancing Properties": As described more fully in the
Examples below, 17 kDa Foam Protein, when added to a product such as water,
milkshakes, soft drinks, alcopops or beer, causes the product to have enhanced
foam
quality. Parameters of foam quality that are enhanced include foam potential
(P),
foam stability (S), and foam half-life (F), as described more fully below.
"Nucleic Acid Sequences Encoding 17 kDa Foam Protein": Nucleic
acid sequences encoding 17 kDa Foam Protein were determined by methods
described more fully in the Examples below. The nucleic acid sequence of an
isolated barley cDNA and its deduced amino acid sequence combined with the
determined sequence of the purified protein are shown in Table 1. Nucleotides
1-57
are back-translated from the determined amino acid sequence, using the codon
usage bias of the barley 1-3,1-4 ~i-glucanase as described by Jensen et al.,
1996
PNAS USA 93:3487-3491. A genomic nucleic acid sequence encoding barley 17
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kDa Foam Protein and its deduced amino acid sequence is shown in Table 2. The
deduced amino acid sequence of the 17 kDa Foam Protein encoded by the cDNA
and genomic sequences show close homology, but are not identical. The given
1336
nucleotide sequence comprises a 522 nucleotide sequence encoding the precursor
17
kDa Foam Protein polypeptide and 674 and 140 nucleotides of 5' and 3' flanking
sequences, respectively. The 3' flanking sequence contains consensus sequences
for
three polyadenylation signals (AATAAA). The deduced amino acid sequence of the
precursor 17 kDa Foam Protein is predicted to have a 19 amino acid signal
peptide
sequence. A genomic nucleic acid sequence encoding a wheat 17 kDa Foam Protein
and its deduced amino acid sequence, showing 85% amino acid sequence homology
to the barley 17 kDa Foam Protein, is shown in Table 4. Additional genes or
nucleic
acid sequences encoding 17 kDa Foam Proteins (homologs) include those
identified,
for example, by one of several standard molecular biology techniques
including:
(1) PCR amplification of nucleic acid libraries (including
genomic DNA and cDNA libraries) constructed from a cereal
plant, using sequence-specific primers based on the
nucleotide sequence of the barley or wheat 17 kDa Foam
Protein genes, or alternatively using degenerate primers back-
translated from the determined deduced amino acid sequence
of 17 kDa Foam Protein from wheat or barley;
(2) RNA-PCR amplification of mRNA prepared from a
developing cereal grain using sequence-specific or degenerate
primers based on the nucleotide or amino acid sequence, of
the barley or wheat 17 kDa Foam Protein and their genes;
(3) The cDNA or genomic sequence may be identified in a library
constructed from a cereal plant by screening the library with a
barley cDNA encoding 17 kDa Foam Protein (for example,
that shown in Table I ) as a probe, using standard
hybridization conditions (SxSSC, Sx Denhardts solution,
0.5% SDS at 65°C) followed by washing at increasing
stringency, with a final 30 minute wash at high stringency
(65°C in 0.2% SSC, 0.5% SDS).
"homologs": As discussed above, a 17 kDa Foam Protein homolog is
defined to include proteins derived from cereal plants having the functional
and
structural characteristics of the described wheat or barley 17 kDa Foam
Proteins.
Homologous nucleic acid sequences hybridize to the described nucleic acid
sequences encoding barley and wheat 17 kDa Foam Protein, under standard and
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stringent hybridization conditions, for example, those recited above.
Homologous
amino acid sequences contain the characteristics of primary structure listed
above.
Sequence Modifications
Applicants recognize, and include with in the scope of their
invention, a nucleic acid sequence encoding 17 kDa Foam Protein which contains
codons that are modified according to optimal codon frequencies for a
particular
cellular host. For example, modification for expression in yeast is preferred
for
production of enhanced 17 kDa Foam Protein in yeast, using known preferred
codon
frequencies for yeast.
Redundancy in the genetic code permits variation in the gene
sequences shown in Tables 1, 2 and 4. In particular, specific codon
preferences are
recognized for a specific host such that the disclosed sequence can be adapted
as
preferred for the desired host. For example, rare codons having a frequency of
less
than about 20% in known sequences of the desired host are preferably replaced
with
higher frequency codons.
Additional sequence modifications are known to enhance protein
expression in a cellular host. These include elimination of sequences encoding
spurious polyadenylation signals, exon/intron splice site signals, transposon-
like
repeats, and other such well characterized sequences which may be deleterious
to
gene expression. The G-C content of the sequence may be adjusted to levels
average for a given cellular host, as calculated by reference to known genes
expressed in the host cell. Where possible, the sequence is modified to avoid
predicted hairpin secondary mRNA structures. The genomic sequence may
additionally be modified by the removal of introns.
Gene delivery
The nucleic acid sequence encoding the 17 kDa Foam Protein is
delivered to host cells, including yeast and plant cells, for transient
transfections or
for incorporation into the cells by known methods. Preferably, the gene is
used to
stably transform plant cells for expression of the protein in vivo.
To accomplish such delivery, the gene containing the coding
sequence for the 17 kDa Foam Protein may be attached to regulatory elements
needed for the expression of the gene in a particular host cell or system.
These
regulatory elements include, for example, promoters, terminators, and other
elements
that permit desired expression of the protein in a particular plant host, in a
particular
tissue or organ of a host such as starchy endosperm, aleurone or embryo
tissues of
the barley kernel, during grain development or germination or in response to a
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particular signal. The 17 kDa Foam Protein gene may additionally be attached
to a
regulatory signal peptide directing its expression into the secretory pathway
within
the cell. In a preferred example, the 17 kDa Foam Protein gene coding sequence
is
attached to regulatory sequences directing its expression into the secretory
pathway
within the starchy endosperm of the developing barley endosperm. The protein
is
also preferentially adapted for production in yeast.
Gene Constructs
The isolated nucleic acid sequence of the invention can be
incorporated into DNA constructs and used to transform or transfect a host
cell.
Many DNA vectors can be used, depending on the host cell and desired
expression.
Examples of suitable vectors include, but are not limited to, self-replicating
or
integration plasmids suitable for expression in prokaryotic or eukaryotic
cells.
A typical gene construct includes a promoter, the coding sequence of
interest, and a terminator sequence coupled in operative association.
Additional
known regulatory elements can also be included in the construct.
Suitable gene constructs for the stable transformation of host cells
include those having constitutive promoters such as the Ubi 1 gene promoter
(Christensen, et al., 1992, Plant Mol. Biol., 18: 675-689) driving expression
of
selectable markers such as the phosphinothricin acetyl transferase gene (bar)
(De
Block, et al., 1987, EMBO J, 6: 2513-2518). Additionally, the plasmid can
include
other gene sequences such as resistance genes (required for the selection and
amplification of host transformed cells), reporter genes or other elements.
Examples
of suitable plant transformation selection systems for cereals or other plants
are
described by Yoder and Goldsbrough, 1994, BiolTechnology, 12: 263-267, and
Tingay, et al., 1997, The Plant Journal 11: 1369-1376 are incorporated herein
by
reference.
Promoters
A DNA construct of the~invention includes a promoter sequence
which may be a "homologous" or "heterologous" promoter. As used herein, the
term
"heterologous" defines a nucleic acid sequence not normally found in the
genome
associated with the 17 kDa Foam Protein-expressing gene. For example, a
heterologous sequence is one derived from a different cell type, different
plant
species, different organism, or one normally associated with a different gene.
A
heterologous promoter is one which does not drive transcription of the 17 kDa
Foam
Protein-expressing gene in its natural, non-transformed genome. In contrast, a
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"homologous" promoter is one normally associated with the I 7 kDa Foam Protein
gene.
A promoter is a DNA sequence that directs the transcription of a
structural gene. Typically, a promoter is located in the S' region of a gene,
proximal
to the transcriptional start site. A promoter may be inducible, increasing the
rate of
transcription in response to an agent, or constitutive, whereby the rate of
transcription is not regulated by an inducing agent. A promoter may be
regulated in
a tissue-specific or tissue-preferred manner, such that it is only active in
transcribing the operably linked coding region in a specific tissue type or
types, for
IO example, plant seeds, leaves, roots, or meristem. An heterologous promoter
may
initiate transcription of an operably linked gene coding sequence at an
earlier time or
developmental stage in a given tissue, than initiated by the native promoter
of the
same gene. Within a given host cell or tissue, certain promoters may drive
transcription more strongly, resulting in a higher accumulation of transcript,
thereby
enhancing synthesis of the gene product.
A promoter useful in the invention is operably linked to a nucleotide
sequence encoding 17 kDa Foam Protein such that transcription of the 17 kDa
Foam
Protein sequence is driven by the promoter. Optionally, the promoter is
operably
linked to a nucleotide sequence encoding a signal peptide, which in turn is
operably
linked to a sequence encoding the mature 17 kDa Foam Protein.
Many different promoters can be used to express the 17 kDa Foam
Protein gene in a host cell. Of particular value in the present invention is a
tissue-
specific promoter which drives gene expression in endosperm tissue of
developing
grain. Examples of suitable endosperm specific promoter sequences include the
promoters of the following genes: B 1 hordein gene (~, hor2-4, GenBank Acc No:
X87232), y-hordein gene (~, hor'y-I, GenBank Acc No: X13508), C hordein gene
(~, hor I-14, GenBank Acc No: M3694I and ~, hor 1-I 7, GenBank Acc No:
X60037); D hordein gene (phor 3-l, GenBank Acc No: X84369); ~i-amylase gene
((3-amyl, GenBank Acc No: D63574) and protein Z gene (Pazl, GenBank Acc No:
X51726).
Additional Regulatory and Targeting Elements
Additional regulatory elements include terminators, polyadenylation
sequences, and nucleic acid sequences encoding signal peptides that permit
localization within a plant cell or secretion of the protein from the cell.
Such
regulatory elements include, but are not limited to, 3'
terminationlpolyadenylation
regions such as those of the Agrobacterium tume~aciens nopaline synthase (nos)
gene (Bevan, et al., 1983, Nucl. Acids Res., 12:369-385); the rubisco rbes
gene from
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Pisum sativum (Coruzzi, et al., 1984, EMBO J., 3:1671-1679); the potato
proteinase inhibitor II (PINII) gene (Keil, et al., 1986,Nucl. Acids. Res.,
14:5641-
5650); and An, et al., 1989, Plant Cell, 1:115-122). Methods for adding or
exchanging these elements with the regulatory elements of the 17 kDa Foam
5 Protein-expressing gene are known.
Gene Transformation Methods
Numerous methods for introducing foreign genes into plants, such as
biological and physical plant transformation protocols, can be used to insert
the 17
10 kDa Foam Protein gene into a plant host. See, for example, Miki, et al.,
1993,
"Procedure for Introducing Foreign DNA into Plants", In: Methods in Plant
Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press,
Inc.,
Boca Raton, pages 67-88. The particular method may vary depending on the host
plant. Suitable methods include chemical transfection methods such as the use
of
calcium phosphate, microorganism-mediated gene transfer such as transfection
using an Agrobacterium-mediated transfection system (Horsh, et al., 1985,
Science,
227:1229-31), electroporation, micro-injection, and biolistic bombardment.
Expression vectors and in vitro culture methods for plant cell or
tissue transformation and regeneration of plants are known and available. See,
for
example, Gruber, et al., 1993, "Vectors for Plant Transformation" In: Methods
in
Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC
Press,
Inc., Boca Raton, pages 89-119.
Agrobacterium-mediated Transformation
The most widely used method for introducing an expression vector
into plants is based on the natural transformation system of Agrobacterium. A.
tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which
genetically
transform plant cells. The Ti and Ri plasmids for A. tumefaciens and A.
rhizogenes,
respectively, include genes responsible for this genetic transformation. See,
for
example, Kado, 1991, Crit. Rev. Plant Sci. 10:1. Descriptions of the
Agrobacterium
vector system and methods for Agrobacterium-mediated gene transfer are
provided
in Gruber, et al., supra; Miki, et al., supra; and Moloney, et al., 1989,
Plant Cell
Reports 8:238. This transformation method has primarily been successful in
transforming dicotyledonous plants. The development of new Agrobacterium
binary
vectors has extended the application of this transformation method to certain
important cereal crops including rice (Hiei, et al., 1994, The Plant Journal
6:271-
282) and maize (Yuji, et al., 1996, Nature Biotechnology 14:745-750) and
barley
(Tingay et al., 1997 supra).
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Direct Gene Transfer
Alternative methods of plant transformation, collectively referred to
as direct gene transfer, have also been developed. A generally applicable
method of
plant transformation is microprojectile-mediated transformation, wherein DNA
is
earned on the surface of microprojectiles measuring about 1 to 4 ~.m in
diameter.
The expression vector is introduced into plant tissues with a biolistic device
that
accelerates the microprojectiles to speeds of 300 to 600 m/s, sufficient to
penetrate
the plant cell walls and membranes. (Sanford, et al., 1987, Part. Sci.
Technol. 5:27;
Sanford, 1988, Trends Biotech 6:299; Sanford, 1990, Physiol. Plant 79:206; and
Klein, et al., 1992, Biotechnology 10:268). The application of this method for
the
transformation of barley has been reported (Wan and Lemaux, 1994, Plant
Physiol.
104:37-48) and is currently one of the preferred methods for the
transformation of
cereals.
Another method for physical delivery of DNA to plants is by
sonication (Zang, et al., 1991, BiolTechnology 9:996). Alternatively, liposome
or
spheroplast fusions have been used to introduce expression vectors into
plants. See,
for example, Deshayes, et al., 1985, EMBO J 4:2731; and Christou, et al.,
1987,
Proc. Natl. Acad. Sci. USA 84:3962. Direct uptake of DNA into protoplasts
using
CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been
reported.
See, for example, Hain, et al., 1985, Mol. Gen. Genet. 199:161; and Draper et
al.,
1982, Plant Cell Physiol. 23:451.
Electroporation of protoplasts and whole cells and tissues has also
been described. See, for example, D'Halluin, et al., 1992, Plant Cell 4:1495-
1505;
and Spencer et al., 1994, Plant Mol. Biol. 24:51-61.
Methods for Expression in Yeast
The foaming properties of beer are enhanced by the addition of 17
kDa Foam Protein to the wort. 17 kDa Foam Protein can be commercially produced
by expression in yeast, either laboratory yeast (e.g.,~5accharomyces
cervisiae.) or in
brewer's yeast (e.g., Saccharomyces carlsbergensis). For such expression, the
nucleic acid sequence encoding 17 kDa Foam Protein is preferably fused to a
yeast
signal peptide and cloned into a yeast expression vector, for example under
the
control of an inducible promotor, and used to transform yeast cells.
Alternatively, a 17 kDa Foam Protein expression cassette including
the 17 kDa Foam Protein coding sequence, yeast signal peptide coding sequence,
and promoter, is stably integrated into the yeast genome. Using yeast cells
containing the 17 kDa Foam Protein expression cassette during fermentation, 17
kDa
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Foam Protein can be directly secreted into the wort. An example of one such
expression system is more fully described below in Example 17.
17 kDa Foam Protein secreted into the yeast cell growth medium can
also be isolated and purified as described in the Example 16 and 17 below, and
added to the product during one or more steps in the brewing process.
Host cells
Suitable host cells for transformation with the nucleic acid sequence
of the invention or its coding sequence include cells that will benefit from
the
expression of enhanced amounts of 17 kDa Foam Protein, and cells that are
useful as
raw materials for the production of foaming products. Host cells may be
adapted for
the production, isolation and purification of large amounts of 17 kDa Foam
Protein
polypeptide. Preferred host cells include plant cells, such as barley or other
cereal
grains, and yeast cells, that are useful in a commercial process to produce
foaming
products, such as beer.
Host cells such as bacterial, yeast, or eukaryotic cell lines are
transformed with the nucleic acid sequence of the invention such that the
transformed cells produce enhanced levels of active 17 kDa Foam Protein. The
active protein is then added to a brewing mixture, for example, to malt, malt
extract,
wort, or clarified product to enhance final product foam quality.
In a preferred embodiment of the invention, the nucleic acid sequence
encoding 17 kDa Foam Protein is used to transform plants whose grain is used
in
fermentation processes, including barley, wheat, sorghum and other cereals.
Other
host cells including bacteria, yeast, and eukaryotic cell lines are
transformed with the
nucleic acid sequence of the invention, and used to produce an 17 kDa Foam
Protein
that can be added to the system during beer production.
17 IZDa Foam Protein Assay Methods
Conventional assays are used to assay 17 kDa Foam Protein and its
gene. For example, transgenie plant cells, callus, tissues, kernels, and
transgenic
plants are tested for the presence of the 17 kDa Foam Protein-expressing gene
by
DNA analysis (Southern blot or PCR), for expression of the gene by immunoassay
(ELISA or Western blot), or for functional protein activity by a foam activity
assay.
Examples of some such conventional methods are shown in the Examples below.
RNA and DNA Analysis of 17 kDa Foam Protein Gene and mRNA
Using standard techniques, transgenic plant cells or tissue can be
assayed for the presence of 17 kDa Foam Protein mRNA transcripts by
hybridization
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to 17 kDa Foam Protein DNA probes. For example, the cDNA sequence encoding
the barley 17 kDa Foam Protein [Sequence ID No.9J is a useful hybridization
probe
for identifying the presence of 17 kDa Foam Protein mRNA in a test sample.
Such
probes are preferably about 10-35 consecutive nucleic acids, but the size may
be
S larger. Transcripts from plant tissue transformed with a construct
comprising the 17
kDa Foam Protein gene fused to heterologous 5' or 3" untranslated region (UTR)
sequences can be selectively detected and quantitated by RNA-PCR using primers
pairs located in the coding region and the 5' or 3' UTR.
A I 7 kDa Foam Protein gene construct, fused to a heterologous
promoter and/or terminator, can be detected in the genome of transformed
tissue by
PCR using primer pairs located in the 17 kDa Foam Protein coding region and
the
heterologous promoter or terminator sequences. The PCR product can be used as
a
hybridization probe for Southern blot analysis of genomic DNA from transformed
plants. Transformed plants are compared with untransformed plants to
distinguish
the introduced constructs from the endogenous l 7 kDa Foam Protein gene.
ELISA Assay for 17 IcDa Foam Protein
Samples, including transgenic cells, tissue, plants, and foaming
products are screened for content of the 17 kDa Foam Protein by immunological
assays, including an Enzyme Linked Immunoassay (ELISA). Polyclonal antibodies
used in an ELISA are, for example, generated against the purified 17 kDa Foam
Protein or as described below for Example 8.
Many variations of ELISA are known. In one representative type of
ELISA, wells of a microtiter plate are coated with anti-I 7 kDa Foam Protein
antibodies. An aliquot of a sample (the antigen) is added in serial dilution
to each
antibody-coated well. Labelled anti-17 kDa Foam Protein antibodies, such as
biotinylated antibodies, are then added to the microtiter plate. The
concentration of
bound labelled (e.g., biotinylated) antibody is determined by the interaction
of the
biotin with streptavidin coupled to peroxidase. The activity of the bound
peroxidase
is easily determined by known methods. The amount of 17 kDa Foam Protein in a
sample is quantitated with reference to the ELISA performed with pure antigen
standards, where the detection range should lie in the range of 0.2 - I O
ng/ml. Any
known method for producing antibodies and using such antibodies in an ELISA
assay can be used to determine the amount of 17 kDa Foam Protein in samples,
and
expressed in transgenic plant cells and tissues of the invention.
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Immunoassay Kits
An immunoassay kit of the invention includes an anti-17 kDa Foam
Protein antibody. Preferably, the kit also contains 17 kDa Foam Protein
standards
for quantitative analysis. Optionally, the kit can include other anti-Foam
Protein
antibodies such as anti-LTP 1 antibodies and/or additional reference
antibodies or
standards.
Use of 17 kDa Foam Protein in the Production of Beer
The invention provides for improvements in the production of
foaming products, such as beer. Improvements in beer are particularly in the
quality
of the beer foam.
A manufacturing process for the production of beer can include the
following processing steps:
malting of grain to produce a malt
2. mashing of malt to produce a sweet wort
3. boiling the sweet wort
4. fermenting the boiled wort to produce a beer
5. clarifying and finishing the beer to produce a beer
product.
In general, beers are manufactured from grains, including barley
grains, which are naturally low in fermentable sugars. Hydrolysis of starch to
sugars
is needed prior to fermentation with yeasts. To effect this hydrolysis, grains
are
wetted and allowed to germinate, during which time the germinating kernels
produce
hydrolytic enzymes (malting). At the end of malting, the malt is kilned and
stored.
The malt is then ground and suspended in water at the start of mashing, during
which the major part of starch hydrolysis occurs to produce the wort. The wont
is
boiled and separated from insoluble materials. The wort is then fermented, for
example by adding yeast to the wort. Fermentation will convert the wont to
beer. To
clarify and finish the product, insolubles are filtered, and other
constituents are
added.
The quality of the foam, e.g., beer foam, depends on both the brewing
process and the raw materials used. In the present invention, the quality of
foam is
enhanced by supplementing the brewing process with 17 kDa Foam Protein added
during one or more processing steps and/or by providing raw materials such as
barley grain or brewer's yeast producing enhanced amounts of 17 kDa Foam
Protein.
Danish Lager beer brewed with adjuncts contains about 10-50 mg/1 of 17 kDa
Foam
Protein. The effect of further addition of 17 kDa Foam Protein is dependent on
the
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composition of the beer and the ratio of the beer's foam components. However,
in
general, addition of about 25 mg/1 or more of 17 kDa Foam Protein is expected
to
have a beneficial effect on the foam properties of the beer.
In a preferred embodiment of the invention, the quality of foam in a
foaming product is enhanced by providing enhanced amounts of 17 kDa Foam
Protein in combination with enhanced amounts of LTP protein.
Transgenic Plants
In a most preferred embodiment of the invention, a combination of
enhanced amounts of 17 kDa Foam Protein and enhanced amounts of LTP 1 are
provided by genetically engineering plants such as barley, rye and wheat to
produce
high amounts of these Foam Proteins.
Production of LTPl has been enhanced in barley grain, by insertion
of the gene sequence encoding LTP 1 (Sandager, 1996, "Engineering of barley
for
1 S enhanced levels of lipid transfer proteins in the seed", M.Sc. Thesis,
Aarhus
University, Denmark No: 890564). In a similar manner, and using methods common
to transformation of plants, transgenic plants producing enhanced levels of 17
kDa
Foam Protein are produced.
Most preferred is a cereal plant, e.g., a barley plant carrying
transgenes encoding both LTP l and 17 kDa Foam Protein. In a preferred method
for
producing such a double transgene, two independent transgenic lines (e.g.
barley) are
produced; one transformed to express enhanced levels of LTP1 and a second
transformed to produce enhanced levels of 17 kDa Foam Protein. The two lines
are
crossed to generate and breed lines which are homozygous for both transgenes,
thereby producing a plant, e.g. barley plant, providing even greater
enhancement of
foam potential and stability than achieved by either transgene alone.
EXAMPLES
The invention may be better understood by reference to the following
examples, which serve to exemplify the invention and are not intended to limit
the
scope of the invention in any way.
AnalXtical procedures
The following Analytical Procedures were used in the Examples.
Foam assays
The Head Hunter, an opto-electrical foam assay system using digital
video image analysis, was used to measure foam potential and foam half-life of
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16
small, degassed samples. This system creates foam on 10 ml samples by a short,
vigorous, standardized shaking procedure. The decay with time of the foam
column
generated in this way is then automatically monitored (Haugsted, et al., 1990
Monatsschr. Brauwissenschaft 43:336-339; Haugsted and Erdal, 1991 Proc. 23rd
EBC Congress (Lisbon): 449-456). The foam potential (P) is the amount of foam
formed initially, in ml per ml sample. The foam half-life (F) is the time, in
seconds,
after which the foam column is reduced to half the initial volume. In the
following
examples, foam assays on the Head Hunter were conducted at least in duplicate.
The Foam Stability Analyser, System Carlsberg, as described in
Rasmussen, 1981 Carlsberg Res. Commun. 46:25-36, was used for foam assays on
bottled beer containing CO2. In this system, 150 g of beer is fully converted
to foam,
and the decay of foam into beer is followed. After an initial lag phase of
about 30
seconds, this decay is a first order process (Hallgren, et al., 1991 J. Am.
Soc. Brew.
Chem. 49:78-86). The foam half-life, in seconds, is then determined.
Determination of C02 in beer
C02 in beer was determined by a titration procedure. Initially, COZ
was absorbed in a carbonate-free solution of sodium hydroxide. Then, COZ was
liberated again via addition of sulphuric acid and forced into a solution of
barium
hydroxide of known concentration. After precipitation of barium carbonate, the
surplus of barium hydroxide was determined by titration with hydrochloric acid
using phenolphtalein as an indicator, and C02 in beer was finally calculated
from
this determination.
Determination of protein content
The protein content was determined from amino acid analyses or by
the dye binding method of Bradford (Bradford, 1976 Anal. Biochem 72:248-254).
SDS-PAGE and Western blots
SDS-polyacrylamide gel electrophoresis (PAGE) was performed
after boiling the samples for 5 minutes in NuPAGE sample buffer from Novex
supplemented with 10 mM dithiothreitol using a Novex XCell IITM mini cell,
NuPAGETM 10% Bis-Tris gels (1.0 mm x 10 wells) and NuPAGE MES-SDS
Running Buffer from Novex. Blotting onto nitrocellulose was then performed in
the
Xcell IITM blot module using the NuPAGETM Transfer Buffer from Novex. After
washing with water, the nitrocellulose was incubated 30 minutes with calf
serum to
block non-specific binding and then incubated overnight with antibodies. The
Promega Western blot AP system (Catalogue No. W3930) was then applied to
detect
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specific antibody binding. A mixture of nitro blue tetrazolium and 5-bromo-4-
chloro-3-indolyl phosphate was used as color development substrate for
alkaline
phosphatase.
Amino acid composition and N-terminal amino acid sequencing
Amino acid compositions were determined on an amino acid analyzer
(LKB, model Alpha PIusTM) after hydrolysis in 6 M HC1 at 110°C for 24
hours in
evacuated tubes.
N-terminal amino acid sequencing was performed on a gas-phase
sequence (Applied Biosystems model 470A), using the program provided by the
company. The phenylthiohydantoin-labelled amino acids from the sequencer were
identified on-line by reversed-phase HPLC using an Applied Biosystems model
120A phenylthiohydantoin analyzer.
Example 1
Isolation of Foam Positive Agents from Beer
To isolate foam positive components from beer, two principally
different approaches can be taken: (a) to isolate the individual components
directly
from beer and investigate their capability of making foam, or (b) to isolate
foam
positive components directly from collapsed beer foam. ~l'he latter strategy
has been
the basis for this work and is described by Se~rensen, et al. 1993 MBAR Techn.
Quart. 30:136-145, Bech, et al. 1995 WO 95/13359, and Bech, et al. 1995 Proc.
25th EBC Congress (Brussels): 561-568.
In a large foam tower (30 cm X 150 cm), foam was produced from 15
liters of beer by sparging with nitrogen, as described in S~rensen, et al.,
1993, MBAR
Techn. Ouart. 30:136-145. The foam fraction was diluted with water and
reintroduced into the foam tower. Two additional flotations were carried out
to get
rid of components which were not foam-active themselves but merely carried
over
with the foam. The third flotate was used in the following studies.
By gel chromatography on Sephadex G75, the foam fractions were
separated according to size into three fractions: a high molecular weight
fraction
(HMW), a low molecular weight fraction (LMW), and a very low molecular weight
fraction. The very low molecular weight fraction consisted of small peptides
and
carbohydrates, free amino acids, and iso-a acids. Foam analyses in the Head
Hunter
revealed that the foam active components are present in the HWM and LMW foam
fractions, while the very low-molecular weight foam fraction does not
contribute to
foam formation nor foam stability. When dissolved in water, the HMW foam
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fraction forms a low but most stable foam, while the LMW foam fraction
provides a
very high foam potential.
The HMW foam fraction contains mainly carbohydrate (90%) but
also some protein Z, a barley albumin. Foam measurements on protein Z isolated
from beer showed that protein Z creates a low but very stable foam when
dissolved
in water. Further, protein Z isolated from beer has a stabilizing effect on
the foam
created by other proteins.
After foaming beer in the foam tower, protein Z was distributed with
approximately 1 /3 in the first flotate and 2/3 in the remanent. In the
following
flotations, most of protein Z from the beer remained in the remanent. Thus,
protein
Z does not concentrate in the foam upon foaming of beer.
Protein Z has also been purified from malt and antibodies have been
raised against this protein. Based on these antibodies a sandwich ELISA for
the
quantification of protein Z has been established. Using these antibodies, an
affinity
column directed towards protein Z has been synthesized and used for selective
removal of protein Z from beer. Removal of protein Z does not influence the
foam
potential, but affects the foam stability, depending on the composition of the
beer.
In beer made from undermodified malt, where storage protein
reserves are only partially degraded, the stability is independent of protein
Z. In
contrast, protein Z is a highly stabilizing factor in beer made from
overmodified
malt, where the protein reserves have been extensively degraded. In
commercially
available beer types, foam stability is reduced to varying degrees when
protein Z is
removed, probably due to varying degrees of modification of the malt. This is
in
good agreement with literature presenting diverging ideas on the significance
of
protein Z for the foam.
The LMW foam fraction consists of 10% carbohydrate and 90%
protein. A major component in LMW foam is the protein Lipid Transfer Protein 1
(LTP1), a barley protein with a molecular weight of 10,000. The LMW foam
fraction also contains substantial amounts of other peptides of which the
major part
can be separated from LTP1 by ion exchange chromatography.
When dissolved in water, LTP 1 from foam (foam-LTPl ) forms a
very high foam which is not particularly stable. Foam-LTP1 also increases foam
potential when dissolved in beer, the effect, however, being not as pronounced
as in
water since beer already contains foam positive agents.
LTP 1 has also been purified from barley (barley-LTP 1 ), and foam
measurements have shown that foam-LTP 1 is considerably more foam active than
is
barley-LTP 1. It was, therefore, of interest to compare the two varieties and
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investigate when the transformation of LTP1 into a more foam active form takes
place.
A comparison of the amino acid compositions indicated that the two
varieties of LTP 1 were almost identical. Foam-LTP l, however, contains more
glutamine/glutamate (Glx) and proline than barley-LTPl. This is probably due
to
traces of hordein peptides in the preparations. The primary structures of the
two
varieties of LTP1 are identical, but NMR analyses revealed that while barley-
LTP1
has a well-defined three-dimensional structure, the NMR-spectrum for foam-LTP1
is characteristic for proteins which are fully or partly denatured.
The molecular mass of barley- and foam-LTPI was determined by
mass spectrometry. Barley-LTP1 has a molecular weight of 9663 daltons, while
foam-LTP1 is heterogeneous with a molecular weight in the range of 9,687-
10,000
daltons.
To investigate the correlation between the amount of LTP 1 and the
foam characteristics of the final beer, antibodies were raised against barley-
and
foam-LTP1. ELISA assays were established for both forms and no cross reaction
was seen between the two forms.
The transformation of barley-LTP 1 to foam-LTP 1 during the
malting and brewing processes was investigated by means of these assays. The
concentration of LTP 1 was unchanged during malting, and no foam-LTP I was
demonstrated in malt extracts.
During mashing, LTPI was extracted immediately after mashing in,
and the yield did not depend on the mashing procedure. In first wort prepared
on a
laboratory scale, no foam-LTP 1 was demonstrated, but in first wort from the
brewhouse, traces of this foam-LTP 1 were seen.
During wort boiling, the concentration of barley-LTP 1 measured by
ELISA was reduced to 10-20% of the initial level, and in parallel, foam-LTPl
was
increased.
During fermentation, there was no further transformation of barley-
LTP 1. The LTP 1 in beer is thus a mixture of barley-LTP 1 (with poor foam-
ability)
and foam-LTP 1. In the foam tower, foam-LTP 1 is transferred quantitatively to
the
foam fraction, while barley-LTP 1 remains in the remanence. In a similar
manner,
when a beer is poured, foam-LTP 1 concentrates in the foam.
An affinity column directed towards barley-LTP I was synthesized
and used for selective removal of barley-LTPI from first wort, which was then
boiled. The foam potential in the boiled LTPI-free wort was considerably lower
than in the reference wont. This experiment thus confirmed the significance of
LTP1
for foam. The remaining foam positive components in the LTP 1-free boiled wont
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were, not unexpectedly, still able to form a considerable foam potential, and
it
should be expected that the quantitative significance of LTP1 is dependent on
the
concentrations of other foam-promoting components.
In addition to foam-LTP1 the LMW foam fraction also contains
S substantial amounts of other peptides of which the major part can be
separated from
LTP 1 by ion exchange chromatography. Amino acid analysis of this pool, "Pool
1 ",
showed high concentrations of proline and glutamate/glutamine, indicating that
it
contains substantial amounts of hordein peptides. When dissolved in water,
Pool 1
contributes to foam potential as well as foam stability. Pool 1 is also able
to increase
10 foam potential when dissolved in beer.
It has been demonstrated that beer brewed from undermodified malt
has good foam characteristics, while overmodified malt results in a poorer
foam. In
foam from beer of undermodified malt, the LMW foam fraction and especially
Pool
1, is considerably larger than normal, which suggests that Pool 1 could be one
of the
15 fractions that vary between brews. Pool 1 was analyzed by SDS-PAGE, and
appeared heterogeneous. The only well-def ned fragment to be identified in the
SDS-PAGE was a peptide with a molecular weight of approximately 17,000
daltons.
20 Example 2
Purification of 17 kDa Foam Protein From Beer
17 kDa Foam Protein was purified from 5 liters of beer by addition of
ammonium sulfate to 55% saturation. After 16 hours at 4° C, the
suspension was
centrifuged and the precipitate was dissolved in water and dialyzed in a
SpectraporT"' membrane against water. The dialysate was passed through a 270
ml
SP-SepharoseT"' column equilibrated with 20 mM sodium acetate, pH 4.5. The
run-through fraction from this column was adjusted to pH 3.9 by addition of
HCl
and the ionic strength was reduced to 0.03 mS by addition of water. 'The
resulting
fraction was subjected to ion exchange chromatography on a 270 ml SP-
SepharoseTM column equilibrated with SmM sodium formate, pH 3.9. ELISA
revealed the 17 kDa Foam Protein to be eluted by a linear gradient from 0 to
0.25 M
NaCI. Fractions containing the 17 kDa Foam Protein were concentrated by vacuum
evaporation and applied to a 350 ml SephadexTM G50 column equilibrated with
20mM sodium acetate, 100 mM sodium chloride, pH 4.9. The resultant fractions
were analyzed by ELISA and those containing the 17 kDa Foam Protein were
pooled, dialyzed against water and lyophilized. SDS-PAGE and Western blots of
the resulting preparations always showed a distinct double band of 17-18 kDa.
See,
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21 - __
for example, Figure 2A, SDS-PAGE, showing foam-LTP 1 (lane A); beer protein
(lane B); and 17 kDa Foam Protein from beer (lane C).
Example 3
Purification of 17 kDa Foam Protein from Barley
17 kDa Foam Protein was isolated from 2 kg barley flour (Alexis) by
extraction with 10 L water at 45°C for 1 hour. The mixture was
centrifuged and
ammonium sulfate (50% saturation) was added to the supernatant to precipitate
protein. After 16 hours at 4°C, the suspension was centrifuged and the
precipitate
was dissolved in water and dialyzed in a Spectrapor membrane against water. 17
kDa Foam Protein was purified from barley essentially as described above for
purification from beer in Example 2. After ion exchange chromatography on SP-
Sepharose pH 3.9, the 17 kDa Foam Protein eluted in two peaks containing a
first
form with an intact peptide chain and a second form with a partly nicked
peptide
chain, respectively. These two protein forms were independently characterized.
Example 4
Purification of 17 lcDa Foam Protein from First Wort
17 kDa Foam Protein was isolated from first wort from a production
scale mashing (Carlsberg Pilsner) using the purification procedure described
above
for Example 2. On SDS-PAGE, the preparation showed a distinct double band of
17-18 kDa.
Example 5
Amino Acid Sec~uencin~ of 17 lcDa Foam Protein
1,7 kDa Foam Protein isolated from barley and beer as described
above was subjected to N-terminal sequencing. 17 kDa Foam Protein from barley
was sequenced 40 cycles while 17 kDa Foam Protein from beer was found to be N-
terminally blocked. According to the amino acid analysis, 17 kDa Foam Protein
contains 7 methionyl residues corresponding to 8 cyanogen bromide fragments.
After chemical cleavage with cyanogen bromide, five of these fragments were
isolated and sequenced and the sequence showed homology with that of the 17
kDa
Foam Protein encoded by the cDNA and genomic clones shown in Tables 1 and 2.
The amino acid sequence of each of these fragments is indicated in the
sequence
Table 1 by underlining, and is listed below.
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SEQ ID FRAGMENT SEQUENCE
NO:
1 L DTTCSQGYGQCQQQPQQQM
2 N TCAAFLQQCSQTPYVQSQM
3 R Q Q C C Q P L A Q I S E Q A
4 R
Q
Q
Q
G
Q
S
F
S
Q
P
Q
Q
Q
V L Q T L P S M C S V N I P Q Y C T T T P C
T T I T P
Example 6
Isolated BarleyNucleic Acid Sequences Encoding 17 kDa Foam Protein
5 The nucleotide sequence of the barley gene coding for the 17 kDa
Foam Protein was determined from a cDNA clone generated by reverse
transcription
and amplification techniques (RT-PCR). The cDNA clone was selectively
amplified from a total RNA population isolated from developing barley
endosperm
tissue cv Alexis, 20 days after anthesis, as described by Rechinger, et al.
1993,
Theor. Appl. Genet. 85:829-840.
The mRNA in the RNA population was reverse transcribed with a
poly dT primer having a BamHI restriction site sequence at the 5'end (5'
ATGGATCCT» 3') [SEQ ID NO: 6]. The reaction mixture (20 ~1), comprising 1
~g total RNA, 1 ~M poly dT primer, 1 mM deoxynucleoside triphosphates, 28U
RNasin (Promega) and 20U M-MuLV reverse transcriptase (Boehringer Mannheim)
in a buffer supplied by the manufacturer, was incubated at 40°C for 30
minutes. The
first strand cDNA was amplified with a sequence-specific, but degenerate
primer,
based on a determined amino acid sequence located near the N-terminus of the
17
kDa Foam Protein (PQQQMN) [SEQ ID NO: 7], having a BamHI restriction site
sequence at the 5'end (5'ATGGATCCICAICAICAIATGAA 3') [SEQ ID NO: 8]
("I" denotes the degeneracy). The reaction mixture ( 100,1) comprised 4 ~1
first
strand cDNA reaction mixture, 0.2 ~,M sequence-specific and polydT primers,
0.2
mM deoxynucleoside triphosphates and 2.SU AmplitaqTM DNA polymerase (Perkin
Elmer) in a buffer supplied by the manufacturer.
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23 _
SEQ ID SEQUENCE
NO:
6 ATGGATCCT~~
7 P Q Q Q M N
8 ATGGATCCICAICAICAIATGAA
The cDNA was amplified in a Perkin Elmer Thermocyler 480 with 30
heating cycles (95°C for 1 minute / 50°C for 1 minute) and then
analysed by agarose
S gel electrophoresis. A cDNA fragment of approximately 580 nucleotides was
isolated, cloned into a plasmid vector and its nucleic acid sequence was
determined
with an AmpliCycleTM Sequencing Kit (Perkin Elmer). The determined and back-
translated nucleic acid sequence [SEQ ID NO: 9] and the determined and deduced
amino acid sequence [SEQ ID NO: 10] are shown below in Table 1. Nucleotides 1 -
57 were back-translated from the determined N-terminal amino acid sequence of
the 17 kDa Foam Protein using the codon bias of the barley 1-3,1-4 [3-
glucanase
according to Jensen et al., 1996 supra.
The deduced amino acid sequence encoded by the partial cDNA clone
shows complete homology with the sequence of N-terminal and cyanogen bromide
I 5 peptide fragments determined for the purified 17 kDa foam polypeptide,
confirming
that the isolated cDNA encodes the 17 kDa Foam Protein. Underlined sequences
in
Table 1 indicate the sequences of the cyanogen bromide fragments. PCR primers
used to amplify the clone are indicated above the sequence. * Denotes stop
codon.
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Table 1
cDNA Encoding 17 kDa Foam Protein'
sense primer ~
S CTCGACACCACCTGCTCCCAAGGCTACGGCCAATGCCAACAACAACCGCAACAACAAATG 60
L D T T C S Q G Y G Q C Q Q Q P Q Q Q M
AACACCTGCGCTGCTTTCCTGCAGCAGTGCAGCCAGACACCATACGTCCAGTCACAGATG 120
N T C A A F L Q Q C S Q T P Y V Q S Q M
TGGCAGGCAAGCGGTTGCCAGTTGATGCGGCAACAATGCTGCCAGCCACTGGCCCAGATC 180
W Q A S G C Q L M R Q Q C C Q P L A Q I
TCGGAGCAGGCTCGGTGCCAGGCCGTCTGTAGCGTGGCACAGGTCATCATGCGGCAACAG 240
IS S E Q A R C Q A V C S V A Q V I M R Q Q
CAAGGGCAGAGTTTCAGTCAGCCTCAGCAGCAGCAATCGCAAAGTTTCGGCCAGCCTCAG 300
Q G Q S F S Q P Q Q Q Q S Q S F G Q P Q
2O CAGCAGGTTCCGGTTGAGATAATAAGGATGGTGCTTCAGACCCTTCCGTCGATGTGCAGC 360
Q Q V P V E I I R M V L Q T I. P S M C S
2S
GTGAACATTCCGCAATATTGCACCACCACCCCGTGCACCACCATCACCCCCACCATCTAC 420
V N I P Q Y C T T T P C T T I T P T I Y
AGCATCCCCATGGCAGCTACCTGTGCCGGTGGTGTCTGCTAAGATCTGTGATGTGCTAGC 480
S I P M A A T C A G G V C
TAGATCGATCACCGTTTAGTTGATCGATGAAAGCTACAAAATAAAAGTGCCATACGTCAT 540
3~ t-polydT primer
CATTGTGTGCCGGTACTATTGCAACTTGGAAATAATAAACCTCTGTTTCTGAATAXAAAAlz
The cDNA clone encoding the 17 kDa Foam Protein was used to
screen a commercially available barley genomic library (Lambda Fix II Barley
cv
3S Igri Genomic Library from Stratagene, Catalog No: 946104) in order to
obtain the
genomic gene sequence. Using standard hybridization and plaque purification
methods described, for example, in Sambrook, Fritsch & Maniatis, 1989,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press Plainview,
NY 2"d Edition, three positive plaques were identified out of 400,000 plaques.
40 One characterized lambda genomic clone contained a hybridizing
Hind III -Not I fragment of about 2800 nucleotides, including the 1336
nucleotide
sequence shown in Table 2 (SEQ ID NO: 11). This genomic sequence contains the
complete coding sequence for a precursor form of the 17 kDa Foam Protein. The
deduced amino acid sequence of this precursor 17 kDa Foam Protein (SEQ ID NO:
4S 12) is predicted to comprise a 19 amino acid signal peptide with a signal
peptide
cleavage site between Ala 19 and Gln 20, according to the signal peptide
algorithm
developed by Nielsen, et al., 1997 Protein Engineering 10: 1-6. The N-terminal
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amino acid of the determined mature 17 kDa Foam Protein amino acid sequence is
Leu 21. This suggests that the actual signal peptide cleavage site is G1n20 1
Leu2l, or
alternatively that the N-terminal GIn20 is cleaved off post-translationally to
give
the amino acid sequence of a mature 17 kDa Foam Protein.
5 The deduced amino acid sequences of the 17 kDa Foam Proteins
encoded by the genomic and cDNA clones show close homology, with six amino
acid missmatches. These sequence differences may reflect heterogeneity between
cultivars or the presence of more than one copy of the gene in the haploid
genome.
The gene 3' flanking sequence of 140 nucleotides contains consensus sequences
for
10 three polyadenylation signals. The gene 5' upstream sequence of 674
nucleotides
contains a TATAA box, shown underlined in Table 2, located 85 nucleotides
upstream of the translation start codon. An endosperm box (TGTAAAG) followed
by a GCN4 box (ATGAGTCAT), shown in bold, located 304 nucleotides upstream
of the translation start, are transcription regulatory elements found in the
promoters
15 of many cereal endosperm storage protein genes (Miiller and Knudsen 1993
The
Plant Journal 4: 343-355).
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26 ..
Table 2
17 kDa Foam Protein Gene Nucleotide and Deduced Amino Acid Sequence
AGCATAGCTCCATGACAATCTTTTACAGGTAAAGGAAAATTTATGAGTCATCAATGCTCT 60
S
ACTGATGCCGTTTGTATTACCAAAGTAGTACAAGGAAAACAAAATCCAAGATAACAAAAC 120
CAGTTTTCAGGAAACAATGAGATGGGAGTGCGGGGCATGCCAATCTGATTTATATCTAAC 180
IO AACTCGTACAAGATAACAAAATGAATTTCACAAAAAGACTCAATCCGGATATACGCTTGA 290
CATGTAAAGTGATCAGTGATGAGTCATATGGATTATCGTGGTCAGGCGCGAGCTGATTTA 300
TATCTAACAACTCGTACAAGATAACAAAATGAATTTCACAAAAAGACTCAATCCAGATAT 360
1S
ACGGTTGACATGTAAAGTGCTCAGTGATGAGTCATATGGATCATCGAGGTCAGACGCGAG 420
CTAACTGACATCTACACGATATGTGTTGAAAAGTATATTTGACGACCATCCAAGATTGGA 480
2O CTTGTAGCCCAACCTAACACAAGTGTGTCAGATGATCAGTTGGAAAAGCACACAAAACCT 540
TTAGCATAGGAACCTACAGATGCAATGCACCAAATGATGCCATGGTAGCTATAAATAGGC 600
CCGCACCATGAAGATCCTCCCTCATCATCCTTCACACAAACACAAGCATCAAAGCAAACT660
2S
TGTAGCCAGCCACCATGAAGACCATGCTGATCCTCGCGCTCATCGCCTTCGCGGCGACCA720
M K T M L I L A L I A F A A T S 1
6
Signal peptide
GCGCCGTTGCACAGCTGGACACTACCTGTAGCCAGGGCTACGGGCAGTGCCAGCAACAGC780
3 A V A Q L D T T C S Q G Y G Q C Q Q Q P 3
O 6
Mature protein
CGCAGCAGCAGATGAACACATGTGCTGCCTTCCTGCAGCAGTGCAGCCGGACACCATACG890
Q Q Q M N T C A A F L Q Q C S R T P Y V 5
6
3S TCCAGTCACAGATGTGGCAGGCAAGCGGTTGCCAGTTGATGCGGCAACAATGCTGCCAGC900
Q S Q M W Q A S G C Q L M R Q Q C C Q P 7
6
CGCTGGCCCAGATCTCGGAGCAGGCTCGGTGCCAGGCCGTCTGTAGCATGGCACAGGTCA960
L A Q I S E Q A R C Q A V C S M A Q V I 9
6
40
TCATGCGGCAACAGCAAGGGCAGAGTTTCACTCAGCCTCAGCAGCAGCAATCGCAAAGTT1020
M R Q Q Q G Q S F T Q P Q Q Q Q S Q S F 116
TCGGCCAGCCTCAGCAGCAGGTTCCGGTTGAGGTAATGAGGATGGTGCTTCAGACCCTTC1080
4S G Q P Q Q Q V P V E V M R M V L Q T L P 136
CGTCGATGTGCAGCGTGAACATCCCGCAATATTGCACCACCACCCCGTGCAGCACCATCA1190
S M C S V N I P Q Y C T T T P C S T I T 156
SO CCCCCACCATCTACAGCATCCCCATGGCAGCTACCTGTGCCGGTGGTGTCTGCTAAGATC1200
P T I Y S I P M A A T C A G G V C * 1
7
4
TGTGATGTGCTAGCTAGATCGATCACCGTTTAGTTGATCGATGAAGAGCTACAAAATAAA 1260
SS AGTGCCATACGTCATCATGTGTGGCCGGTACTATTGCAACTTGGAAATAATAAACCTCTG 1320
TTTCTGAATAAAGCTT 1336
* Denotes stop codon terminating the open reading frame.
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27
Analysis of the amino acid sequence of the 17 kDa Foam Protein
identified the novel 17 kDa Foam Protein as a member of the barley hordein
storage
protein family with particularly close homology with the y-hordein class,
based on
protein sequence alignment with the members of the sulfur-rich B- and y-
hordein
S polypeptides.
Alignment of the amino acid sequence of 17 kDa Foam Protein (el
hordein) encoded by the cDNA clone, with members of the sulfur-rich B- and y-
hordein polypeptide families found in barley, Hordeum vulgare (GenBank
accession
numbers Blhor, X03103; B2hor, X87232; B3hor, X53691; y2hor, X13508; y3 hor,
X72628) is shown in Table 3. Cysteine residues are shown in bold and amino
acid
residues in the 17 kDa Foam Protein showing homology to other members of the
sulfur-rich hordein polypeptides of barley are underlined.
Eight cysteine residues highly conserved among the monomeric ~y-
type gliadin and hordein were also conserved in the 17 kDa Foam Protein
sequence,
indicating that the protein may also assume a globular structure, stabilized
by 4
disulphide bridges (Miiller and Wieser, 1997, J. Cereal Science, 26:169-176)
as
proposed in Figure 1. In contrast to other sulfur-rich cereal storage
proteins, the 17
kDa Foam Protein lacks an N-terminal proline/glutamine rich repetitive domain,
and hence has been classified by Applicants as a new class of hordein, namely
El
hordein. The C-terminal domain of the 'y-type storage proteins found in wheat,
which are highly homologous to y hordein, are known to assume compact
structures
with a marked surface hydrophobicity (Popineau and Pineau, 1988, Lebensmittel-
Wissenschaft and Technologie, 21:112-117) which may contribute to the foam
properties of the 17 kDa Foam Protein.
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28
Table 3
Alignment of sulfur rich hordein polypeptides
1 50
S B2hor .......... .......QQQPFP..QQP.IPQQPQPYPQQP.QPYP.Q..
B3hor .......... .......QQQPFP..QQP.FPQQPQPYPQQP.QPYP.QQP
Blhor .......... .......QQQPFP..QQP.IPQQPQPYPQQP.QPYP.QQP
y2hor ....EMQVNP SVQVQPTQQQPYPESQQPFISQSQQQFPQPQ.QPFP.QQP
y3hor ITTTTMQFNP SGLELERPQQLFPQWQP..LPQQPPFLQQEPEQPYPQQQP
51 100
B2hor .......... .............QPFPPQQAFPQQPPF..WPQQPFPQQP
B3hor FQPQQPFPQQ TIPQQPQPYPQ..QPFPPQQEFPQQPPF..WPQQPFPQQP
Blhor FPPQQPFPQQ PVPQQPQPYPQ..QPFPPQQPFPQQPPF..WQQKPFPQQP
IS y2hor QQPFPQSQQQ CLQQPQHQFPQPTQQFPQRPLLPFTHPFLTFPDQLLPQPP
y3hor LPQQQPFPQQ PQLPHQHQFPQQL....PQQQFPQQMPLQ..PQQQFPQQM
101 150
B2hor PF.GLQQPIL SQQQPCTPQQTPLPQGQLYQTLLQLQIPYVQPSI....LQ
2O B3hor PF.GLQQPIL SQQQPCTPQQTPLPQGQLYQTLLQLQIPYVHPSI....LQ
Blhor PF.GLQQPIL SQQQPCTPQQTPLPQGQLYQTLLQLQIQYVHPSI....LQ
y2hor HQ.SFPQPPQ SYPQP.PLQPFPQPPQQKYPEQPQQPFPWQQPTIQLYLQQ
y3hor PLQPQQQPQF PQQKPFGQYQQPLTQQPYPQ...QQPLAQQQPSIEE..QH
Elhor ............................LDT _QQP.....
_TCS_QGYGQC QQ
Q
2S _ _
151 200
B2hor QLNPCKVFLQ QQCS...PVRMPQLIA...RSQMLQQSSCHVLQQQCCQQL
B3hor QLNPCKVFLQ QQCS...PVRMPQLIA...RLQMLQQSSCHVLQQQCCQQL
Blhor QLNPCKVFLQ QQCS...PVPVPQRIA...RSQMLQQSSCHVLQQQCCQQL
3O y2hor QLNPCKEFLL QQCR...PVSLLSYI....WSKIVQQSSCRVMQQQCCLQL
y3hor QLNLCKEFLL QQCTLDEKVPLLQSVISFLRPHISQQNSCQLKRQQCCQQL
Elhor QMNTCAAFL. QQCSQTPYVQ..........SQMWQASGCQLMRQQCCQPL
201 250
3S B2hor PQIPEQFRHE AIRAIVYSIFLQEQPQQSVQGASQPQQQLQEEQVGQCYFQ
B3hor PQISEQFRHE AIRAIVYSIFLQEQPQQSVQGVSQTQQQLQQEQVGQCSFQ
Blhor PQIPEQFRHE AIRAIVYSIFLQEQPQQLVEGVSQPQQQLWPQQVGQCSFQ
y2hor AQIPEQYKCT AIDSIVHAIFMQQGQRQGVQIVQ................Q
y3hor ANINEQSRCP AIQTIVHAIVMQQQVQQQVGHG................FV
4O slhor AQISEQARCQ AVCS.VAQVIMRQQQGQSF.....................
251 300
B2hor QPQPQQLGQP .....QQVPQSVFLQPHQIAQLEATNSIALRTLPTMCNVN
B3hor QPQPQQLGQA .....QQVPQSVFLQPHQIAQLEATTSIALRTLPRMCNVN
4S Blhor QPQPQQVGQQ .....QQVPQSAFLQPHQIAQLEATTSIALRTLPMMCSVN
y2hor QPQPQQVGQC .....VLVQGQGVVQPQQLAQMEAIRTLVLQSVPSMCNFN
y3hor QSQLQQLGQG MPIQLQQQPGQAFVLPQQQAQFKVVGSLVIQTLPMLCNVH
Elhor .SQPQQ.... ......QQSQS.FGQPQQQVPVEIIR.MVLQTLPMVCSVN
S~ 301 323
B2hor VPLY..DIMP FGVGTRVGV* [SEQ ID N0:
13J
B3hor VPLY..DIMP PDFWH*....... [SEQ ID NO:
14]
Blhor VPLY..RILR .GVGPSVGV*... [SEQ ID N0:
15]
y2hor VPPNCSTIKA PFVGVVTGVGGQ* [SEQ ID NO:
16]
SS y3hor VPPYCSPFGS MATGSGGQ*.... [SEQ ID NO:
17]
elhor IPQYCTTTPC TTITPTIYSIPMAATCAGGVC* ID N0:
[SEQ 10]
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Example 7
Isolated Wheat Nucleic Acid Sequence Encoding 17 lcDa Foam Protein
The nucleotide sequence of a wheat gene coding for a homologue of
the barley 17 kDa Foam Protein was determined from a genomic DNA fragment
S amplified by PCR. Wheat grain (Triticum aestivum L.) cv Husar was germinated
and
grown in the dark for 6 days and the etiolated leaves harvested for the
preparation of
genomic DNA using a Plant DNA Isolation Kit from Boehringer Mannheim. Wheat
genomic DNA (0.5 p,g) was PCR amplified with degenerate sense and antisense
oligonucleotide primers based on the deduced amino acid sequence of the barley
17
kDa Foam Protein:
SEQ ID SEQUENCE TYPE
NO:
18 5' G T I G C I C A I I T I G A Y Sense Primer
A C I A C 3'
19 5' G C I C A I G T I G C I G C C Antisense
A T 3'
Primer
The genomic DNA was amplified with 2.5 pmol of each primer and
AmpliTaqTM DNA polymerase (Perkin Elmer) in a reaction mixture provided by the
manufacturer, using a 'touch-down' thermocycling program (95°C for 0.45
minutes,
54°C [ -1°C /cycle ( 40°C) ] for 0.45 minutes and
72°C for 2 minutes for 15 cycles;
95°C for 0.45 minutes, 40°C for 0.45 minutes and 72"C for 2
minutes for 25 cycles;
72°C for 6 minutes). The PCR product of about 500 nucleotides was
isolated,
cloned in a pCR 2.1-TOPO plasmid vector (Invitrogen) and sequenced with an
AmpliCycleTM sequencing Kit (Perkin Elmer) on an Applied Biosystems 373 DNA
sequencer.
The nucleotide sequence of the amplified genomic DNA, based on
the consensus sequence of four independent PCR clones, was found to encode a
homolog of the barley 17 kDa Foam Protein. The nucleic acid sequence [SEQ ID
NO: 20] and deduced amino acid sequence [SEQ ID NO: 21 ] of the wheat 17 kDa
Foam Protein are shown in Table 4. The wheat and barley 17 kDa Foam Protein
amino acid sequences, deduced from their respective genomic sequences, showed
85
homology. Sequence alignment of the nucleic acids encoding wheat and barley
I7 kDa Foam Protein homologs indicates that the wheat amplified genomic
fragment, including the primers, encodes the last 2 residues of the predicted
signal
peptide and extends to within 4 amino acids of the C--terminus of the mature
barley
17 kDa Foam Protein sequence. The high degree of sequence homology between the
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wheat and barley 17 kDa Foam Protein homologues is consistent with recognition
of
a wheat 17 kDa polypeptide by the anti-barley 17 kI)a Foam Protein antibodies
described in Example 19.
Table 4
Wheat Gene Encoding 17 kDa Foam Protein
SENSE PRIMER
lO GTGGCGCAGCTGGATACTACATGTAGCCATGGCTATGGGCAATGCCAGCAGCAGCCGCAA 60
V A Q L D T T C S H G Y G Q C Q Q Q P Q 20
CAGCAGGTGAACACATGCGCTGCTCTCCTGCAGCAGTGCAGCCCGACACCATATGTCCAG 120
IS Q Q V N T C A A L L Q Q C S P T P Y V Q 4 0
2S
TCACAGATGTGGCAGGCAAGCGGTTGCCAGGTGATGCGGCAACAGTGCTGCCAGCCGCTG 180
S Q M W Q A S G C Q V M R Q Q C C Q P L 6 0
GCCCAGATCTCGGAGCAGGCTCGGTGCCAAGCTGTCTGTAGCGTGGCCCATGTCATCATG 240
A Q I S E Q A R C Q A V C S 'V A H V I M B 0
CGACAGCAGCAAGGGCAAAGTTTCAGTCAGCCTCAGCAACAACAAGTGCAAAGTTTCGGT 300
R Q Q Q G Q S F S Q P Q Q Q Q V Q S F G 100
3O CAGCCACATCAGCAGGTTCCGGTTGAGATAACGAGGATGGTGCTTCAGACCCTTCCATCG 360
Q P H Q Q V P V E I T R M V L Q T L P S 1 2 0
GTCTGCAGCGTGAACATCCCGCAATATTGCGCCACCACCCCATGCAGCACCATCTTTCAG 420
3S V C S V N I P Q Y C A T T P .. S T I F Q 140
ANTISENSE PRIMER
ACCCCCTACAACATCCCTATGGCCGCCACCTGCGC 455
T P Y N I P M A A T C A 152
Example $
Foam Capacity of Isolated 17 lcDa Foam Protein
17 kDa Foam Protein was isolated from beer and from barley as
4S described for Examples 2 and 3. Head Hunter foam assays were performed on
the
isolated proteins dissolved in distilled water at concentrations of 0.25 or
O.SO mg/ml.
The data shown below in Table S demonstrates that the protein isolated from
beer
was able to produce a high and stable foam, whereas the protein isolated from
barley
produced only a moderate foam with very little stability. The foam capacity of
the
SO barley-form of 17 kDa was only slightly affected by the presence or absence
of nicks
in the peptide chain.
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Table 5
Protein 0.25 m ml 0.50 m ml
17 kDa Foam foam potentialfoam stabilityfoam potentialfoam
Protein (ml/10 ml) (seconds) (ml/10 ml) stability
seconds
17 kDa Foam 7.0 not not not
Protein from measurable determined determined
barley
a)
17 kDa Foam 5.0 not 8.2 not
Protein from measurable measurable
barley
b)
17 kDa Foam 9.8 154 14.3 199
Protein from
beer
a) intact peptide-chain b) peptide-chain partly nicked
Foam assays were also performed on beer supplemented with 17 kDa
Foam Protein that had been isolated from beer. The 17 kDa Foam Protein was
supplemented either alone or in combination with LTP 1 that had been isolated
from
beer foam (Bech, et al., 1995, supra). A lager beer, Carlsberg Pilsner,
naturally
containing 30 mg/1 of the foam-type of LTP 1 and 25 mg/1 of 17 kDa Foam
Protein
was diluted 1:1 with a solution of 4% ethanol in water and used as basis for
these
experiments. After addition of purified 17 kDa Foam Protein and/or LTP1 of the
foam-type, the levels of these two proteins were approximately twice the
levels
found in the original beer prior to dilution with the ethanol-protein
solution. As
shown below in Table 6, addition of 17 kDa Foam Protein alone had hardly any
effect on the foam potential (P), whereas addition of foam-LTP 1 alone had
some
effect. However, addition of both proteins simultaneously resulted in the
greatest
improvement in foam potential, suggesting that the ratio of the foam
components is
important. In this study, none of the additions had any significant impact on
the
foam stability (S).
Table 6
Sample Foam Potential Foam stability
(mUlO (seconds)
ml)
Reference (beer dilutedI 1.7 +/- 0.3 181 +/- 12
1: I )
Reference + 17 kDa 1 I .5 +/- 0.8 141 +/- 47
protein
Reference + foam-type 12.8 +/- 0.6 168 +/- S 1
LTP 1
Reference + 17 kDa 13.5 +/- 0.3 186 +/-22
Foam
Protein + foam-type
LTP 1
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32
Example 9
Antibodies Specific to 17 kDa Foam Protein
Two rabbits were immunized with LMW, the low molecular weight
fraction obtained from beer foam as described above. The immunization was
performed by Dako, Glostrup, Denmark, according to the standard immunization
scheme of this company.
The rabbits received 250 pg LMW foam preparation at each injection.
A volume of 20 ml serum was obtained from each animal approximately two months
after the first injection, and then at monthly intervals. Serum obtained from
the
second bleeding of one of the animals (batch no. 1897), was used throughout
the
experiments described below.
Antibodies of the immunoglobulin G class (IgG) were purified from
other serum components by affinity chromatography on Hi-Trap Protein A-
Sepharose (Pharmacia, Uppsala, Sweden) according to the manufacturer's
instructions.
Apart from antibodies recognizing 17 kDa Foam Protein, the pool of
antibodies thus obtained also contained antibodies recognizing LTP1 of the
foam-
type, which is a prominent Foam Protein in beer. Antibodies recognizing LTPl
were removed from the IgG pool by affinity chromatography on a small column
containing LTP 1 of the foam-type covalently attached to CNBr-activated
Sepharose
according to the manufacturer's instructions. When the remaining antibodies
were
used in Western blots of beer protein, only 17 kDa Foam Protein was stained as
a
distinct double hand (See Figure 2B, lane A foam-LTP-1; lane B, beer; lane C,
17
kDa Foam Protein from beer).
The antibodies specific to 17 kDa Foam Protein were used as coating
reagent in ELISA assays, performed as described below for Example 10.
Biotinylated antibodies used in this assay were prepared using the complete
IgG
fraction of serum no. 1897.
A small affinity column was prepared by covalently coupling
antibodies specific to 17 kDa Foam Protein to CNBr-activated Sepharose
(Pharmacia, Uppsala, Sweden) according to the manufacturer's directions. This
column was used for selective removal of 17 kDa Foam Protein from solutions as
described below for Example 13.
Example 10
ELISA Assay for Quantification of 17 kDa Foam Protein
Prior to setting up an ELISA procedure for quantification of 17 kDa
Foam Protein, 10 mg antibodies, obtained as described above for Example 8,
were
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biotinylated as described in Bech, et al., 1995 WO 95/13359. An ELISA
procedure
was then established, based on a non-competitive double antibody sandwich-
technique. This assay format comprises five steps:
1 ) coating polystyrene wells with anti- 17 kDa Foam Protein antibodies
and blocking residual binding sites;
2) incubation of samples containing I 7 kDa Foam Protein in coated
wells;
3) incubation with biotinylated antibodies;
4) incubation with a conjugate of streptavidin and horseradish
peroxidase; and
5) incubation with a substrate for horseradish peroxidase.
The assay was performed in polystyrene wells arranged in strips of
twelve, and the strips were placed in frames each containing eight strips
(Nunc
Immuno Module C12 MaxisorpT'" from Life Technologies, Denmark).
In step 1, the specific 17 kDa Foam Protein antibodies were diluted to
2 ~g/ml in PBS I 0 (phosphate buffered saline: I 0 mM sodium phosphate pH 7.3,
150
mM NaCI). 200 ~1 aliquots were added to each well and incubated at 4°C
for 16-20
hours. After this, the wells were emptied and washed S times with PBST (PBS10
supplemented with 0.01 % Tween 20) (Merck, Darmstadt, Germany). Residual
binding sites on the polystyrene surface were then blocked by adding 200 ~l
BSA/PBST (PBSIO supplemented with bovine serum albumin (BSA), 1 g/1, and
0.05% Tween 20) to each well. The wells were incubated at 37°C for 1
hour,
emptied and washed five times as described above. The wells could then be
stored
at -20°C for up to three months before use.
In step 2, samples containing I7 kDa Foam Protein or standards of
purified 17 kDa Foam Protein were diluted appropriately in BSA/PBST, and 200
~.l
aliquots were incubated in the coated wells for I hour at ambient temperature
(20-
24°C). At least two separate dilutions were made of all samples, and
all dilutions
were assayed in at least three wells. After incubation, the plates were
emptied and
washed.
In step 3, biotinylated antibodies were diluted to 1 ~,g/ml in
BSA/PBST. 200 ~l aliquots were incubated in the wells for I O minutes at
ambient
temperature. After this, the wells were emptied and washed.
In step 4, a conjugate of streptavidin and horseradish peroxidase
(SIGMA) was diluted to 0.25 ~,g/ml in BSA/PBST. 200 ~,l aliquots were
incubated
in the wells for 10 minutes at ambient temperature. After this, the wells were
emptied and washed.
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34
In step 5, a substrate was prepared containing 3.3',5.5'-
tetramethylbenzidine (TMB), 100 ~tg/ml, and Hz02, 0.015%, in phosphate-citrate
buffer pH 5Ø 200 ~l aliquots were incubated in the wells for 5 minutes at
ambient
temperature. After this, the enzyme reaction was stopped by addition of 100
~,1 S N
HCI to each well, and the absorba.nce of the wells at 450 nm was measured in a
spectrophotometer matching the 96-well plates (Perkin-Elmer Lambda Reader).
Each series of analyses included a set of 17 kDa Foam Protein
standards prepared from "Pool 1 ", a fraction of beer foam rich in this
protein. One
preparation of Pool 1, obtained from a commercial lager beer (Carlsberg
Pilsner),
was used in the experiments described below. The total protein content in this
Pool
1 was determined by amino acid analysis, and standards were prepared in the
range
0-33 ng Pool 1 protein/ml. A standard curve was made by plotting the
absorbance
of a well versus the concentration of Pool 1 protein, and the content of 17
kDa Foam
Protein in a sample was then quantified in arbitrary units, AU, by comparison
with
this curve (reaction of 1 mg pool 1 protein/1= 1 AU).
Comparison of the ELISA reactivity of the "Pool 1" standard used in
these experiments with the reactivities of 17 kDa Foam Protein purified from
barley,
first wort or beer allowed conversion of the arbitrary units to precise
quantification
of 17 kDa Foam Protein in mg/I. Preparations of 17 kDa Foam Protein isolated
from
barley (with or without nicks in the peptide chain) or from first wort had
practically
the same reactivity. On a mg basis, these preparations reacted about 6.2 times
stronger than the preparation of Pool 1 used in these experiments, whereas 17
kDa
Foam Protein isolated from beer reacted about 4.2 times stronger than Pool 1
(See
Table 7). The approximate content of 17 kDa Foam Protein in barley extracts or
first wort was thus be obtained in mg/1 by dividing the content estimated in
AU with
6.2, whereas the content in beer was obtained by dividing with 4.2.
The final assay was highly specific to 17 kDa Foam Protein. Only
very slight reactions with other proteins purified from beer, barley or malt
could be
observed (See Table 7).
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35 -
Table 7
Protein Source Reactivity in ELISA
(AU)
17 kDa Foam Proteinsbarley 5.9-6.2
17 kDa Foam Proteinbbarley 6.2-6.4
17 kDa Foam Protein first wort 6.1-6.2
I7 kDa Foam Protein beer 4.0-4.3
"Pool 1 " beer foam = 1
LTP 1 beer foam 0.004-0.010
protein Z beer 0.001
LTP1 barley < 0.001
protein Z malt < 0.001
aintact peptide chain bpeptide-chain partly nicked
Example 11
ELISA Assa~rs for Quantification of Other Beer Proteins
ELISA procedures for detection and quantitation of the foam-type
and the barley-type LTP1 proteins have been described elsewhere (Bech, et al.,
1995
supra). These assays were used to quantify the two types of LTP I in the
experiments described below.
In order to quantify protein Z, this protein was purified from malt
essentially as described in the literature (Hejgaard, 1982 Physiol. Plant
54:174-182)
and used for immunization of rabbits using the methods described for Example
9. A
non-competitive ELISA procedure of the sandwich--type was then established for
protein Z, essentially as described for 17 kDa Foam Protein in Example I0. The
preparation of protein Z used for immunization was also used as standard in
the
ELISA assays.
All ELISA quantifications were based on comparison of the reaction
of samples with the reaction of purified standard proteins. One preparation of
each
standard protein was used for the experiments described below. However,
various
preparations of foam-type LTP 1 have previously been shown to vary somewhat in
ELISA reactivity (Beck, et al., 1995 supra). Further, protein Z was difficult
to
isolate from beer, and it was thus not possible directly to compare the
reactivity of
protein Z in beer with the reactivity of the preparation from malt used as
standard in
the assays.
Therefore, the ELISA determinations of both foam-type LTPI,
barley-type LTP 1 and protein Z were verified by means of three small affinity
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36 -
columns. These columns contained antibodies specific to either protein
covalently
coupled to CNBr-activated Sepharose according to the manufacturer's
instructions.
SDS-PAGE and Western blots were used to demonstrate that these columns could
quantitatively remove the respective beer proteins from small aliquots of
beer. After
elution of each column with acetic acid, the amount of eluted protein was
determined by amino acid analysis, and the concentration of the respective
proteins
in the original beer sample was calculated based on these determinations.
A set of 22 beers varying in original gravity from 7.3 to 15.9 and with
widely different levels of beer proteins were used for testing the
correspondence
between the two methods of quantification. Some beers were brewed with 30-40%
maize grits as adjunct, and some were all-malt beers. For all three proteins,
the
ELISA results agreed well with the quantifications based on affinity
chromatography. The slope of straight lines obtained as best fit when plotting
analytical values obtained by affinity chromatography vs. values obtained by
ELISA
were 0.78 for the barley-type LTP, 0.90 for foam-type LTP and 0.82 for protein
Z.
Thus, affinity chromatography gave slightly lower results than ELISA for all
three
proteins, probably due to a slight loss of material during the chromatographic
procedure.
Example 12
Transfer of 17 lcDa Foam Protein to Foam During Flotation
Foam was produced from 15 liters of lager beer by sparging with
nitrogen gas in a foam tower overnight at a rate of 450 ml/minute (Serensen et
al.,
1993 supra). The nitrogen gas was saturated with water vapor before
introduction
into the foam tower. After collapse, the foam collected at the outlet was
diluted to
the original volume with distilled water and reintroduced into the foam tower.
A
second and a third flotation was performed as described above. After each
flotation
step, aliquots of the foam, termed the flotate, and of the remaining unfoamed
liquid,
termed the remanent, were collected for analysis.
The content of 17 kDa Foam Protein in flotates and remanents and in
the original lager beer was determined by ELISA assays performed as outlined
in
Example 10. During the first flotation, at least 75% of the 17 kDa Foam
Protein
found in the beer was transferred to the foam, whereas only 25% or less
remained in
the unfoamed liquid. During repeated flotations, only very small amounts of 17
kDa
Foam Protein were found in the remanent, whereas the 17 kDa Foam Protein found
in foam from a flotation was transferred almost quantitatively to a subsequent
foam
fraction (see Figure 3).
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37
Flotation of 200 ml aliquots of various lager beers in a small scale
foam tower demonstrated that the percentage of 17 kDa Foam Protein left in the
remanent during the first flotation could be even less than about 25% for some
beers,
occasionally only about 10%.
Example 13
Selective Removal of 17 kDa Protein Severely Reduces Foam Potential
A small affinity column was prepared by covalent coupling of
antibodies specific to 17 kDa Foam Protein to CNBr-activated Sepharose
according
to the manufacturer's directions. A volume of 90 ml of a standard lager beer
from a
production plant (Carlsberg Pilsner) was repeatedly passed through the column,
and
the content of 17 kDa Foam Protein, foam-type LTP, barley-type LTP and protein
Z
was determined after each passage of the column by means of ELISA assays. The
foaming capacity of the sample was determined after each passage by use of
HeadHunter equipment.
It was demonstrated that during each passage, the level of 17 kDa
Foam Protein was reduced. In contrast, the content of protein Z and both types
of
LTP1 remained completely unaffected by this procedure. The foam stability of
the
beer was drastically reduced when only minor amounts of 17 kDa Foam Protein
were removed, and also the foam potential was somewhat diminished. (See Figure
4).
Example 14
Content of 17 kDa Foam Protein in Beer and Correlation to Foam Half-Life
The content of 17 kDa Foam Protein in 50 Danish lager beers was
determined by means of the ELISA procedure described in Example 10. The beers
were obtained from a variety of Danish breweries and included both all-malt
types
and beer brewed with maize grits as adjunct. They were collected during the
first
three months of 1997 and were at that time all within the day of latest
purchase.
The samples were further analyzed by ELISA procedures for the
foam-type of LTP 1, the barley-type of LTP 1 and for protein Z as described
above
for Example 10. The content of COZ was determined as described in the section
on
analytical procedures. The foam half-life (F) of the: bottled beer was
determined on
the Foam Stability Analyzer. The content of all tested beer proteins,
including 17
kDa Foam Protein, varied widely within the material. The average concentration
of
17 kDa Foam Protein and other beer proteins in 50 Danish lager beers is shown
below in Table 8.
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Table 8
Protein Average concentrationConcentration
range
17 kDa Foam Protein25 9-35
LTP1, foam- a 40 20-74
LTP1, bade - a 14 3-23
rotein Z 24 5-59
The content of COZ in beer is known to influence foam half-life
determinations made on the Foam Stability Analyzer. Therefore, the beers were
grouped according to their content of COZ before investigating if any
relationships
existed between foam half-life and content of beer proteins.
For beers with a low or medium content of C02 (4.8-5.0 g/1 or 5.1-
5.3 g/1, respectively), rather weak correlations could be found between foam
half-
life and 17 kDa Foam Protein and between foam half-life and LTP 1 of the foam-
type. However, better correlations were observed between foam half-life and
the
total amount of 17kDa Foam Protein and foam-type LTP 1 in the samples (Figures
7
A and 7B). Highly carbonated beers (5.4-5.6 g COz/liter) had generally a high
foam
half-life, and no significant correlation between half-life and any beer
proteins
could be demonstrated for this group of beers (Figure 7C). Protein Z and
barley-
type LTP1 did not correlate with foam half-life, neither alone nor in
combinations.
Example 15
Enhanced Accumulation of 17 lcDa Foam Protein
in the Developing or Germinating Barley Grain
The foaming properties of a beer may be enhanced by the use of
barley malt, genetically engineered to contain an elevated content of the 17
kDa
Foam Protein, as a raw material in the beer brewing process. The 17 kDa Foam
Protein is found in endosperm of mature grain and as a member of the hordein
storage protein family is synthesized during grain development. The
accumulation of
17 kDa Foam Protein in the developing endosperm is enhanced, for example, by
the
insertion of additional copies of the 17 kDa Foam Protein gene into the barley
genome, under the transcriptional control of its native promoter or,
alternatively,
under the control of any one of the various previously characterized endosperm
specific promoters, e.g. hordein gene promoters. In one example, the sequence
encoding the mature 17 kDa Foam Protein is cloned downstream of the promoter
of
the D hordein gene (Hor3, GenBank Accession number: X84368), as shown in
Figure 9.
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Since proteins homologous in sequence to 17 kDa Foam Protein are
found in wheat (Table 4) and in rye (see Example 19, below) (Rocher, et al.,
1996,
Biochem. Biophys. Acta. 1295: I3-22), and may be found in other cereals, it is
assumed that these homologs could equally be used to enhance the foaming
properties of beer. As shown for the wheat protein in Example 7, the 17 kDa
Foam
Protein cDNA clone or other probes may be used to screen out gene sequences
encoding 17 kDa Foam Protein homologs from cDNA or genomic libraries
constructed from cereals such as rye, wheat or rice. Alternatively, PCR
amplification techniques may be used to amplify homologous 17 kDa Foam Protein
gene sequences from genomic DNA prepared from these cereals. Similar
transformation expression cassettes, namely promoter, signal peptide-encoding
sequences and terminator, can be used to express transgenes encoding 17 kDa
Foam
Protein homologues in the developing barley grains or in other cellular hosts.
Example 16
I7 kDa Foam Protein Expression in Yeast
The foaming properties of beer may be enhanced by the addition of
17 kDa Foam Protein or its homologues to the wort. 17 kDa Foam Protein or a
homolog is produced on an industrial scale by expression of the 17 kDa Foam
Protein gene in either laboratory yeast (e.g. Saccharomyces cerevisiae), or
brewers
yeast (e.g. Saccharomyces carlsbergensis). For example, the 17 kDa Foam
Protein
coding sequence, fused to an appropriate sequence encoding a yeast signal
peptide,
is cloned into a self-replicated yeast expression plasmid under the
transcriptional
control of an inducible promoter and transformed and maintained in yeast under
selective pressure. The I7 kDa Foam Protein secreted into the yeast growth
medium
can subsequently be purified according to protocols similar to those described
in
Example 2. Alternatively, the 17 kDa Foam Protein polypeptide expression
cassette
(yeast promoter + yeast signal peptide coding sequence + 17 kDa Foam Protein
coding sequence) can be stably integrated into the yeast genome. 17 kDa Foam
Protein secreted into the yeast growth medium may similarly be purified as
described
in Example 2 and subsequently added to the wort. Stable integration of the 17
kDa
Foam Protein expression cassette into brewers yeast allows the secretion of
the 17
kDa Foam Protein directly into the wort during fermentation.
One example of a self-replicating yeast expression vector is shown in
Figure 5, which comprises the PRB 1 promoter (derived from the S. cerevisiae
PRB
1 gene encoding protease B), the PGK terminator (derived from the S.
cerevisiae
PGK gene encoding phosphoglycerate kinase) and the mature 17 kDa Foam Protein
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coding sequence inserted downstream of the sequence encoding the B. macerans
( 1-3, 1-4)-(3-glucanase signal peptide.
The 17 kDa Foam Protein yeast expression vector is derived from
pBl-L-MH(A16-M) (Meldgaard, et al., 1995, Glycoconjugate J. 12: 380-390)
S using the following cloning steps. The plasmid, pB 1-L-MH(A16-M), is first
linearized with BgIII and the site is blunt ended. The plasmid is then
digested with
BstEII to excise the (I-3, 1-4)-(3-glucanase coding sequence, which is then
replaced with the 17 kDa Foam Protein coding sequence amplified from the 17
kDa
Foam Protein cDNA using the following primers:
SEQ SEQUENCE TYPE
ID
NO:
''
22 f T ACA CTG GTG ACC ACA TTT GGG Sense Primer
TTT
TCY TTG ATT TTT TCT GTA AGC GCT
TTA
GCG CTC GAC ACC TGC TCC CAA 3'
23 5' CAG ATC TTA GCA GAC ACC ACC GGC Antisense
3' Primer
The PCR product is then digested with BstEII (BstEII site underlined) prior
to the replacement cloning step. The 17 kDa Foam Protein yeast expression
vector
is transformed into an appropriate Leu- yeast strain and cultivated in
fermentors in
1 S SC medium without Leu as described by Meldgaard, et al., 1995, supra.
Example 17
Integration of 17 kDa Foam Protein Gene into S. carlsbergensis Brewer's Yeast
To make a DNA construction suitable for integration of a hybrid 17
kDa Foam Protein gene into S carlsbergensis brewer's yeast, the yeast
expression
plasmid shown in Figure 5 may be used as a basis. The plasmid is restriction
digested with EcoRI, and the large fragment is purified. Into this fragment is
ligated
a 2.0 kb EcoRI DNA fragment from pCH216 (Hadfield, 1994 In: Molecular
Genetics of Yeast. A Practical Approach. Johnston, J.R. (ed.). Oxford
University
Press, Oxford, UK, pp. 17-48) containing the APTI gene, conferring resistance
to
the antibiotic 6418. The APTI gene is transcribed from a yeast PGKl promoter
which makes it usable as a dominant selectable marker in brewer's yeasts at a
concentration of 30 mg/ml on YPD plates (rich medium). The resulting plasmid
construct (see Figure 6) is devoid of yeast 2 ~ origin of replication
sequences,
making self-replication in yeast impossible. Thus, stable transformation of
brewer's
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yeast with this construct requires the integration of the whole plasmid into
the yeast
genome.
Integration of the plasmid is obtained through employment of the first
part ('loop-in') of the method for replacement of chromosome segments in yeast
described by Scherer and Davis, 1979, PNAS USA 76:4951-495. To obtain
integration at a certain location in the genome, the plasmid needs to be
linearized in
one of the yeast DNA sequences that it harbors (Ort~-Weaver, et al., 1981 PNAS
USA 78:6354-6358). Linearization is, therefore, performed through restriction
digestion at the unique NgoAIV site (with any of the isoschizomers NgoAIV,
NaeI,
NgoMI or MroNI) in the PRBI gene, and the resulting linear DNA used to
transform
S. carlsbergensis brewer's yeast to 6418-resistance.
6418-resistant yeast clones are then checked for integration of the
plasmid containing the 17 kDa Foam Protein expression cassette at the proper
location in the wildtype PRBI gene. The functionality of the latter gene will
not be
changed, as none of the gene is missing after proper integration.
Example 18
17 kDa protein in different bariey cultivars
The content of 17 kDa protein in 25 samples of malting barley was
determined. Two samples were both of the variety Optima, which differed in
total
protein content, whereas the rest of the samples represented different
varieties. Most
varieties were grown in Denmark during 1997, but a few were obtained from
China
or Thailand.
Of each variety, about 6 g of kernels were ground to a fine flour in a
laboratory mill, and 5 g of flour were extracted with 20 ml water for 1 hour
at 0°C.
17 kDa Foam Protein was then analyzed in the crude supernatants by the ELISA
procedure described in Example 10.
The content of 17 kDa Foam Protein varied widely within this set of
samples. About 8 times more 17 kDa Foam Protein was extracted from the
cultivar
Maresi than from Polygena and Optima under the specified extraction conditions
(Table 9).
The results for the two samples of Optima indicate that the content of
17 kDa Foam Protein may be linked to total protein content within a variety.
One of
these samples had about 50% more total protein than the other and also about
50%
more 17 kDa Foam Protein. However, among the cultivars of this investigation,
there is no correlation between 17 kDa Foam Protein and total protein. The
ratio of
I7 kDa Foam Protein in the samples varies from approximately 0.5-2 mg 17 kDa
protein/g total protein (Table 9).
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Table 9
Content of 17 lcDa Foam Protein and total protein content in 26 barley samples
Variety, Country 17 kDa Foam total protein,17 kDa 'Foam
Protein, mg/kgg/kg barleyProtein /total
barle rotein; m
Amadea Denmark 132 lI8 1.12
An ora Denmark 124 __132 0.94
Bartok Denmark 308 83 _ 3.71
Brenda Denmark 204 _ 84 2.43
Carlsber Denmark 92 104 0.88
II
Chariot Denmark 212 116 1.83
Hanka Denmark 188 121 1.55
Madonna Denmark 116 96 1.21
Maresi Denmark 400 86 4.65
Marina Denmark 68 99 0.69
O tuna Denmark 48 91 0.53
Papas Denmark 196 105 1.85
Pol ena Denmark 52 89 0.58
Proctor Denmark 276 106 2.60
Re ae Denmark 262 86 3.05
Sultane Denmark 64 95 0.67
Thurin Denmark 168 99 1.70
a
O tuna Denmark 76 132 0.58
Mentor China 164 145 1.13
Gan i no.lChina 124 133 0.93
BRB 2 Thailand 88 169 0.52
BRB 9 Thailand 276 142 1.94
Morex Thailand 168 154 1.09
Maud Denmark 192 96 2.00
Alexis Denmark 172 91 1.89
Caminant Denmark 412 103 4.00
Example 19
Reaction of barley, wheat and rye extracts with antibodies to 17 IcDa protein
Samples of barley (cv. Maud), wheat, and rye (cv. not known) were
extracted as described in Example 18. The crude supernatants were analyzed by
SDS-PAGE (reducing conditions) followed by Western blotting with antibodies to
17 kDa Foam Protein as described in Analytical Procedures.
On Western blotting, the barley extract gave a double band with a
molecular mass of approximately 17-18 kDa (Figure 8, lane C). The identity of
the
two bands from the barley extract was investigated by blotting from SDS-PAGE
onto a PVDF membrane, staining shortly with Comassie blue stain and cutting
out
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each of the two bands separately. The N-terminal sequence of the first 4-8
amino
acids of the proteins corresponding to the two bands was then determined as
described in Analytical Procedures. Both bands gave a sequence identical to
the N-
terminal sequence of 17 kDa Foam Protein (Table 1 ) and therefore probably
represent isoforms of 17 kDa Foam Protein.
The rye extract (Figure 8, lane A) gave three major bands at
approximately 15, 17 and 20 kDa, whereas the wheat extract (lane B) gave two
bands at approximately 17 and 20 kDa. Apart from these bands, faint bands were
observed at higher molecular mass from the rye and wheat extracts. Thus, the
antibodies to 17 kDa Foam Protein from barley recognize components in wheat
and
rye with approximately the same molecular mass. The amino acid sequence and/or
the tertiary structure of these components are expected to be very similar to
the 17
kDa Foam Protein from barley.
Example 20
Transformation of Barley with 17 kDa Foam Protein
A transformation cassette useful for introducing enhanced amounts of 17
lcDa Foam Protein into barley is shown in Figure 9. The cassette, which
includes the
hor 3-1 promoter; the 17 kDa Foam Protein coding sequence; and the NOS
terminator, is transformed into barley. Transformed lines, identified by PCR
screening for the presence of transgene, are grown to maturity. Transformed
lines
expressing enhanced levels of 17 kDa Foam Protein in the grain are identified
by
using the ELIZA assay described in Example 10, and the selected lines are bred
to
homozygosity for the transgene. Homozygous GMO barley lines expressing
enhanced levels of 17 kDa Foam Protein are then crossed with GMO barley
expressing enhanced levels of LTP1. Sandager (1996, supra) describes GMO
barley
lines transformed with a chimeric LTP1 gene cassette (Chi26 promoter-Ltpl or
Hor3-1 promoter-Ltp 1 ) which accumulate enhanced levels of LTP 1 in the
mature
grain. Hybrid GMO barley lines carrying both LTP1 and 171cDa Foam Protein
transgenes are then used as a preferred barley for the production of beer and
other
foaming products.
Example 21
Isolated Rye Nucleic Acid Sequences Encoding 17 kDa Foam Proteins
The nucleic acid sequences of three rye genes encoding homologues
of the barley and wheat 17 kDa Foam Protein have been determined from cDNA and
genomic clones, generated by RT-PCR and PCR amplification respectively.
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The cDNA was selectively amplified from a total RNA population
isolated from developing rye (Secale cereale) endosperm tissue harvested 25
days
after anthesis, as described by Rechinger, et al. supra. The total RNA was
reverse
transcribed with two antisense primers, whose sequence was based on conserved
nucleotide sequences located at the 3' end of the coding sequences of the
barley and
wheat 17 kDa Foam Protein genes:
SEQ ID SEQUENCE TYPE
NO:
24 5' G T A G C T G C C A T G G G G Antisense
A T 3' Primer
25 5' C A A T A T T G C G G G A T G Antisense
T T C 3' Primer
The reverse transcription reactions with 1 mg RNA were performed
at 55°C using the TitanTM One Tube RT-PCR System supplied by Boehringer
Mannheim GmbH. A single sense primer, with a sequence based on a conserved
nucleotide sequence at the 5' end of the barley and wheat 17 kDa Foam Protein
coding sequences, was included in both RT-PCR reactions.
'SEQ ID SEQUENCE TYPE
NO:
26 5' G G A C A C T A C C T G T A G Sense Primer
C C A 3'
The PCR amplification step was 30 thermocyles (94°C for 30
seconds / 60°C for 30 seconds / 68°C for 30 seconds) followed by
7 minutes at 68°C
and the amplification products were analysed by agarose gel electrophoresis.
cDNA
fragments of approximately 440 and 380 nucleotides were isolated, cloned into
a
pCR 2.1-TOPO plasmid vector (Invitrogen) and the nucleotide sequence of the
inserts determined with an AmpliCycleTM Sequencing Kit (Perkin Elmer) on an
Applied Biosystems 373 DNA sequencer. The nucleotide sequence of the 369 and
435 nucleotide inserts revealed two distinct cDNA sequences [SEQ ID N0:27) and
[SEQ ID N0:28J, which share close homology to the barley and wheat 17 kDa Foam
Protein coding sequences.
Rye genomic sequences encoding homologues of the barley 17 kDa
Foam Protein were determined from genomic DNA fragments amplified by PCR.
Rye grain were germinated and grown in the dark for 6 days and genomic DNA was
prepared from the etiolated leaves using a Plant DNA Isolation Kit from
Boehringer
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Mannheim GmbH. Rye genomic DNA (0.1 mg) was amplified with 25 pmol each of
the sense primer [SEQ ID N0:26J and the antisense primer [SEQ ID N0:24J, using
native Pfu DNA polymerase (Stratagene) in a reaction mixture recommended by
the
manufacturer. The PCR amplification was performed using a 'touch-down'
thermocycling program (95°C for 0.45 minutes, 68 °C [-I
°C / cycle (54 °C) ] for 0.45
minutes and 72 °C for 2 minutes for 15 cycles; 95 °C for 0.45
minutes, 54 °C for
0.45 minutes and 72 °C for 2 minutes for 25 cycles; 72 °C for 6
minutes). The
amplification products were analysed by agarose gel electrophoresis and DNA
fragments of approximately 440 nucleotides were isolated, cloned into a pCR-
BIuntII TOPO vector (Invitrogen) and sequenced as described for the cDNA
fragments. The nucleotide sequences of the cloned fragments revealed three
distinct
gene sequences present in the rye genome, which all showed homology to the
barley
17 kDa Foam Protein gene. Two of the rye gene sequences [SEQ ID N0:29] and
[SEQ ID N0:3IJ showed identity to the cDNA clones, [SEQ ID N0:27] and [SEQ
ID N0:28J respectively, while a third had a distinct sequence [SEQ ID N0:33].
Alignment of the nucleotide sequences of the rye genes with the
barley 17 kDa Foam Protein gene indicates that the amplified rye sequences,
including the primers, encode homologues of the I 7 kDa Foam Protein starting
at
the second residue of the mature barley I7 kDa Foam Protein and extending to 7
residues short of the C-terminus. The amino acid sequences of the deduced rye
17
kDa Foam Protein homologues are aligned with the barley and wheat I7 kDa Foam
Protein sequences in Table 10, where the rye gene shown in SEQ ID N0:29
encodes
the rye polypeptide shown in SEQ ID N0:30, the rye gene shown in SEQ ID N0:31
encodes the rye polypeptide shown in SEQ ID N0:32, and the rye gene shown in
SEQ ID N0:33 encodes the rye polypeptide shown in SEQ ID N0:34. Amino acid
identity is represented by ellipses (....), amino acid deletions are
represented by a
dashed line ( ), the alignment end is represented by an asterisk (*). The N-
terminal amino acid sequence of the deduced rye 17 kDa Foam Proteins shown in
SEQ ID NOS:32 and 34 are almost identical to that of two unidentified rye
polypeptides of 15 and I 8 kDa, previously detected in a rye grain extract
(Rocker et
al., 1996, BBA 1295: I3-22). The rye 17 kDa Foam Protein shown in SEQ ID
N0:34 is I I amino acid residues smaller than the protein shown in SEQ ID
N0:32
due to a 33 nucleotide deletion in its gene. The conserved cysteine residues
found in
the barley and wheat 17 kDa Foam Protein homologues are also conserved in all
the
rye homologues. The close homology between the rye and barley I7 kDa Foam
Protein homologues is consistent with the recognition of rye polypeptides of
17 by
the anti-barley 17 kDa Foam Protein antibodies described in Example 19.
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Table 10
Alignment of Barley, Rye and Wheat 17 kDa Foam Proteins
$ 10 20 30 40 50
SEQID No:lO LDTTCSQGYG QCQQQPQQQM NTCAAFLQQC SRTPYVQSQM WQASGCQLMR
SEQID No:30 ......... ..... - .. .......... .P........ ..........
SEQID No:32 ......... ...L.-.... .......... .......... ..........
SEQID No:34 ......... ...L.-.... .......... .......... ..........
SEQID No:21 ......H... .........V .....L.... .P........ .......V..
60 70 80 90 100
SEQ1D No:lO QQCCQPLAQI SEQARCQAVC SMAQVIMRQQ QGQSFTQPQQ QQSQSFGQPQ
SEQID No:30 .......... ........I. .V......R. ...IYG.... ..G.......
SEQID No:32 .......... .......... .V........ .....G.... ..G.......
SEQID No:34 .......... .......... .V........ .....G.... . -_______
SEQID No:21 .......... .......... .V.H...... .....5.... ..V......H
110 120 130 140 150
2O SEQID No:lO QQVPVEVMRM VLQTLPSMCS VNIPQYCTTT PCSTITPTIYS IPMAATCAGG
SEQID No:30 ......I... .......... .......... ..R...Q.P.I F....*
SEQID No:32 ....I.IT.. .......... .........I .......AV.. .....*
SEQID No:34 - ..I.IT.. .......... .........I .......AV.. .....*
SEQID No:21 ......IT.. .......V.. .......A.. .....FQ.P.N ........*
152
SEQ ID No : 10 VC
Table 11
Nucleotide Sequence of Rye PCR Product (369 bp) [SEQ ID N0:27]
10 20 30 90 50 60
3S GGACACTACCTGTAGCCAGGGCTACGGGCAATGCCAGCAGCAGCAGATGAACACATGCGC
70 80 90 100 110 120
CGCTTTCCTGCAACAGTGCAGCCCTACACCATATGTCCAGTCACAGATGTGGCAAGCAAG
130 190 150 160 170 180
CGGTTGCCAGTTGATGCGGCAACAGTGCTGCCAGCCGCTGGCCCAGATCTCGGAGCAGGC
190 200 210 220 230 240
TCGGTGCCAGGCCATCTGTAGCGTGGCACAAGTCATCATGCGGCGGCAGCAAGGGCAAAT
250 260 270 280 290 300
TTATGGCCAGCCTCAGCAGCAGCAAGGGCAAAGTTTTGGACAGCCTCAGCAACAGGTTCC
310 320 330 340 350 360
4S GGTTGAGATAATGAGGATGGTGCTTCAGACCCTTCCGTCGATGTGCAGCGTGAACATCCC
370 380 390 900 410 420
GCAATATTG...................................................
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Table 12
Nucleotide Sequence of Rye PCR Product (435 bp) [SEQ ID N0:28]
S 10 20 30 40 50 60
GGACACTACCTGTAGCCAGGGCTACGGGCAATGCCAACTGCAGCAGCAGCAGATGAACAC
70 80 90 100 110 120
ATGCGCTGCTTTCCTGCAGCAGTGCAGCCGGACACCATATGTCCAGTCACAGATGTGGCA
130 140 150 160 170 180
IO GGCAAGCGGTTGCCAGTTGATGCGGCAACAGTGCTGCCAGCCGCTGGCCCAGATCTCGGA
190 200 210 220 230 240
GCAGGCTCGGTGCCAGGCCGTCTGTAGCGTGGCACAGGTCATCATGCGGCAGCAGCAAGG
250 260 270 280 290 300
GCAAAGTTTTGGCCAGCCTCAGCAGCAGCAAGGGCAAAGTTTCGGCCAGCCTCAGCAGCA
IS 310 320 330 390 350 360
GGTTCCGATTGAGATAACGAGGATGGTGCTTCAGACCCTTCCGTCGATGTGCAGCGTGAA
370 380 390 400 410 420
CATCCCGCAATATTGCACTACCATCCCATGCAGCACCATCACCCCTGCCGTCTACAGCAT
430 440 450 460 470 480
2O CCCCATGGCAGCTAC.............................................
Table 13
2S Rye Genomic PCR Product (429 bp) (SEQ ID N0:29]
10 20 30 40 50 60
GGACACTACCTGTAGCCAGGGCTACGGGCAATGCCAGCAGCAGCAGATGAACACATGCGC
70 80 90 100 110 120
3O CGCTTTCCTGCAACAGTGCAGCCCTACACCATATGTCCAGTCACAGATGTGGCAAGCAAG
130 140 150 160 170 180
CGGTTGCCAGTTGATGCGGCAACAGTGCTGCCAGCCGCTGGCCCAGATCTCGGAGCAGGC
190 200 210 220 230 240
TCGGTGCCAGGCCATCTGTAGCGTGGCACAAGTCATCATGCGGCGGCAGCAAGGGCAAAT
3S 250 260 270 280 290 300
TTATGGCCAGCCTCAGCAGCAGCAAGGGCAAAGTTTTGGACAGCCTCAGCAACAGGTTCC
310 320 330 340 350 360
GGTTGAGATAATGAGGATGGTGCTTCAGACCCTTCCGTCGATGTGCAGCGTGAACATCCC
370 380 390 400 410 420
4O GCAATATTGCACCACCACCCCATGCAGAACCATCACTCAGACCCCCTACATCTTCCCCAT
430 490 450 460 970 480
GGCAGCTAC...................................................
4S Table 14
Rye Deduced Amino Acid Sequence (SEQ ID N0:30]
20 30 40 50 60
SO DTTCSQGYGQ CQQQQMNTCA AFLQQCSPTP YVQSQMWQAS GCQLMRQQCC QPLAQISEQA
70 80 90 100 110 120
RCQAICSVAQ VIMRRQQGQI YGQPQQQQGQ SFGQPQQQVP VEIMRMVLQT LPSMCSVNIP
130 190 150 160 170 180
QYCTTTPCRT ITQTPYIFPM AA........ ..........
SS
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Table 15
Rye Genomic PCR Product (435 bp) [SEQ ID N0:31]
10 20 30 40 50 60
GGACACTACCTGTAGCCAGGGCTACGGGCAATGCCAACTGCAGCAGCAGCAGATGAACAC
70 80 90 100 110 120
ATGCGCTGCTTTCCTGCAGCAGTGCAGCCGGACACCATATGTCCAGTCACAGATGTGGCA
130 190 150 160 170 180
lO GGCAAGCGGTTGCCAGTTGATGCGGCAACAGTGCTGCCAGCCGCTGGCCCAGATCTCGGA
190 200 210 220 230 240
GCAGGCTCGGTGCCAGGCCGTCTGTAGCGTGGCACAGGTCATCATGCGGCAGCAGCAAGG
250 260 270 280 290 300
GCAAAGTTTTGGCCAGCCTCAGCAGCAGCAAGGGCAAAGTTTCGGCCAGCCTCAGCAGCA
310 320 330 340 350 360
GGTTCCGATTGAGATAACGAGGATGGTGCTTCAGACCCTTCCGTCGATGTGCAGCGTGAA
370 380 390 400 410 420
CATCCCGCAATATTGCACTACCATCCCATGCAGCACCATCACCCCTGCCGTCTACAGCAT
430 490 450 460 470 480
2O CCCCATGGCAGCTAC.............................................
Table 16
Rye Deduced Amino Acid Sequence [SEQ ID N0:32]
10 20 30 40 SO 60
DTTCSQGYGQ CQLQQQQMNT CAAFLQQCSR TPYVQSQMWQ ASGCQLMRQQ CCQPLAQISE
70 80 90 100 110 120
3O QARCQAVCSV AQVIMRQQQG QSFGQPQQQQ GQSFGQPQQQ VPIEITRMVL QTLPSMCSVN
130 190 150 160 170 180
IPQYCTTIPC STITPAVYSI PMAA...... .......... .......... ..........
Table 17
Rye Genomic PCR Product (402 bp) [SEQ ID N0:33]
10 20 30 90 50 60
GGACACTACC TGTAGCCAGG GCTACGGGCA ATGCCAACTG CAGCAGCAGC AGATGAACAC
4O 70 80 90 100 110 120
ATGCGCTGCTTTCCTGCAGCAGTGCAGCCGGACACCATATGTCCAGTCACAGATGTGGCA
130 190 150 160 170 180
GGCAAGCGGTTGCCAGTTGATGCGGCAACAGTGCTGCCAGCCGCTGGCCCAGATCTCGGA
190 200 210 220 230 240
4S GCAGGCTCGGTGCCAGGCCGTCTGTAGCGTGGCACAGGTCATCATGCGGCAGCAGCAAGG
250 260 270 280 290 300
GCAAAGTTTCGGCCAGCCTCAGCAGCAGGTTCCGATTGAGATAACAAGGATGGTGCTTCA
310 320 330 340 350 360
GACCCTTCCGTCGATGTGCAGCGTGAACATCCCGCAATATTGCACTACCATCCCATGCAG
SO 370 380 390 900 410 420
CACCATCACCCCTGCCGTCTACAGCATCCCCATGGCAGCTAC..................
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
49 -
Table 18
Rye Deduced Amino Acid Sequence [SEQ ID N0:34]
$ 10 20 30 40 50 60
DTTCSQGYGQ CQLQQQQMNT CAAFLQQCSR TPYVQSQMWQ ASGCQLMRQQ CCQPLAQISE
70 80 90 100 110 120
QARCQAVCSV AQVIMRQQQG QSFGQPQQQV PIEITRMVL(~ TLPSMCSVNI PQYCTTIPCS
130 190 150 160 170 180
lO TITPAVYSIP MAA....... .......... .......... .......... ..........
The present invention should not be considered limited to the
particular examples described above, but rather should be understood to cover
all
1 S aspects of the invention as fairly set out in the attached claims. Various
modifications, equivalent processes, as well as numerous structures to which
the
present invention may be applicable will be readily apparent to those of skill
in the
art to which the present invention is directed upon review of the instant
specification.
20 The specification includes reference to many patents and literature
citations, each of which is hereby incorporated by reference for all purposes,
as if
fully set forth.
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
SEQUENCE LISTING
<110> Carlsberg Research Laboratory
<120> 17 kDa FOAM PROTEIN
<130> 11225.9W001
<190> New Filing
<141> 1999-09-03
<150> 60/115,756
<151> 1999-O1-13
<150> 09/146,703
<151> 1998-09-03
<160> 34
<170> PatentIn Ver. 2.0
<210> 1
<211> 20
<212> PRT
<213> Hordeum vulgare
<400> 1
Leu Asp Thr Thr Cys Ser Gln Gly Tyr Gly Gin Cys Gln Gln Gln Pro
1 5 10 15
Gln Gln Gln Met
20
<210> 2
<211> 20
<212> PRT
<213> Hordeum vulgare
<400> 2
Asn Thr Cys Ala Ala Phe Leu Gln Gln Cys Ser Gln Thr Pro Tyr Val
1 5 10 15
Gln Ser Gln Met
20
<210> 3
<211> 15
<212> PRT
<213> Hordeum vulgare
<900> 3
Arg G1n Gln Cys Cys Gln Pro Leu Ala Gln Ile Ser Glu Gin Ala
1 5 10 15
<210> 4
<211> 14
<212> PRT
<213> Hordeum vulgare
<400> 9
Arg G'_n Gln Gln Gly Gln Ser Phe Ser Gln Pro Gln G1n G~n
1 5 10
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
<210>
<211>
<212> ?RT
<213> -ordeum vulgare
<400>
Val L~~ G1n Thr Leu Pro Ser Met Cys Ser Val Asn Ile Pro Gln Tyr
1 5 10 15
Cys Tar Thr Thr Pro Cys Thr Thr Ile Thr Pro
20 25
<210> 6
<211> 25
<212> DNA
<213> Unknown
<220>
<223> Description of Unknown Organism: None - poly DT
primer with S' Bam ti I restriction size
<400> 6
atgga=cctt tttttttttt ttttt 25
<210> 7
<211> 6
<212> PRT
<213> Hordeum vulgare
<400> 7
Pro G~.~n G1n Gln Met Asn
1 5
<210> 8
<211> 19
<212> DNA
<213> Yordeum vulgare
<400> 8
atggatccca cacaatgaa 19
<210> 9
<211> 611
<212> DNA
<213> Hordeum lgare
vu
<220>
<221> CDS
<222> (1)..(959)
<400> 9
ctc gac acc tgctcccaaggc tacggccaa tgccaacaa caaccg
acc 48
Leu Asp Thr CysSerGlnGly TyrGlyGln CysGlnGln GlnPro
Thr
1 ~ 10 15
caa caa atg aacacctgeget getttect:gcagcagtge agceag
caa 96
Gln Gln Met AsnThrCysAla AlaPheLeu GlnGlnCys SerGln
Gln
20 25 30
aca cca gtc cagtcacagatg tggcaggca agcggttgc cagttg
tac ~4~
Thr Pro Val GlnSerGlnMet TrpGlnAla SerGlyCys GlnLeu
Tyr
2
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
35 90 45
atg egg caa caa tgc tgc cag eea etg gcc cag atc teg gag cag get 192
Met Arg Gln Gln Cys Cys Gln Pro Leu Ala Gln Ile Ser Glu Gln Ala
50 55 60
cgg tgc cag gcc gtc tgt agc gtg gca cag gtc atc atg cgg caa cag 240
Arg Cys Gln Ala Val Cys Ser Val Ala Gln Val Ile Met Arg Gln Gln
65 70 75 80
caa ggg cag agt ttc agt cag cct cag cag cag caa tcg caa agt ttc 288
Gln Gly Gln Ser Phe Ser Gln Pro Gln Gln Glr_ G1n Ser Gln Ser Phe
85 90 95
ggc cag cct cag cag cag gtt ccg gtt gag ata ata agg atg gtg ctt 336
Gly Gln Pro Gln Gln Gln Val Pro Val Glu Ile Ile Arg Met Val Leu
100 105 I10
cag acc ctt ccg tcg atg tgc agc gtg aac att ccg caa tat tgc acc 389
Gln Thr Leu Pro Ser Met Cys Ser Val Asn Ile Pro Gln Tyr Cys Thr
115 120 125
acc acc ccg tgc acc acc atc acc ccc acc atc tac agc atc ccc atg 932
Thr Thr Pro Cys Thr Thr Ile Thr Pro Thr Ile Tyr Ser Ile Pro Met
130 135 140
gca get ace tgt gcc ggt ggt gtc tge taagatctgt gatgtgctag 479
Ala Ala Thr Cys Ala Gly Gly Val Cys
145 150
ctagatcgat caccgtttag ttgatcgatg aaagctacaa aataaaagtg ccatacgtca 539
tcattgtgtg ccggtactat tgcaacttgg aaataata~a cctctgtttc tgaataaaaa 599
aaaaaaaaaa as 611
<210> 10
<211> 153
<212> PRT
<213> Hordeum vulgare
<400> 10
Leu Asp Thr Thr Cys Ser Gln Gly Tyr Gly G1n Cys Gln Gln Gln Pro
1 5 10 15
Gln Gln Gln Met Asn Thr Cys Ala Ala Phe Leu Gln Gln Cys Ser Gln
20 25 30
Thr Pro Tyr Val Gln Ser Gln Met Trp Gln A-_a Ser Gly Cys Gln Leu
35 40 45
Met Arg Gln Gln Cys Cys Gln Pro Leu Ala Gln Ile Ser Glu Gln Ala
50 55 60
Arg Cys Gln Ala Val Cys Ser Val Ala Gln Val Ile Met Arg Gln Gln
65 70 75 80
Gln Gly Gln Ser Phe Ser G1n Pro Gln Gln G:Ln Gln Ser Gln Ser Phe
85 90 95
Gly Gln Pro Gln Gln Gln Val Pro Val Glu Ile Ile Arg Met Val Leu
100 105 110
Gln Thr Leu Pro Ser Met Cys Ser Val Asn Ile Pro Gln Tyr Cys Thr
115 120 125
3
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
Thr Thr Pro Cys T~:r Thr Ile Thr Pro Thr Ile Tyr Ser ile Pro Met
130 135 190
Ala Aia Thr Cys Aia Gly Gly Val Cys
145 150
<210> 11
<211> 1336
<212> DNA
<213> Hordeum vulgare
<220>
<221> CDS
<222> (675)..(1193)
<220>
<221> sig_peptide
<222> (675)..(731)
<400> 11
agcatagctc catgacaatc ttttacaggt aaaggaaaat ttatgagtca tcaatgctct 60
actgatgccg tttgtattac caaagtagta caaggaaaac aaaatccaag ataacaaaac 120
cagttttcag gaaacaatga gatgggagtg cggggcatgc caatctgatt tatatctaac 180
aactcgtaca agataacaaa atgaatttca caaaaaga~~t caatccggat atacgcttga 240
catgtaaagt gatcagtgat gagtcatatg gattatcgtg gtcaggcgcg agctgattta 300
tatctaacaa ctcgtacaag ataacaaaat gaatttcaca aaaagactca atccagatat 360
acggttgaca tgtaaagtgc tcagtgatga gtcat~a~gga ,tcatcgaggt cagacgcgag 420
ctaactgaca tctacacgat atgtgttgaa aagr~-vu'_tt gacgaccatc caagattgga 480
cttgtagccc aacctaacac aagtgtgtca gatc;at~:agt tggaaaagca cacaaaacct 540
ttagcatagg aacctacaga tgcaatgcac caaatgat:gc catggtagct ataaataggc 600
ccgcaccatg aagatcctcc ctcatcatcc ttcacacaaa cacaagcatc aaagcaaact 660
tgtagccagc cacc atg aag acc atg ctg atc ctc gcg ctc atc gcc ttc 710
Met Lys Thr Met Leu Ile heu P.la Leu Iie Ala Phe
1 5 10
gcg gcg acc agc gcc gtt gca cag ctg gac act acc tgt agc cag ggc 758
Ala Aia Thr Ser Ala Val Ala Gln Leu Asp 1'hr Thr Cys Ser Gln Gly
15 20 25
tac ggg eag tgc cag eaa cag ceg cag cag cag atg aac aca tgt get 806
Tyr Gly Gln Cys Gln Gln Gln Pro G1n Gln Gln Met Asn Thr Cys Ala
30 35 40
gcc ttc ctg cag cag tgc agc cgg aca cca tac gtc cag tca cag atg 854
Ala Phe Leu Gln Gln Cys Ser Arg Thr Pro Tyr Va1 Gln Ser Gln Met
45 50 '_>5 60
tgg cag gca agc ggt tgc cag ttg atg cgg caa caa tgc tgc cag ccg 902
Trp Gln Ala Ser Gly Cys Gln Leu Met Arg Gln G1n Cys Cys Gln Pro
65 70 75
ctg gcc cag atc tcg gag cag get cgg tgc c:ag gcc gtc tgt agc atg 95C
Leu A1a Gln Ile Ser Glu Gln Ala Arg Cys G'n Ala Val Cys Ser Met
80 8 90
4
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/D1597
gca cag gtc atc atg cgg caa cag caa ggg cag agt ttc act cag cct 998
Ala Gln Val Ile Met Arg Gln Gln G1n Gly Gln Ser Phe Thr Gln Pro
95 100 105
cag cag cag caa tcg caa agt ttc ggc cag cct cag cag cag gtt ccg
1046
Gln Gln Gln Gln Ser Gln Ser Phe Gly Gln Pro Gln Gln Gln Val Pro
110 115 120
gtt gag gta atg agg atg gtg ctt cag acc ctt ccg tcg atg tgc agc
1094
Val Glu Val Met Arg Met Val Leu Gln Thr Leu Pro Ser Met Cys Ser
125 130 135 140
gtg aac atc ccg caa tat tgc acc acc acc ccg tgc agc acc atc acc
1192
Val Asn Ile Pro Gln Tyr Cys Thr Thr Thr Pro Cys Ser Thr Ile Thr
145 150 155
ccc acc atc tac agc atc ccc atg gca get acc tgt gcc ggt ggt gtc
1190
Pro Thr I1e Tyr Ser Ile Pro Met Ala Ala Thr Cys Ala Gly Gly Val
160 165 170
tgc taagatctgt gatgtgctag ctagatcgat caccgtttag ttgatcgatg
1243
Cys
aagagctaca aaataaaagt gccatacgtc atcatgtgtg gccggtacta ttgcaacttg
1303
gaaataataa acctctgttt ctgaataaag ctt
1336
<210> 12
<211> 173
<212> PRT
<213> Hordeum vulgare
<400> 12
Met Lys Thr Met Leu Ile Leu Ala Leu Ile Ala Phe A1a Ala Thr Ser
1 5 10 15
Ala Val Ala Gln Leu Asp Thr Thr Cys Ser Gln Gly Tyr Gly Gln Cys
20 25 30
Gln Gln Gln Pro Gln Gln Gln Met Asn Thr Cys A1a A1a Phe Leu Gln
35 40 95
G1n Cys Ser Arg Thr Pro Tyr Val Gln Ser Gln Met Trp Gln Ala Ser
50 55 60
Gly Cys Gln Leu Met Arg Gln Gln Cys Cys G:Ln Pro Leu Ala Gln Ile
65 70 75 80
Ser G1u Gln Ala Arg Cys Gln Ala Val Cys Ser Met A1a Gln Val Ile
85 9C 95
Met Arg Gln Gln Gln Gly Gln Ser Phe Thr G1n Pro Gln G1n Gln G1n
100 105 110
Ser Gln Ser Phe Gly Gin Pro Gln Gln G1n Val Pro Va1 Glu Va1 Met
115 120 125
5
CA 02341760 2001-03-O1
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Arg Me. Val Leu Gln Thr Leu Pro Ser Met Cys Ser Val As.-: Ile Pro
13J 135 140
Gln Tar Cys Thr Thr Thr Pro Cys Ser Thr Ile Thr Pro Thr Ile Tyr
145 150 155 160
Ser I_2 Pro Met Ala Ala Thr Cys Ala Gly Gly Val Cys
165 170
<210> 13
<211> 252
<212> PRT
<223> Horde~.:m vulgare
<400> 13
Gln Gl.n Gln Pro Phe Pro G1n Gln Pro Ile Pro Gln Gln Pro Gln Pro
1 5 10 15
Tyr Pro Gln Gln Pro Gln Pro Tyr Pro Gln Gln Pro Phe Pro Pro Gln
20 25 3C
Gln Ala Phe Pro Gln Gln Pro Pro Phe Trp Pro Gln Gln Pro Phe Pro
35 40 45
Gln G'_:~ Pro Pro Phe Gly Leu Gln Gln Pro Ile Leu Ser Gi:~ Gln Gln
50 55 60
Pro Cys Thr Pro Gln Gln Thr Pro Leu Pro Gln Gly Gln Leu Tyr Gln
65 70 75 80
Thr Leu Leu Gln Leu Gln Ile Pro Tyr Val Gln Pro Ser Ile Leu Gln
85 90 95
Gln Leu Asn Pro Cys Lys Val Phe Leu Gln Gln Gln Cys Ser Pro Val
100 105 110
Arg Met Pro Gln Leu Ile Ala Arg Ser Gln Met Leu Gln Gln Ser Ser
115 120 125
Cys His Val Leu Gln Gln G1n Cys Cys Gln Gln Leu Pro Gln Ile Pro
130 135 190
Glu Gln Phe Arg His Glu A1a Ile Arg Ala Ile Va1 Tyr Ser Ile Phe
145 150 155 160
Leu Gln Glu Gln Pro Gln Gln Ser Val Gln Gly Ala Ser Gln Pro Gln
165 170 175
Gln Gln Leu Gln Glu Glu Gln Val Gly Gln Cy:> Tyr Phe Gln Gln Pro
180 185 190
Gln Pro Gln G1n Leu Gly G1n Pro Gln Gln Va'~. Pro Gln Ser Val Phe
195 200 205
Leu Gln Pro His Gln Ile Ala Gln Leu Glu Ala Thr Asn Se. I1e Ala
210 215 220
Leu Arg Thr Leu Pro Thr Met Cys Asn Val Asn Val Pro Leu Tyr Asp
225 230 235 240
Ile Met Pro Phe Gly Val Gly Thr Arg Val Gly Val
245 250
<210> 14
6
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/~1597
<211> 271
<212> PRT
<213> Hordeum vulgare
<400> 14
Gln Gln Gln Pro Phe Pro G1n Gln Pro Phe Pro Gln Gln Pro Gln Pro
1 5 10 15
Tyr Pro Gln Gln Pro Gln Pro Tyr Pro Gin Gln Pro Phe Gln Pro Gln
20 25 30
G1n Pro Phe Pro Gln Gln Thr Ile Pro Gln Gln Pro Gln Pro Tyr Pro
35 40 45
Gln Gln Pro Phe Pro Pro Gln Gln Glu Phe Pro Gin Gln Pro Pro Phe
50 55 60
Trp Pro Gln Gln Pro Phe Pro Gln Gln Pro Pro Phe Gly Leu Gln Gln
65 70 75 80
Pro Ile Leu Ser Gln G1n Gln Pro Cys Thr Pro Gin Gln Thr Pro Leu
85 90 95
Pro Gln Gly Gln Leu Tyr Gln Thr Leu Leu G.ln Leu G1n Ile Pro Tyr
100 105 I10
Val His Pro Ser Ile Leu Gln Gln Leu Asn Pro Cys Lys Val Phe Leu
115 120 125
Gln Gln Gln Cys Ser Pro Val Arg Met Pro G1n Leu Ile Ala Arg Leu
130 135 140
Gln Met Leu Gln Gln Ser Ser Cys His Val Leu G1n Gln Gln Cys Cys
I45 150 155 160
Gln Gln Leu Pro Gln Ile Ser Glu Gln Phe Arg His Glu Ala Ile Arg
165 170 175
Ala Ile Val Tyr Ser Ile Phe Leu Gln Glu Gln Pro Gln Gln Ser Val
180 185 190
Gln Gly Val Ser Gln Thr Gln Gln Gln Leu Gln G1n G1u Gln Val Gly
195 200 205
Gln Cys Ser Phe Gln Gln Pro Gln Pro Gln Gl.n Leu Gly Gln Ala Gln
210 215 220
Gln Val Pro Gln Ser Val Phe Leu Gln Pro His Gln Ile Ala Gln Leu
225 230 235 240
Glu Ala Thr Thr Ser Ile Ala Leu Arg Thr Leu Pro Arg Met Cys Asn
245 250 255
Val Asn Val Pro Leu Tyr Asp Ile Met Pro Pro Asp Phe Trp His
260 265 270
<210> 15
<211> 273
<212> PRT
<213> Hordeum vulgare
<900> 15
Gln Gln Gln Pro Phe Pro Gin Gln Pro Ile P:ro Gln Gln Pro Gln Pro
1 5 10 15
7
CA 02341760 2001-03-O1
WO PCT/IB99/Ot597
00/14237
TyrPro GlnGlnPro G1nProTyr ProGlnGln ProPhePro ProGln
20 25 30
GlnPro PheProGln GlnProVal ProGlnGln ProGlnPro TyrPro
35 40 45
GlnPro PheProPro GlnGlnPro PheProGln GlnProPro PheTrn
_
50 55 60
GlnGln LysProPhe ProGlnGln ProProPhe GlyLeuGln GlnPro
65 70 75 80
IleLeu SerGlnGln GlnProCys ThrProGln GlnThrPro LeuPro
85 90 95
GlnGly GlnLeuTyr GlnThrLeu LeuGlnLeu G'_.~.IleGln TyrVal
100 105 110
HisPro SerIleLeu GlnGlnLeu AsnProCys LysValPhe LeuGln
115 120 125
GlnGln CysSerPro ValProVal ProGlnArg IleAlaArg SerGln
130 135 14~
MetLeu GlnGlnSer SerCysHis ValLeuG.LnGlr~GlnCys CysG1n
145 150 155 160
GlnLeu ProGlnIle ProGluGln PheArgHis G'_uAlaIle ArgAla
165 17G 175
IleVal TyrSerIle PheLeuGln GluGlnPro GinGlnLeu ValGlu
180 185 190
GlyVal SerGlnPro GlnGlnGln LeuTr,Pro GlnGinVal GlyGln
195 200 205
CysSer PheGlnGln ProGlnPro GlnGinVii GiyGlnGln GlnGln
210 215 2?~
ValPro GlnSerAla PheLeuGln ProHisGln IieAlaGln LeuGlu
225 230 235 290
AlaThr ThrSerIle AlaLeuArg ThrLeuPro MetMetCys SerVal
245 250 255
AsnVal ProLeuTyr ArgIleLeu ArgGlyVal GlyProSer ValGly
260 265 270
Val
<210> 16
<211> 286
<212> PRT
<213> Hordeum vulgare
<400> 16
Glu Met Gln Val Asn Pro Ser Val Gln Val Gln Pro Thr Gln Gln Gln
1 5 10 1.5
Pro Tyr Pro Glu Ser Gln Gln Pro Phe Iie Ser C:ln Ser Gln Gln Gln
20 25 30
Phe Pro G1n Pro Gln G1n Pro Phe Pro Gln Gln Pro Gln Gln Pro Phe
35 ~ 90 45
g
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/Q1597
Pro Gln Ser G1n Gln Gln Cys Leu Gln Gln Pro Gln His Gln Phe Pro
50 55 60
Gln Pro Thr Gln Gln Phe Pro Gln Arg Pro Leu Leu Pro Phe Thr His
65 70 75 80
Pro Phe Leu Thr Phe Pro Asp Gln Leu Leu Prc Gln Pro Pro His Gln
85 90 95
Ser Phe Pro Gln Pro Pro Gln Ser Tyr Pro Gln Pro Pro Leu Gln Pro
100 I05 110
Phe Pro Gln Pro Pro Gln Gln Lys Tyr Pro Glu Gln Pro Gln Gln Pro
115 120 125
Phe Pro Trp Gln Gln Pro Thr Ile Gln Leu Tyr Leu Gln Gln Gln Leu
130 135 140
Asn Pro Cys Lys Glu Phe Leu Leu Gln Gln Cys Arg Pro Val Ser Leu
145 150 155 160
Leu Ser Tyr Ile Trp Ser Lys Ile Val Gln Gln Ser Ser Cys Arg Val
165 I70 175
Met Gln Gln Gln Cys Cys Leu Gln Leu Ala Gln I1e Pro Glu Gln Tyr
180 185 190
Lys Cys Thr Ala Ile Asp Ser Ile Val His Ala Ile Phe Met Gln Gln
195 200 205
Gly Gln Arg Gln Gly Val Gln Ile Val Gln Gln Gln Pro Gln Pro Gln
21U 215 220
Gln Val Gly Gln Cys Val Leu Val Gln Gly Gln Gly Val Val Gln Pro
225 230 23'_i 240
Gln Gln Leu Ala Gln Met Glu Ala Ile Arg Thr Leu Val Leu Gln Ser
295 250 255
Val Pro Ser Met Cys Asn Phe Asn Val Pro Pro Asn Cys Ser Thr Ile
260 265 270
Lys Ala Pro Phe Val Gly Val Val Thr Gly Val Gly Gly Gln
275 280 285
<210> 17
<211> 288
<212> PRT
<213> Hordeum vulgare
<900> 17
Ile Thr Thr Thr Thr Met Gln Phe Asn Pro Se:r Gly Leu Glu Leu Glu
1 5 10 15
Arg Pro Gln Gln Leu Phe Pro Gln Trp Gln Pro Leu Pro Gln Gln Pro
20 25 30
Pro Phe Leu Gln G1n GIu Pro Glu Gln Pro Tyr Pro Gln Gln Gln Pro
35 40 45
Leu Pro G1n Gln G1n Pro Phe Pro Gln Gln Pro Gln Leu Pro His Gln
50 55 60
His Gln Phe Pro Gln G1n Leu Pro Gln Gln Gin Phe Pro Gln G1n Met
9
CA 02341760 2001-03-O1
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65 70 75 80
Pro Leu Gln Pro Gln Gln Gln Phe Pro Gln Gln Met Pro Leu Gln Pro
85 90 95
Gln Gln Gln Pro Gln Phe Pro Gln Gin Lys Pro Phe Gly Gln Tyr Gln
100 105 110
Pro Leu Thr Gln Gln Pro Tyr Pro Gln Gln Gln Pro Leu Ala Gln Gln
115 120 125
Gln Pro Ser Ile Glu Glu Gln His Gln Leu Asn Leu Cys Lys Glu Phe
130 135 140
Leu Leu Gln Gln Cys Thr Leu Asp Glu Lys Val Pro Leu Leu Gln Ser
145 150 155 160
Val I1e Ser Phe Leu Arg Pro His Ile Ser Gln Gln Asn Ser Cys Gln
165 170 175
Leu Lys Arg Gln Gln Cys Cys Gln Gln Leu Ala Asn Ile Asn Glu Gln
180 185 190
Ser Arg Cys Pro Ala Ile G1n Thr Ile Val His Ala Ile Val Met Gln
195 200 205
Gln Gln Val Gln Gln Gln Val Gly His Gly Phe Val Gln Ser Gln Leu
210 215 220
Gln Gln Leu Gly Gln Gly Met Pro Ile Gln Leu Gln Gln Gln Pro Gly
225 230 235 240
Gln Ala Phe Val Leu Pro Gln Gln Gln Ala Gln Phe Lys Val Val Gly
245 250 255
Ser Leu Val I1e Gln Thr Leu Pro Met Leu Cys Asn Val His Val Pro
260 265 270
Pro Tyr Cys Ser Pro Phe Gly Ser Met Ala Thr Gly Ser Gly Gly Gln
275 280 285
<210> 18
<211> 20
<212> DNA
<213> Hordeum vulgare
<400> 18
gtngcncann tngayacnac 20
<210> 19
<211> 17
<212> DNA
<213> Hordeum vulgare
<400> 19
gcncangtng cngccat 17
<210> 20
<211> 455
<212> DNA
1~
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
<213> Triticum aestivum
<400> 20
gtggcgcagc tggatactac atgtagccat ggctatgggc aatgccagca gcagccgcaa 60
cagcaggtga acacatgcgc tgctctcctg cagcagtgca gcccgacacc atatgtccag 120
tcacagatgt ggcaggcaag cggttgccag gtgatgcggc aacagtgctg ccagccgctg 180
gcccagatct cggagcaggc tcggtgccaa gctgtctgta gcgtggccca tgtcatcatg 240
cgacagcagc aagggcaaag tttcagtcag cctcagcaac aacaagtgca aagtttcggt 300
cagccacatc agcaggttcc ggttgagata acgaggatgg tgcttcagac ccttccatcg 360
gtctgcagcg tgaacatccc gcaatattgc gccaccaccc catgcagcac catctttcag 420
accccctaca acatccctat ggccgccacc tgcgc 455
<210>
21
<211>
152
<212>
PRT
<213>
Triticum
aestivum
<400>
21
Val Ala Leu AspThrThr CysSerHisGly TyrGlyGln CysGln
Gln
1 5 10 15
Gln Gln Gln GlnGlnVal AsnThrCysAla AlaLeuLeu GlnGln
Pro
20 25 30
Cys Ser Thr ProTyrVal GlnSerGlnMet TrpGlnAla SerGly
Pro
35 90 45
Cys Gln Met ArgGlnGln CysCysGlnPro LeuAlaGln IleSer
Val
50 55 60
Glu Gln Arg CysGlnAla ValCysSerVa.lAlaHisVal IleMet
Ala
65 70 75 80
Arg Gln Gln GlyGlnSer PheSerGlnPro GlnGlnGln GlnVal
Gln
85 90 95
Gln Ser Gly GlnProHis GlnGlnValPro ValGluIle ThrArg
Phe
100 105 110
Met Val Gln ThrLeuPro SerValCysSer ValAsnIle ProGln
Leu
115 120 125
Tyr Cys Thr ThrProCys SerThrIlePhe GlnThrPro TyrAsn
Ala
130 135 140
Ile Pro Ala AlaThrCys Ala
Met
145 150
<210>
22
<211>
73
<212>
DNA
<213> ulgare
Hordeum
v
<400> 22
tacactggtg accacatttg ggttttcytt gattttttct gtaagcgctt tagcgctcga 60
cacctgctcc caa 73
11
CA 02341760 2001-03-O1
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<210> 23
<211> 24
<212> DNA
<213> Hordeum vulgare
<900> 23
cagatcttag cagacaccac cggc 24
<210> 24
<211> 17
<212> DNA
<213> Synthetic
<400> 24
gtagctgcca tggggat 17
<210> 25
<211> 18
<212> DNA
<213> Synthetic
<400> 25
caatattgcg ggatgttc
18
<210> 26
<211> 18
<212> DNA
<213> Synthetic
<400> 26
ggacactacc tgtagcca 18
<210> 27
<211> 369
<212> DNA
<213> Secale cereale
<400> 27
ggacactacc tgtagccagg gctacgggca atgccagcag cagcagatga acacatgcgc 60
cgctttcctg caacagtgca gccctacacc atatgtccag tcacagatgt ggcaagcaag 120
cggttgccag ttgatgcggc aacagtgctg ccagccgctg gcccagatct cggagcaggc 180
tcggtgccag gccatctgta gcgtggcaca agtcatcatg cggcggcagc aagggcaaat 240
ttatggccag cctcagcagc agcaagggca aagttttgga cagcctcagc aacaggttcc 300
ggttgagata atgaggatgg tgcttcagac ccttccgtcg atgtgcagcg tgaacatccc 360
gcaatattg 369
<210> 28
<211> 935
<212> DNA
<213> Secale cereale
<900> 28
ggacactacc tgtagccagg gctacgggca atgccaactg cagcagcagc agatgaacac 60
12
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
atgcgctgct ttcctgcagc agtgcagccg gacaccatat gtccagtcac agatgtggca 120
ggcaagcggt tgccagttga tgcggcaaca gtgctgccag ccgctggccc agatctcgga 180
gcaggctcgg tgccaggccg tctgtagcgt ggcacaggtc atcatgcggc agcagcaagg 240
gcaaagtttt ggccagcctc agcagcagca agggcaaagt ttcggccagc ctcagcagca 300
ggttccgatt gagataacga ggatggtgct tcagaccctt ccgtcgatgt gcagcgtgaa 360
catcccgcaa tattgcacta ccatcccatg cagcaccatc acccctgccg tctacagcat 420
ccccatggca gctac 435
<220> 29
<211> 429
<212> DNA
<213> Secalecereale
<220>
<221> CDS
<222> (2)..(927)
<400> 29
g gac act c t 49
ac tg agc
cag
ggc
tac
ggg
caa
t:gc
cag
cag
cag
cag
atg
Asp Thr r s y n ln
Th Cy Ser Tyr Gln Met
Gln Gly Gln
Gl Gln G
C;ys
Gl
1 5 10 15
aac aca gccgetttcctg caacagtgcagc cctacacca tatgtc 97
tgc
Asn Thr AlaAlaPheLeu GlnGlnCysSer ProThrPro TyrVal
Cys
20 25 30
cag tca atgtggcaagca agcggttgccag ttgatgcgg caacag 145
cag
Gln Ser MetTrpGlnAla SerGlyCysGln LeuMetArg GlnGln
Gln
35 40 95
tgc tgc ccgctggcccag atctcggagcag getcggtgc caggcc 193
cag
Cys Cys ProLeuAlaG1n IleSerGluGln AlaArgCys GlnAla
Gln
50 55 60
atc tgt gtggcacaagtc atcatgcggcgg cagcaaggg caaatt 241
agc
I1e Cys ValAlaGlnVal IleMetArgArg G1nGlnGly GlnIle
Ser
65 70 75 80
tat ggc cctcagcagcag caagggcaaagt tttggacag cctcag 289
cag
Tyr Gly ProGlnGlnGln GlnGlyGlnSer PheG1yGln ProGln
Gln
85 90 95
caa cag ccggttgagata atgaggatggtg cttcagacc cttccg 337
gtt
Gln Gln ProValGluIle MetArgMetVal LeuGlnThr LeuPro
Val
100 105 110
tcg atg agcgtgaacatc ccgcaatattgc accaccacc ccatgc 385
tgc
Ser Met SerValAsnIle ProGlnTyrCys ThrThrThr ProCys
Cys
115 120 125
aga acc actcagaccccc tacatcttcccc atggcaget ac 929
atc
Arg Thr ThrGlnThrPro TyrIlePhePro MetAlaAla
Ile
130 135 190
<210> 30
<211> i42
<212> PRT
13
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
<213> Secale cereale
<900> 30
Asp Thr Thr Cys Ser Gln Gly Tyr Gly Gln Cys Gln Gln Gln Gln Me~
1 5 10 15
Asn Thr Cys Ala Ala Phe Leu Gln Gln Cys Ser Pro Thr Pro Tyr Va_
20 25 30
G1n Ser Gln Met Trp Gln Ala Ser Gly Cys Gln Leu Met Arg Gln G1.-:
35 40 45
Cys Cys Gln Pro Leu Ala Gln Ile Ser Glu Gln Ala Arg Cys Gln Ala
50 55 60
I1e Cys Ser Val Ala Gln Val Ile Met Arg Arg G1n Gln Gly Gln Ile
65 70 75 8C
Tyr Gly Gln Pro Gln Gln Gln Gln Gly Gln Ser Phe Gly Gln Pro Gln
85 90 95
Gln Gln Val Pro Val Glu Ile Met Arg Met Val Leu Gln Thr Leu Pro
100 105 110
Ser Met Cys Ser Val Asn Ile Pro Gln Tyr Cys Thr Thr Thr Pro Cys
115 120 125
Arg Thr Ile Thr Gln Thr Pro Tyr Ile Phe Pro Met Ala Ala
130 135 140
<210> 31
<211> 935
<212> DNA
<213> Secale cereale
<220>
<221> S
CD
<222> )..(933)
(2
<400>
31
g gac t t 49
ac acc agc
tg cag
ggc
tac
ggg
caa
tgc
caa
ctg
cag
cag
cag
Asp r r y n ln
Th Th Cys G1 C:ys Gln
Ser Gln
Gln Leu
Gly Gln
Tyr G
Gl
1 5 1 0 15
cag aacaca tgcgetgetttc ctgcagcag tgcagccgg acacca 97
atg
Gln AsnThr CysAlaAlaPhe LeuGlnGln CysSerArg ThrPro
Met
20 25 30
tat cagtca cagatgtggcag gcaagcggt tgccagttg atgcgg 195
gtc
Tyr GlnSer GlnMetTrpGln AlaSerGly CysGlnLeu MetArg
Val
35 40 45
caa tgctgc cagccgctggcc cagatctcg gagcagget cggtgc 193
cag
Gln CysCys GlnProLeuAla GlnIleSer GluGlnAla ArgCys
Gln
50 55 60
cag gtctgt agcgtggcacag gtcatcatg cggcagcag caaggg 291
gcc
Gln ValCys SerValAlaGln ValIleMet ArgGlnGln GlnGly
Ala
65 70 75 80
caa tttggc cagcctcagcag cagcaaggg caaagtttc ggccag 289
agt
Gln PheGly GlnProG1nGln GlnGlnGly GlnSerPhe GlyGln
Ser
85 90 95
cct cag cag cag gtt ccg att gag ata acg agg atg gtg ctt cag acc 337
14
CA 02341760 2001-03-O1
W O PCT/IB99/1597
00/14237
ProGln GinGln ValProIleGlu I1eThr Arq_MetValLev GlnThr
100 105 110
cttccg tagatg tgcagcgtgaac atcccg caatattgcact accatc 385
LeuPro SerMet CysSerValAsn IlePro GlnTyrCysThr ThrIle
115 120 125
ccatgc agcacc atcacccctgcc gtctac agcatccccatg gcaget 433
ProCys SerThr IleThrProAla ValTyr SerIleProMet AlaAla
130 135 190
ac 435
<210> 32
<211> 144
<212> PRT
<213> Secale cereale
<400> 32
Asp Thr Thr Cys Ser Gln Gly Tyr Gly Gln Cy:~ Gln Leu Gln Gln Gln
1 5 10 15
Gln Met Asn Thr Cys Ala Ala Phe Leu Gln Gln Cys Ser Arg Thr Pro
20 25 3C
Tyr Val Gln Ser Gln Met Trp Gln Ala Ser Gly Cys Gln Leu Met Arg
35 40 45
Gln Gln Cys Cys Gln Pro Leu Ala Gln Ile Ser Glu Gln Ala Arg Cys
50 55 60
Gln Ala Val Cys Ser Val Ala Gln Val Ile Met Arg Gln Gln Gln Gly
65 70 75 80
Gln Ser Phe Gly Gln Pro Gln Gln Gln Gln Gly Gln Ser Phe Gly Gln
85 90 95
Pro Gln Gln Gln Val Pro Ile G1u Ile Thr Arg Met Val Leu Gln Thr
100 105 ~ 110
Leu Pro Ser Met Cys Ser Val Asn Ile Pro Gln Tyr Cys Thr Thr Ile
115 120 125
Pro Cys Ser Thr Ile Thr Pro Ala Val Tyr Ser Ile Pro Met Ala Ala
130 135 140
<210> 33
<211> 402
<212> DNA
<213> Secale cereale
<220>
<221> CDS
<222> (2)..(400)
<400> 33
g gac act acc tgt agc cag ggc tac ggg caa tgc caa ctg cag cag cag 49
Asp Thr Thr Cys Ser Gln Gly Tyr Gly Gln Cys Gln Leu Gln Gln G1n
1 5 10 15
cag atg aac aca tgc get get ttc ctg cag cag tgc agc cgg aca cca 97
Gln Met Asn Thr Cys Ala Ala Phe Leu Gln Gln Cys Ser Arg Thr Pro
20 25 30
15
CA 02341760 2001-03-O1
WO 00/14237 PCT/IB99/01597
tat gtc cag tca cag atg tgg cag gca agc gc3t tgc cag ctg atg cgg 145
Tyr Val Gln Ser Gln Met Trp Gln Ala Ser Gly Cys G1n Leu Met Arg
35 40 45
eaaeagtgetgeeag eegetg geecagatetcg gagcagget cggtge 193
GlnGlnCysCysGln ProLeu AlaGlnIleSer GiuGlnAla ArgCys
50 55 60
caggccgtctgtagc gtggca caggtcatcatg cggcagcag caaggg 241
GlnA1aValCysSer ValAla GlnValIleMet ArgGlnGln GlnGly
65 70 75 80
caaagtttcggccag cctcag cagcaggttccg attgagata acaagg 289
GlnSerPheGlyGln ProGln GlnGlnValPro I1eGluIle ThrArg
85 90 95
atggtgcttcagacc cttccg tcgatgtgcagc gtgaacatc ccgcaa 337
MetValLeuGlnThr LeuPro SerMetCysSer ValAsnIle ProGln
100 105 110
tattgcactaccatc ccatgc agcaccatcacc cctgccgtc tacagc 385
TyrCysThrThrIle ProCys SerThrIleThr ProAlaVal TyrSer
115 120 125
atccceatggeaget ac 902
IleProMetAlaAla
130
<210>
34
<211> 3
13
<212> T
PR
<213> calecer eale
Se
<400>
34
Asp ThrCys SerGlnGly TyrGlyGln CysG1nLeu GlnGlnGln
Thr
1 5 lU 15
Gln AsnThr CysAlaAla PheLeuGln GinCysSer ArgThrPro
Met
20 25 30
Tyr GlnSer GlnMetTrp GlnAlaSer GiyCysGln LeuMetArg
Val
35 40 45
Gln CysCys GlnProLeu AlaGlnIle SerGluGln AlaArgCys
Gln
50 55 60
Gln ValCys SerValAla GlnValI1e MetArgGln GlnGlnGly
Ala
65 70 75 80
Gln PheGly GlnProGln GlnGlnVal FroIleGlu IleThrArg
Ser
85 90 95
Met LeuGln ThrLeuPro SerMetCys SerValAsn I1eProGln
Val
100 105 110
Tyr ThrThr IleProCys SerThrile ThrProAla ValTyrSer
Cys
115 120 125
Ile Pro Met A1a Ala
130
16
