Note: Descriptions are shown in the official language in which they were submitted.
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PROTEINS AND PEPTIDES
This invention relates to antimicrobial proteins and peptides, processes for
their
manufacture and use, and DNA sequences encoding them.
In this context, antimicrobial proteins and peptides are defined as proteins
and
peptides possessing antifungal and/or antibacterial activity and/or antiviral
activity.
Activity includes a range of antagonistic effects such as partial inhibition
or death.
A wide range of antimicrobial proteins with activity against plant pathogenic
fungi
have been isolated from certain plant species. We have previously described a
class of
antifungal proteins capable of isolation from radish and other plant species.
These
proteins are described in the following publications which are specifically
incorporated
herein by reference: International Patent Application Publication Number
W093/05153
published 18 March 1993; Terras FRG et al, 1992, J Biol Chem, 267:15301-15309;
Terras et al, 1993, FEBS Lett, 316:233-240; Terras et al, 1995, Plant Cell,
7:573-588.
The class includes Rs-AFP 1 (antifungal protein 1 ), Rs-AFP2, Rs-AFP3 and Rs-
AFP4
from Raphanus sativus and homologous proteins such as Bn-AFP 1 and Bn-AFP2
from
Brassica napus, Br-AFP1 and Br-AFP2 from Brassica rang, Sa-AFP1 and Sa-AFP2
from
Sinanis alba, At-AFP1 from Arabidopsis thaliana, Dm-AMP1 and Dm-AMP2 from
Dahlia merckii, Cb-AMP l and Cb-AMP2 from Cnicus benedictus, Lc-AFP from
Lathyrus cicera, Ct-AMP1 and Ct-AMP2 from Clitoria ternatea. The proteins
specifically
inhibit a range of fungi and may be used as fungicides for agricultural or
pharmaceutical
or preservative purposes.
It has been proposed that this class of antimicrobial proteins should be named
plant defensins (Terras F.R.G. et al 1995, Plant Cell, 7 573-583) and these
proteins have
in common a similar motif of conserved cysteines and glycines (Broekaert W.F.
et al
1995, Plant Physiol. 108 1353-1358).
As used herein the term "plant defensin" is used to denote those proteins
having
antimicrobial activity, especially antifungal activity or antifungal and
antibacterial activity
and also having the following characteristic structural features: a cysteine
residue at
positions 4, 15, 21, 25, 36, 45, 47 and 51; disulphide bridge formation
between the
cysteines at positions 4 and 51, 15 and 36, 21 and 45 and 25 and 47; an
aromatic amino
acid residue 4 amino acids upstream from the cysteine at position 15, a
glycine residue 2
amino acids upstream from the cysteine at position 15, a glutamic acid residue
7 amino
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acids upstream from the cysteine at position 36, and a glycine residue 2 amino
acids
upstream of the cysteine at position 36 wherein the positions of the cysteine
residues are
defined relative to the amino acid sequence of Rs-AFP1 as shown in SEQ ID NO
1, and
homologues, active variants and derivatives thereof.
QKLCERPSGTWSGVCGNNNACKNQCINLEKARHGSCNYVFPAHKCICYFPC
(SEQ ID NO 1 )
In some plant defensins the segments between positions l and 3, 5 and 14, 26
and 35 and
37 and 44 may vary in length by one to three amino acids but this does not
affect the
overall characteristic cysteine motif described above. This characteristic
structural feature
of the plant defensins can be summarised as follows:
...C......a.G.C.....C...C...E....G.C........C.C...C (SEQ ID NO 2)
where a is an aromatic amino acid (F,W,Y), C represents cysteine, E represents
glutamic
acid
and G is glycine and unspecified amino acids or groups of amino acids are
represented by
stops.
The expression "homologues" as used herein refers to any peptide which has
some
amino acids in common with the given sequence. Suitably at least 60% of the
amino
acids will be similar, more suitably at least 70%, preferably at least 80%,
more preferably
at least 90% and most preferably at least 95%. 96%, 97% or 98% of amino acids
will be
similar to the corresponding amino acid in the given sequence.
2o As used herein the term "similar" is used to denote sequences which when
aligned
have similar (identical or conservatively replaced) amino acids in like
positions or
regions, where identical or conservatively replaced amino acids are those
which do not
alter the activity or function of the protein as compared to the starting
protein. For
example, two amino acid sequences with at least 85% similarity to each other
have at
least 85% similar (identical or conservatively replaced) amino acid residues
in a like
position when aligned optimally allowing for up to 3 gaps, with the proviso
that in respect
of the gaps a total of not more than 15 amino acid residues is affected. The
degree of
similarity may be determined using methods well known in the art (see, for
example,
Wilbur, W.J. and Lipman, D.J. "Rapid Similarity Searches of Nucleic Acid and
Protein
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Data Banks." Proceedings of the National Academy of Sciences USA 80, 726-730
(1983)
and Myers E.and Miller W. "Optimal Alignments in Linear Space". Comput. Appl.
Biosci. 4:11-17(1988)). One programme which may be used in determining the
degree of
similarity is the MegAlign Lipman-Pearson one pair method (using default
parameters)
which can be obtained from DNAstar Inc, 1228, Selfpark Street, Madison,
Wisconsin,
53715, USA as part of the Lasergene system.
Amino acids which differ from the basic sequence may be conservatively or non-
conservatively substituted. A conservative substitution is to be understood to
mean that
the amino acid is replaced with an amino acid with broadly similar chemical
properties.
In particular conservative substitutions may be made between amino acids with
the
following groups:
(a) Alanine, Serine, Glycine and Threonine;
(b) Glutamic acid and Aspartic acid;
(c) Arginine and Lysine;
(d) Asparagine and Glutamine;
(e) Isoleucine, Leucine, Valine and Methionine;
(f) Phenylalanine, Tyrosine and Tryptophan.
In general, more conservative than non-conservative substitutions will be
possible
without destroying the antimicrobial properties of the compounds. Suitable
homologues
may be determined by testing antimicrobial properties of the peptide using
routine
methods, for example as illustrated hereinafter.
The term "variant" as used herein includes experimentally generated variants
or
members of a family of related naturally-occurring peptides as may be
identified by
molecular genetic techniques. Such techniques are described for example in US
Patent
No. 5,605,793, US Patent No. 5,811,238 and US Patent No 5,830,721, the content
of
which is incorporated herein by reference. In essence this technique involves
expression
of the parental gene in a microbial expression system such as Escherichia
coli. The
particular system selected must be validated and calibrated to ensure that
biologically
active peptides are expressed, which may be readily achieved using an in vivo
bioassay.
The gene, or preferably a collection of related genes from different species,
may
be subject to mutagenic polymerase chain reaction (PCR) as is known in the
art.
Fragmentation of the products and subsequent repair using PCR leads to a
series of
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chimeric genes reconstructed from parental variants. These chimeras are then
expressed
in the microbial system which can be screened in the usual way to determine
active
mutants, which may then be isolated and sequenced. Reiteration of this
molecular
evolution DNA shuffling cycle may lead to progressive enhancement of the
desired gene
properties. The advantage of a technique of this nature is that it allows a
wide range of
different mutations, including multi-mutation block exchanges, to be produced
and
screened.
Other variants may be identified or defined using bioinformatics systems. An
example of such a system is the FASTA method of W.R. Pearson and D.J. Lipman
PNAS
(1988) 85:2444-2488. This method provides a rapid and easy method for
comparing
protein sequences and detecting levels of similarity and is a standard tool,
used by
molecular biologists. Such similar sequences may be obtained from natural
sources,
through molecular evolution or by synthetic methods and comparisons made using
this
method to arrive at "opt scores" which are indicative of the level of
similarity between the
proteins.
Particular variants of the invention will comprise antimicrobial proteins with
an
amino acid sequence with a FASTA opt score (as defined in accordance with
FASTA
version 3.0t82 November 1, 1997) against any one of the sequences of the
antimicrobial
proteins of the invention described herein as follows. Variants of the
invention will
2o comprise antimicrobial proteins with an amino acid sequence with a FASTA
opt score (as
defined in accordance with FASTA version 3.0t82 November 1, 1997) of greater
than or
equal to 300 against Rs-AFP1 or 2.
The term "derivative" relates to antimicrobial proteins which have been
modified
for example by using known chemical or biological methods. The expression
"protein or
peptide derived from a plant defensin" used herein includes derivatives. A
particular
derivative is one in which cysteine residues are replaced by a-aminobutyric
acid.
Further examples of antifungal plant defensins are described in International
Patent
Application Publication Number W095/18229 published 6 July 1995 which is
specifically incorporated herein by reference. These examples include Hs-AFP1,
an
3o antifungal protein capable of isolation from seeds of Heuchera species and
Ah-AMP1, an
antimicrobial protein capable of isolation from seeds of Aesculus
hippocastanum. The
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proteins specifically inhibit a range of fungi and may be used as fungicides
for
agricultural or pharmaceutical or preservative purposes.
The primary structures of the two antifungal protein isoforms capable of
isolation
from radish seeds, Rs-AFP 1 (SEQ ID NO 1 ) and Rs-AFP2 (SEQ ID NO 3), only
differ at
two positions: the glutamic acid residue (E) at position 5 in Rs-AFP1 is a
glutamine
residue (Q) in Rs-AFP2, and the asparagine residue (N) at position 27 in Rs-
AFP1 is
substituted by an arginine residue (R) in Rs-AFP2. As a result, Rs-AFP2 has a
higher net
positive charge (+2) at physiological pH. Although both Rs-AFPs are 94%
identical at
the amino acid sequence level, Rs-AFP2 is two- to thirty-fold more active than
Rs-AFP 1
on various fungi and shows an increased salt-tolerence.
The proteins Rs-AFP3 and Rs-AFP4 are found in radish leaves following
localized
fungal infection. The induced leaf proteins are homologous to Rs-AFP 1 and Rs-
AFP2
and exert similar antifungal activity in vitro.
It has previously been shown (W097/21814 and W097/21815) that peptides
derived from the regions defined herein of the Rs-AFP plant defensins exhibit
antimicrobial activity. W097/21814 discloses that some specific mutations to
the wild-
type sequence may be made in the peptides whilst retaining activity which may
in fact be
enhanced. Such peptides may be easier to synthesise than the full length plant
defensin
while retaining antifungal or antifungal and antibacterial activity. DNA
sequences
encoding the peptides may also be more suitable for transformation into
biological hosts.
In a first aspect the invention provides an antimicrobial protein or peptide
derived
from a plant defensin characterised in that said protein or peptide comprises
one or more
of the following replacement amino acid residues selected from the group
consisting of:
(i) a tryptophan residue at position 32;
(ii) a valine, leucine, isoleucine, tryptophan, phenylalanine, lysine,
arginine,
tyrosine, methionine, cysteine or histidine residue at position 34;
(iii) an isoleucine, tryptophan, lysine, arginine, valine, leucine,
phenylalanine
or histidine residue at position 35;
(iv) a tryptophan residue at position 36;
(v) a tryptophan, glycine, threonine, tyrosine, glutamine, lysine, arginine,
phenylalanine or histidine residue at position 37;
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(vi) a leucine, isoleucine, tryptophan, phenylalanine, valine or cysteine
residue
at position 38;
(vii) a leucine, isoleucine, tryptophan, phenylalanine, methionine, lysine,
arginine, tyrosine or histidine residue at position 39;
(viii) a tryptophan residue at position 40;
(ix) an isoleucine, a tryptophan, phenylalanine, serine, threonine, tyrosine,
glutamine, asparagine, lysine, arginine, histidine at position 41; and/or
(x) a valine, leucine, isoleucine, tryptophan, phenylalanine, tyrosine,
asparagine, lysine, arginine, serine or threonine residue at position 42:
1o where said amino acid residues are not found naturally at said positions in
the
antimicrobial protein or peptide, with the proviso that the antimicrobial
proteins do not
comprise only a replacement arginine residue at position 37, 39 or 42.
Suitably, where the antimicrobial proteins comprise any basic amino acid
residue at
position 39, they include at least one further replacement residue. Suitably,
the same
IS criteria applies to the peptides derived from the proteins.
In other embodiments, where the antimicrobial proteins or peptides comprise
any
of the above mentioned replacements at positions 37, 39 or 42, they suitably
include at
least one further replacement.
Suitably also, the protein or peptide of the invention has enhanced
antimicrobial
2o activity as compared to the plant defensin from which they are derived.
The amino acid positions mentioned above correspond to the amino acid
positions
found in the full length amino acid sequence of Rs-AFP2 as shown in SEQ ID NO
3 or to
the equivalent positions in defensins from different sources when optimally
aligned with
the Rs-AFP2 sequence.
QKLCQRPSGTWSGVCGNNNACKNQCIRLEKARHGSCNYVFPAHKCICYFPC
(SEQ ID NO 3)
The equivalent position will be readily apparent to the man skilled in the art
when the
sequences are optimally aligned to maximise sequence similarity and
colineaxity relative
to the characteristic pattern of the cysteine residues in the defensin
sequence. The
equivalent position will be readily apparent to the man skilled in the art
based on the
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positioning of amino acid residues relative to the characteristic pattern of
cysteine
residues in the defensin sequence.
The replacement amino acid residue at position 34 is preferably selected from
the
group consisting of a valine, leucine, isoleucine, tryptophan, phenylalanine,
methionine,
cysteine, lysine, histidine or tyrosine residue, more preferably from the
group consisting
of a valine, leucine, isoleucine, tryptophan, phenylalanine, methionine,
lysine or histidine
residue and is most preferably selected from the group consisting of a valine,
leucine or
isoleucine residue.
The replacement residue at position 35 is preferably selected from the group
1o consisting of a valine, leucine, isoleucine, trytophan, phenylalanine,
lysine, arginine, or a
histidine residue, and most preferably selected from the group consisting of a
leucine,
isoleucine, arginine, histidine or a phenylalanine residue.
The replacement residue at position 37 is preferably selected from the group
consisting of a tryptophan, glycine, threonine, tyrosine, glutamine, lysine,
arginine,
15 histidine, or phenylalanine residue and more preferably from the group
consisting of a
tryptophan, tyrosine, lysine, arginine, or histidine residue and is most
preferably selected
from the group consisting of a tryptophan, arginine or histidine residue.
The replacement residues at position 38 are preferably selected from the group
consisting of a leucine, isoleucine, tryptophan, phenylalanine, cysteine or a
valine residue;
20 more preferably from the group consisting of a leucine, tryptophan or
phenylalanine
residue.
The replacement residue at position 39 is preferably selected from the group
consisting of a leucine, isoleucine, tryptophan, phenylalanine, methionine,
lysine,
arginine, histidine, or tyrosine residue, more preferably from the group
consisting of a
25 tryptophan, lysine, arginine or a histidine residue and is most preferably
selected from the
group consisting of an arginine or a histidine residue.
The replacement residue at position 41 is preferably selected from the group
consisting of an isoleucine, tryptophan, phenylalanine, serine, threonine,
tyrosine,
glutamine, asparagine, lysine, arginine or histidine residue, and is more
preferably
30 selected from the group consisting of an isoleucine, tryptophan,
phenylalanine, tyrosine,
asparagine, lysine, arginine or histidine residue and is most preferably
selected from the
group consisting of a tryptophan, phenylalanine, lysine, arginine or histidine
residue.
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The replacement residue at position 42 is preferably selected from the group
consisting of a valine, leucine, isoleucine, tryptophan, phenylalanine,
serine, threonine,
tyrosine, asparagine, lysine or arginine residue and is more preferably
selected from the
group consisting of a tryptophan, phenylalanine, tyrosine, lysine or an
arginine residue
and is most preferably selected from the group consisting of a lysine residue
or an
arginine residue.
Preferably, the protein or peptide of the invention includes at least one
replacement in group (iii), (iv), (v), (vi), (vii), (viii), (ix) or (x) above,
and most preferably
at least one of the replacements listed in group (iii), (iv),(v), (vii), (ix)
or (x) above.
to In this and all further aspects of the invention the antimicrobial protein
or peptide
derived therefrom is preferably a modified plant defensin selected from the
group
Rs-AFPl, Rs-AFP2, Rs-AFP3, Rs-AFP4, Br-AFP1, Br-AFP2, Bn-AFPI, Bn-AFP2,
Sa-AFP 1, Sa-AFP2 and At-AFP 1 and Hs-AFP 1, Ah-AMP I and Dm-AMP 1 which are
fully described in Published International Patent Applications Nos. WO
93/05153 and
WO 95/18229 the teachings of which are incorporated herein by reference, Aly-
AFP and
Alf AFP which are fully described in Published International Patent
Applications Nos.
WO 97/37024 and WO 98/26083 the teachings of which are incorporated herein by
reference. The antimicrobial protein or peptide derived therefrom is more
preferably a
plant defensin selected from the group Rs-AFP 1 or Rs-AFP2, and is most
preferably
2o derived from Rs-AFP2.
In a particularly preferred embodiment of the invention the plant defensin is
Rs-
AFP I or especially Rs-AFP2.
In a further preferred embodiment of the invention, where the proteins and
peptides
contain one or more cysteines, these may be replaced by an alpha-aminobutyric
acid
group.
It is particularly preferred that the peptides and proteins of the invention
are
derived from plant defensins having substantially similar activity to Rs-AFP2,
and which
show at least 40%, 50%, 60%, 70%, 80%, or 85% sequence similarity, more
preferably at
least 90% sequence similarity and most preferably at least 95% sequence
similarity to Rs-
3o AFP2.
Antimicrobial proteins which show sequence similarity to the Rs-AFP2 protein
include the proteins Rs-AFPI, Rs-AFP3, Rs-AFP4, Br-AFP1, Br-AFP2, Bn-AFP1,
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Bn-AFP2, Sa-AFP1, Sa-AFP2 and At-AFP1 and Hs-AFP2, Ah-AMP1 and Dm-AMP1.
Further sequence information on the above-mentioned proteins is provided in
Published
International Patent Applications Nos. WO 93/05153 and WO 95/18229 the
teachings of
which are incorporated herein by reference.
For the purpose of the present invention a conservative replacement is defined
as
one which does not alter the activity/function of the protein when compared
with the
unmodified protein.
The antimicrobial peptides of the invention are preferably at least six amino
acid
residues long, more preferably greater than 10 amino acid residues long
preferably 12
~o amino acids long, most preferably 19 amino acids or longer, especially 20
amino acids in
length. Short peptides will contain at least one modified residue as described
above.
Where this includes modified residues 37, 39 or 42, the peptide suitably
contains more
than one such modification.
In a particularly preferred embodiment of the first aspect the peptide is
derived
from the beta-2 strand/turn/beta-3 strand region of a plant defensin, as
defined by the
three-dimentional structure characterization of these proteins (Bruix et al.
1995
Biochemistry 32, 715-724; Fant et al. 1998, J. Mol. Biol. 279, 257-270. Fant
et al.,
(1999), Proteins: Structure, Function, and Genetics 37 (3), 388-403).
The beta-2 strand/turn/beta-3 strand region of a plant defensin may be
determined
2o by analysis of the primary amino acid sequence information and generally is
predicted to
be located between the fourth and the eighth cysteine residue. For example in
Rs-AFP1
and Rs-AFP2 this occurs between positions 21 to 51 of the sequence, and more
precisely
at positions 30 to 51 of the sequence. The antimicrobial peptides of the
invention are
preferably derived from position 21 to 51 of the Rs-AFP2 sequence, preferably
from
position 30 to 51 of the sequence, more preferably from position 30 to 49 or
position 32
to 43 of the Rs-AFP2 sequence.
The number of replacement residues within a protein or peptide according to
the
invention is preferably no greater than 10 i.e l, 2, 3, 4, 5, 6, 7, 8, 9, or
10 replacement
residues and is more preferably from 1 to 6 residues i.e. l, 2, 3, 4, 5, or 6
replacement
residues.
We have found that the antimicrobial proteins and peptides according to the
invention show activity and are particularly useful against a broad spectrum
of fungi and
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have particularly advantageous antifungal activity in the presence of salts as
evidenced by
their activity in SMF+ medium. The proteins and peptides of the invention may
also be
useful in combating bacterial infections. This is described in more detail in
the
accompanying examples and figures.
An antimicrobial protein or peptide according to the invention may be
manufactured from its known amino acid sequence by chemical synthesis using
standard
peptide chemistry, or produced within a suitable organism (for example, a
micro-organism or plant) by expression of recombinant DNA. The antimicrobial
peptide
is useful as a fungicide and may be used for agricultural or pharmaceutical or
other
1o applications. The antimicrobial peptides and proteins may be used in
combination with
one or more of the antimicrobial proteins or with one or more other
antimicrobial peptides
of the present invention.
Knowledge of its primary structure enables manufacture of the antimicrobial
protein, peptide, or parts thereof, by chemical synthesis using standard
peptide chemistry.
15 It also enables production of DNA constructs encoding the antimicrobial
peptide or
protein.
The invention further provides a DNA sequence encoding an antimicrobial
peptide
or protein according to the invention. The DNA sequence may be predicted from
the
known amino acid sequence and DNA encoding the peptide or protein may be
2o manufactured using a standard nucleic acid synthesiser.
The DNA sequence encoding the antimicrobial peptide or protein may be
incorporated into a DNA construct or vector in combination with suitable
regulatory
sequences (promoter, terminator, transit peptide, etc). For some applications,
the DNA
sequence encoding the antimicrobial peptide or protein may be inserted within
a coding
25 region expressing another protein to form an antimicrobial fusion protein
or may be used
to replace a domain of a protein to give that protein antimicrobial activity.
The DNA
sequence may be placed under the control of a homologous or heterologous
promoter
which may be a constitutive or an inducible promoter (stimulated by, for
example,
environmental conditions, presence of a pathogen, presence of a chemical). The
transit
3o peptide may be homologous or heterologous to the antimicrobial protein and
will be
chosen to ensure secretion to the desired organelle or to the extra cellular
space. The
transit peptide is preferably that naturally associated with the antimicrobial
protein of
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interest. Such a DNA construct may be cloned or transformed into a biological
system
which allows expression of the encoded peptide or protein or an active part of
the peptide
or protein. Suitable biological systems include micro-organisms (for example,
bacteria
such as Escherichia coli, Pseudomonas and endophytes such as Clavibacter ~
subsp.
cynodontis (Cxc); yeast; viruses; bacteriophages; etc), cultured cells (such
as insect cells,
mammalian cells) and plants. In some cases, the expressed peptide or protein
may
subsequently be extracted and isolated for use.
An antimicrobial peptide or protein according to the invention is useful for
combating fungal and bacterial diseases in plants. The invention further
provides a
1 o process of combating microbial infection whereby microbes are exposed to
an
antimicrobial peptide or protein according to the invention. The antimicrobial
peptide or
protein may be used in the form of a composition, for example in combination
with a
suitable carrier or diluent. For example, for agricultural use, compositions
of the
invention may be in the form of either a dilute composition which is ready for
immediate
use, or a concentrated compositions which require dilution before use, usually
with water.
Liquid compositions may contain other conventional components such as surface-
active
agents, dispersants etc.
Solid compositions may be in the form of granules, or dusting powders
wherein the active ingredient is mixed with a finely divided solid diluent,
e.g. kaolin,
2o bentonite, kieselguhr, dolomite, calcium carbonate, talc, powdered
magnesia. Fuller's
earth and gypsum. They may also be in the form of dispersible powders or
grains,
comprising a wetting agent to facilitate the dispersion of the powder or
grains in liquid.
Solid compositions in the form of a powder may be applied as foliar dusts.
In a preferred embodiment the invention provides a method of combating fungal
infection by exposing said fungi to an antimicrobial peptide according to the
invention.
In a further preferred embodiment the invention provides a method of combating
bacterial
infection by exposing said bacteria to an antimicrobial peptide according to
the invention.
For pharmaceutical applications, the antimicrobial peptide or protein
(including
any product derived from it) may be used as a fungicide to treat mammalian
infections
3o (for example, to combat yeasts such as Candida). Suitably, the peptide or
protein is in
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the form of a composition comprising a carrier or diluent, which will be a
pharmaceutically acceptable carrier or diluent as is conventional in the art.
Pharmaceutical compositions of the invention may be in a form suitable for
oral
use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily
suspensions,
emulsions, dispersible powders or granules, syrups or elixirs), for topical
use (for example
as creams, ointments, gels, or aqueous or oily solutions or suspensions), for
administration by inhalation (for example as a finely divided powder or a
liquid aerosol),
for administration by insufflation (for example as a finely divided powder) or
for
parenteral administration (for example as a sterile aqueous or oily solution
for
l0 intravenous, subcutaneous, intramuscular or intramuscular dosing or as a
suppository for
rectal dosing). For further information on Formulation the reader is referred
to Chapter
25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman
of
Editorial Board), Pergamon Press 1990.
An antimicrobial peptide or protein (including any product derived from it )
according to the invention may also be used as a preservative (for example, as
a food or
cosmetic additive). Again, suitable compositions may comprise acceptable
carriers or
diluents.
For agricultural applications, the antimicrobial peptide or protein may be
used to
2o improve the disease-resistance or disease-tolerance of crops either during
the life of the
plant or for post-harvest crop protection. Pathogens exposed to the peptide or
proteins are
inhibited. The antimicrobial peptide or protein may eradicate a pathogen
already
established on the plant or may protect the plant from future pathogen attack.
The
eradicant effect of the peptide or protein is particularly advantageous.
Exposure of a plant pathogen to an antimicrobial peptide or protein may be
achieved in various ways, for example:
(a) The isolated peptide or protein may be applied to plant parts or to the
soil
or other growth medium surrounding the roots of the plants or to the seed of
the plant
before it is sown using standard agricultural techniques (such as spraying).
3o The peptide or protein may have been extracted from plant tissue or
chemically
synthesised or extracted from micro-organisms genetically modified to express
the
peptide or protein. The peptide or protein may be applied to plants or to the
plant growth
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medium in the form of a composition comprising the peptide or protein in
admixture with
a solid or liquid diluent and optionally various adjuvants such as surface-
active agents.
Solid compositions may be in the form of dispersible powders, granules, or
grains.
(b) A composition comprising a micro-organism genetically modified to
express the antimicrobial peptide or protein may be applied to a plant or the
soil in which
a plant grows.
(c) An endophyte genetically modified to express the antimicrobial peptide
or protein may be introduced into the plant tissue (for example, via a seed
treatment
process).
l0 An endophyte is defined as a micro-organism having the ability to enter
into
non-pathogenic endosymbiotic relationships with a plant host. A method of
endophyte-enhanced protection of plants has been described in a series of
patent ,
applications by Crop Genetics International Corporation (for example,
International
Application Publication Number W090/13224, European Patent Publication Number
EP-125468-B1, International Application Publication Number W091/10363,
International Application Publication Number W087/03303). The endophyte may be
genetically modified to produce agricultural chemicals. International Patent
Application
Publication Number W094/16076 (ZENECA Limited) describes the use of endophytes
which have been genetically modified to express a plant-derived antimicrobial
peptide or
protein.
(d) DNA encoding an antimicrobial peptide or protein may be introduced
into the plant genome so that the peptide or protein is expressed within the
plant body (the
DNA may be cDNA, genomic DNA or DNA manufactured using a standard nucleic acid
synthesiser).
Exposure of a plant pathogen to an antimicrobial composition comprising an
antimicrobial peptide or protein according to the invention plus an
antimicrobial protein
may be achieved by delivering the protein as well as the peptide or protein as
described
above. For example, both one of the above-mentioned peptides or proteins
according to
the invention plus Rs-AFP2 or Rs-AFP 1 could be simultaneously applied to
plant parts or
simultaneously expressed within the plant body. We have discovered a
synergistic effect
when peptides derived from Rs-AFP2, such as derivatives having cysteine
residues
replaced with a.-aminobutyric acid, and/or in particular those having
replacement residues
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according to the invention are mixed with the full length natural protein and
this forms a
further aspect of the invention. This is described more fully in the examples
herein.
Plant cells may be transformed with recombinant DNA constructs according to a
variety of known methods (A~robacterium Ti plasmids, electroporation,
microinjection,
microprojectile gun, etc). The invention extends to a plant cell transformed
with a DNA
construct according to the invention. The transformed cells may then in
suitable cases be
regenerated into whole plants in which the new nuclear material is stably
incorporated
into the genome. Both transformed monocotyledonous and dicotyledonous plants
may be
obtained in this way, although the latter are usually more easy to regenerate.
Some of the
1o progeny of these primary transformants will inherit the recombinant DNA
encoding the
antimicrobial peptide or protein(s).
The invention further provides a plant having improved resistance to a
microbial
pathogen and containing recombinant DNA which expresses an antimicrobial
peptide or
protein according to the invention. Such a plant may be used as a parent in
standard plant
15 breeding crosses to develop hybrids and lines having improved microbial
resistance.
In a preferred embodiment the invention further provides a plant having
improved
resistance to a fungal pathogen and containing recombinant DNA which expresses
an
antimicrobial peptide or protein according to the invention.
In a further preferred embodiment the invention further provides a plant
having
20 improved resistance to a bacterial pathogen and containing recombinant DNA
which
expresses an antimicrobial peptide or protein according to the invention.
Recombinant DNA is DNA, preferably heterologous, which has been introduced
into the plant or its ancestors by transformation. The recombinant DNA encodes
an
antimicrobial peptide or protein expressed for delivery to a site of pathogen
attack (such
25 as the leaves). The DNA may encode an active subunit of an antimicrobial
peptide or
protein.
A pathogen may be any fungus growing on, in or near the plant. In this
context,
improved resistance is defined as enhanced tolerance to a fungal pathogen when
compared to a wild-type plant. Resistance may vary from a slight increase in
tolerance to
3o the effects of the pathogen (where the pathogen in partially inhibited) to
total resistance so
that the plant is unaffected by the presence of pathogen (where the pathogen
is severely
inhibited or killed). An increased level of resistance against a particular
pathogen or
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resistance against a wider spectrum of pathogens may both constitute an
improvement in
resistance. Transgenic plants (or plants derived therefrom) showing improved
resistance
are selected following plant transformation or subsequent crossing.
Where the antimicrobial peptide or protein is expressed within a transgenic
plant
or its progeny, the fungus is exposed to the peptide or protein at the site of
pathogen
attack on the plant. In particular, by use of appropriate gene regulatory
sequences,
the peptide or protein may be produced in vivo when and where it will be most
effective.
For example, the peptide or protein may be produced within parts of the plant
where it is
not normally expressed in quantity but where disease resistance is important
(such as in
the leaves).
Examples of genetically modified plants which may be produced include field
crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco,
sugarbeet, cotton,
Soya. maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples,
pears,
strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.
The invention will now be described by way of example only, with reference to
the following drawings wherein:
Figure 1 : shows a histogram analysis of the antifungal activity measured
against the
fungus Fusarium culmorum of peptides from Rs-AFP with replacement residues at
positions 32 to 37 in buffer'/Z PDB. The antifungal activity is expressed as %
relative to
the activity of the reference peptide *RHGSCNYVFPAH#.
Figure 2 : shows a histogram analysis of peptides from Rs-AFP with replacement
residues at positions 38 to 43 in buffer '/z PDB. The antifungal activity is
expressed as
relative to the activity of the reference peptide *RHGSCNYVFPAH#.
Figure 3 : shows a histogram analysis of peptides from Rs-AFP with replacement
residues
at positions 32 to 37 in buffer SMF+ pHS. The antifungal activity is expressed
as
relative to the activity of the reference peptide *RHGSCNYVFPAH#.
Figure 4 : shows a histogram analysis of peptides from Rs-AFP with replacement
residues
at positions 38 to 43 in buffer SMF+ pHS. The antifungal activity is expressed
as
relative to the activity of the reference peptide *RHGSCNYVFPAH#.
Figure 5 : shows a histogram analysis of peptides from Rs-AFP with replacement
residues
at positions 38 to 43 in buffer SMF+ pH7. The antifungal activity is expressed
as
relative to the activity of the reference peptide *RHGSCNYVFPAH#.
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Figure 6 : shows a histogram analysis of peptides from Rs-AFP with replacement
residues
at positions 32 to 47 in buffer SMF+ pH7. The antifungal activity is expressed
as
relative to the activity of the reference peptide *RHGSCNYVFPAH#.
EXAMPLES
Materials. N-methylpyrrolidone (NMP) and piperidine were peptide synthesis
grade and obtained from Perkin Elmer/ABI (Warrington, UK). Dimethylformamide
(DMF), dicyclohexylcarbodiimide (DCC), N-hydroxybenzotriazole (HOBt),
diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), thioanisole (TA),
ethanedithiol
(EDT), dimethylsulfoxide (DMSO), and dimethylamino pyridine (DMAP) were pro-
analysis grade and were obtained from Merck (Darmstad, Germany). Diethylether
was
purified over a column of activated basic aluminumoxide and DIEA was distilled
twice
over ninhydrin and potassium hydroxide before use. Amino acid derivatives and
resins
were obtained from Saxon Biochemicals (Hannover, Germany).
For analytical HPLC we used two Waters pumps model 510, a Waters gradient
controller model 680, a Waters WISP 712 autoinjector, and a Waters 991
photodiode
array detector. Products were analysed in a linear gradient from water with
0.1 % TFA to
60% acetonitrile/water with 0.1 % TFA in 60 min on a Waters Delta Pak C 18-1
OOA
(3.9x150 mm, 5 Vim) column at lml/min. Preparative HPLC was carried out using
a
Waters Prep 4000 liquid chromatograph, equipped with a Waters RCM module with
two
PrepPak cartridges plus guard cartridge (40x210mm or 25x210mm) filled with
delta-Pak
C18-100A (15 pm) material. Peptides were detected at 230 nm using a Waters 486
spectrophotometer with a preparative cell. Amino acid analysis was performed
using a
Waters Pico-Tag system, after hydrolysis in a Pico-Tag workstation using 6N HC
1 with
1% phenol at 150 °C for 1 hour, and derivatization with
phenylisothiocyanate.
PEPSCAN-split. Radiation grafted polyethylene pins were functionalized with
hydroxyl groups. Boc-(3-alanine was coupled using DCC and DMAP as catalyst,
the Boc
group was removed with TFA and after careful washing Fmoc-2,4-dimethoxy-4'
(carboxymethyloxy)-benzhydrylamine (Rink Linker, Bachem, Laufelfingen,
Switzerland)
was coupled using the DOC/HOBt method. Next, 240 dodecapeptides from AFP2 were
synthesized simultaneously using standard Fmoc-chemistry and overnight
couplings with
DCC/HOBt as coupling method. After coupling of the last amino acid the Fmoc
group
was removed with 30% piperidine/DMF and the peptides were acetylated with
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aceticanhydride. After washing and drying, the peptides were deprotected and
cleaved
from the pins with TFA/phenol/TA/water/EDT 10/0.75/0. 5/0.5/0.25. The cleavage
mixtures were evaporated, extracted twice with diethylether, and lyophilized
twice from
water. This procedure yields peptides up to about I mg with a C-terminal
amide.
Multi-synthesis (MPS). A Hamilton Microlab 2200 was programmed to deliver
washing solvents and reagents to a rack with 40 individual 4m1 columns with
filter,
containing 30 pmol of resin for peptide synthesis. The columns were drained
after each
step by vacuum. The next coupling cycle was based on Fmoc chemistry using
double
coupling steps:
1 o I . NMP wash ( 1 ml)
2. 30% (v/v) piperidine/NMP (3 min, O.SmI)
3. 30% (v/v) piperidine/NMP (17 min, O.SmI)
4. NMP wash (5xlml)
5. double coupling (2x30 min)
6. NMP wash (2x 1 ml)
Coupling step: Fmoc-amino acid in NMP (0.4M, 0.25m1), 0.22m1 of 0,45M
HBTU/HOBt in DMF, and 0.2m1 of 2M DIEA in NMP were transferred to the reaction
vessel and allowed to react for 30 min. Then the reaction mixture was drained
and the
coupling procedure was repeated once.
After the coupling of the last amino acid, the Fmoc group was removed with 30%
piperidine/NMP, the peptides were washed, acetylated in 30 min using
NMP/acetic
anhydride/DIEA 10/1/0.1, washed again, dried, and deprotected and cleaved in 2
hr with
I .5m1 of TFA/phenol/TA/water/EDTA 10/0.75/0.5/0.5/0.25. The cleavage mixture
was
filtered, the resin was washed with O.SmI TFA, and the peptide was
precipitated by
adding I3m1 hexane/diethylether I/1. After centrifugation the precipitate was
extracted
again with hexane/diethylether. The precipitate was dried and lyophilized from
water/acetonitrile I/I.
Bioassays. The data (IC50) shown in the tables were all concentrations in
pg/ml
3o that give 50% growth inhibition of mycelium grown from spore suspensions of
Fusarium
culmorum after 72 hours at room temperature in a medium of half strength
potato
dextrose broth ( I /2 PDB, from Difco) at pH 5.8 or medium SMF + pH5 or SMF +
pH7.
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Bioassays were performed as described in Terras et al. 1992. J. Biol. Chem.
267: 15301-
15309). Medium SMF+pH5 consists of medium SMF (Cammue et al. 1992, J. Biol.
Chem 267: 2228-2233) with addition of 1mM CaCl2, 50mM KCl and IOmM MES (final
pH adjusted to 5.0). Medium SMF+pH7 is identical to SMF+pH5 except that IOmM
MES is replaced by IOmM. Tris and that the pH is adjusted to 7Ø
Example 1
It has previously been demonstrated that synthetic peptides corresponding to
parts
of the sequence of plant defensins show antifungal activity (De Samblanx et
al. Peptide
Research, 1996, 9: 262-268 and Published International Patent Applications
W097/21814
to and W097/21815).
We have chosen to synthesise a 12-mer peptide corresponding to the RsAFP2
sequence from position 32 to 43. This peptide with sequence RHGSCNYVFPAH (SEQ
ID NO 4) was found to inhibit 50% of the growth of F. culmorum in three
different
media, '/2 PDB, SMF+pH5 and SMF+pH at concentration 57~g/ml, 400~g/ml and
400~g/ml, respectively. In order to optimise the antifungal potency of this
peptide, amino
acid replacement net tests were performed whereby every residue at each
position was
replaced by any of 19 other amino acids. The antifungal activity of these
substitution
variants was determined against F. culmorum in the media'/2 PDB, SMF+pH5 and
SMF+pH7. The activity data are presented in figures 1 to 6 where thay are
expressed
2o relative to the antifungal activity of the reference peptide RHGSCNYVFPAH.
(SEQ ID
NO 4). As can be seen in figures 1 to 6, the activity of some of the
substitution variants
was increased up to 20-fold compared to that of the reference peptide.
Example 2
We have found that improved antifungal activity can be observed in longer (>19
residues) peptide derivatives of RsAFP2 by replacing the cysteines by alpha-
isoaminobutyric acid. We have now synthesised a 20-mer peptide corresponding
to the
RsAFP2 sequence from position 30 to 49 whereby each of the three cysteines was
substituted by alpha-aminobutyric acid. This peptide is called MBNO1.
3o MBNO1 inhibits growth of F. culmorum by 50% in media'/z PDB, SMF+pHSand
SMF+pH7 at concentration of 5.8, 21.8 and 70 ~g/ml, respectively (table 1).
Variants of
MBNO1 were synthesised in which either one, two, three, four or six residues
were
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substituted. The substitutions were chosen based on the data obtained for the
12-mer
series described above. The antifungal activity of the 20-mer variants against
F.
culmorum determined in three different media ( 1 /2 PDB, SMF+pH5 and SMF+pH7)
is
presented in table 1. Some of the peptides in particular peptides MBY32,
MBY33,
MBZO1, MBZ02 and MBY10 showed strongly improved activity in all three media
relative to MBNO1.
Table 1. Antifungal activity of single- and mufti-substitution peptides of Rs-
AFP2
(30-49).
Code SEQ __ Peptide sequence IC50
value
(pg/ml)
in
medium:
ID '/z SMF SMF+pH7
NO PDB +
pH5
MBNO1 5 *KARHGSBNYVFPAHKBIBYF# 5.8 21.8 70.0
control (n=4) (n=4)
one
substitution
MBY01 6 *KARHGRBNYVFPAHKBIBYF# 7.4 16.2 38.9
MBY02 7 *KARHGSBRYVFPAHKBIBYF# 8.9 16.7 38.3
MBY03 8 *KARHGSBWYVFPAHKBIBYF# 11 17.9 36.9
MBY04 9 *KARHGSBNFVFPAHKBIBYF# 9.8 21.8 79.7
MBY05 10 *KARHGSBNLVFPAHKBIBYF# 7.3 53.6 >100
MBY06 I1 *KARHGSBNYRFPAHKBIBYF# 7.1 10.5 78.8
MBY07 12 *KARHGSBNYVWPAHKBIBYF# 5.0 100 >100
MBY08 13 *KARHGSBNYVFPYHKBIBYF# 5.4 90 81.6
MBY09 14 *KARHGSBNYVFPKHKBIBYF# 3.8 33.4 80.6
two
substitutions
MBY25 15 *KARHGSBRLVFPAHKBIBYF# 5.5 8.3 33.7
MBY26 16 *KARHGSBRYVWPAHKBIBYF# 4.7 15.2 38.7
MBY27 17 *KARHGSBRYRFPAHKBIBYF# 6.5 4.8 21.4
MBY28 18 *KARHGSBNLVFPKHKBIBYF# 4.9 5.8 78.0
MBY29 19 *KARHGSBWYRFPAHKBIBYF# 5.3 9.1 19.7
MBY30 20 *KARHGSBNFVFPYHKBIBYF# 4.9 74.1 73.2
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Code SEQ Peptide sequence IC50
value
(~g/ml)
in
medium:
ID '/z SMF SMF+pH7
NO PDB +
pH5
three
substitutions
MBY31 21 *KARHGSBRFRFPAHKBIBYF# 4.7 4.6 13.7
(n=4)
MBY32 22 *KARHGSBRKRFPAHKBIBYF# 3.7 3.4 9.6
(n=4) (n=4)
MBY33 23 *KARHGSBRYRFPKHKBIBYF# 3.8 3.2 8.5
(n=4) (n=4)
MBY34 24 *KARHGSBRLRFPAHKBIBYF# 4.7 4.0 15.5
(n=4) (n=4)
MBZO1 25 *KARHGRBRYRFPAHKBIBYF# 4.7 4.1 9.8
(n=4) (n=3) (n=4)
MBZ02 26 *KARHGRIWYRFPAHKBIBYF# 5.1 4.6 9.3
(n=4) (n=4) (n=4)
MBZ03 27 *KARHGSBWLRFPAHKBIBYF# 5.5 7.5 19.0
four
substitutions
MBZ04 28 *KARHGSBRLRFPYHKBIBYF# 4.9 5.1 15.5
MBZ05 29 *KARHGSBWLRFPKHKBIBYF# 4.8 4.8 15.6
MBZ06 30 *KARHGSBRLRWPAHKBIBYF# 4.8 4.9 15.7
six
substitutions
MBY10 31 *KARHGRBRFRWPYHKBIBYF# 4.8 3.7 5.9
(n=4) (n=4)
Multiple Peptide Synthesis at 30 umol scale:
10
* = acetyl
# = amide
B = alpha-aminobutyric acid; substitution amino acids are underlined.
Synergistic effects of combinations of Rs-AFP2 and peptide MBO06 or peptide
MBY10
We have done a checkerboard titration of Rs-AFP2 and two peptides: MBQ06 of
SEQ ID NO 5 (identical to MBNO1 see above) or peptide MBY10 (identical to
MBN01
except for the following substitutions; Ser35=>Arg, Asn37=>Arg, Tyr38=>Phe,
Va139=>Arg, Phe40=>Trp, A1a42=>Tyr). Thus MBY10 is of SEQ ID NO 31 shown
above.
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Native protein and peptide were diluted in a microtitre plate and tested for
antifungal activity against Fusarium culmorum in various media. In Table II
synergy
scores are indicated as a function of the molar amounts of native protein and
peptide in
each well. Synergy has been tested in three media: '/2 PDB, SMF+pH5 and
SMF+pH7.
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Table II Synergistic effects of combinations of Rs-AFP2 and peptide
MBQ06 or peptide MBY10, in media'/Z PDB, SMF+pH5 and SMF+pH7
'/z Rs-AFP2
PDB
697 348 174 87.1 43.5 21.7 10.8 5.442.71 1.35 0.679
MBY10
2990
1490
749
374
187 * * * * 0 +++ + 0 0 0 0
93.6 * * * * 0 0 0 0 0 0 0
68 * * * * 0 0 0 0 0 0
'/2 RsAFP2
PDB
697 348 174 87.1 43.5 21.7 10.8 5.442.71 1.35 0.679
MBQ06
3350
1670 * * * * * + + + + + 0
838 * * * * +++ +++ + 0 0 0 0
419 * * * * +++ + 0 0 0 0 0
209 * * * * ++ 0 0 0 0 0 0
104 * * * * + 0 0 0 0 0 0
52.3 * * * * 0 0 0 0 0 0
SMF+ Rs-AFP2
pH5 697 348 174 87.1 43.5 21.7 10.8 5.442.71 1.35 0.679
MBY
2990 * * * 0 0 0 0 0 0 0 0
1490 * * * + + 0 + 0 0 0 0
749 * * + ++ + 0 0 0 0 0 0
374 * * 0 + + 0 0 0 0 0 0
187 * * + 0 0 0 0 0 0 0 0
93.6 * * + 0 0 0 0 0 0 0 0
68 * * 0 + 0 0 0 0 0 0
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SMF+ Rs-AFP2
pH5 697 348 174 87.143.5 21.7 10.85.442.71 1.350.679
MBQ06
3350 + + ++ ++++++++ +++ + 0 0 0 0
1670 0 0 ++ ++ 0 0 0 0 0 0 0
838 0 0 ++ + 0 0 0 0 0 0 0
419 0 0 ++ 0 0 0 0 0 0 0 0
209 0 0 + 0 0 0 0 0 0 0 0
104 0 0 + 0 0 0 0 0 0 0 0
52.3 0 0 + 0 0 0 0 0 0 0
SMF+ Rs-AFP2
pH7 697 348 174 87.143.5 21.7 10.85.442.71 1.350.679
MBY
2990 * * * * + 0 0 0 0 0 0
1490 * * * * + + 0 0 0 0 0
749 * * * * + 0 0 0 0 0 0
374 * * * * ++ + 0 0 0 0 0
187 * * 0 0 + + 0 0 0 0 0
93.6 * * 0 0 + 0 0 0 0 0 0
68 * * 0 0 0 0 0 0 0 0
SMF+ RsAFP2
pH7 697 348 174 87.143.5 21.7 10.85.442.71 1.350.679
MBQ06
3350 * * 0 0 0 0 0 0 0 0 0
1670 * * + 0 0 0 0 0 0 0 0
838 * * + 0 0 0 0 0 0 0 0
419 * * 0 0 0 0 0 0 0 0 0
209 * * 0 0 0 0 0 0 0 0 0
104 * * ++ 0 0 0 0 0 0 0 0
52.3 * * + 0 0 0 0 0 0 0
Amounts of protein/peptide are indicated in pmol/100u1. Test fungus was
Fusarium
culmorum (2 x 10" spores.ml)
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0: No synergy effect was observed.
*: The sum of the percentage growth inhibition of the native protein and the
peptide of the individual inhibitory activity of one of them was above 80%.
+: The growth inhibition was 20% - 40% (+), 40% - 60% (++), 60% - 80%
(+++), 80%,- 100% (++++) more than the sum of the individual growth
inhibition percentages of Rs-AFP2 and MBY10 or MBQ06.
Synergy was scored as growth inhibition more than 20% higher than the sum of
Rs-AFP2 and peptide or of one of the individual components. Peptide MBY10
showed
to activity in all media, whereas peptide MBQ06 only showed inhibitory
activity in'/2 PDB.
In all cases some combinations of peptide (MBY10 or MBQ06) and Rs-AFP2
resulted in
additional inhibitory activity. MBQ06 that did not have inhibitory activity in
SMF+
media was still able to increase growth inhibition in combination with sub-
inhibitory
amounts of Rs-AFP2.
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1
SEQUENCE LISTING
<110> Zeneca Limited
Posthuma, Geertruida A
Schaaper, Wilhelmus MM
Sijtsma, Lolke
Amerongen, Aart V
Fant, Franky
Borremans, Frans AM
<120> Proteins and Peptides
<130> PPD 50287/W0
<140>
<141>
<150> GB 9918155.4
<151> 1999-OS-02
<160> 32
<170> PatentIn Ver. 2.1
<210>1
<211>51
<212>PRT
<213>Raphanus sativus
<220>
<221> DISULFID
<222> (4)..(51)
<220>
<221> DISULFID
<222> (15)..(36)
<220>
<221> DISULFID
<222> (21)..(45)
<220>
<221> DISULFID
<222> (25)..(47)
CA 02378432 2001-12-19
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2
<400> 1
Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly
1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys Ala Arg
20 25 30
His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys Tyr
35 40 45
Phe Pro Cys
<210> 2
<211> 51
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Characteristic
structural feature of plant defensins
<220>
<221> SITE
<222> 1..3, 5..10, 12, 14, 16..20, 22..24, 26..28
<223> Xaa is an unspecified amino acid or group of amino
acids
<220>
<221> SITE
<222> 30..33, 35, 37..44, 46, 48..50
<223> Xaa is an unspecified amino acid or group of amino
acids
<220>
<221> SITE
<222> (11)
<223> Xaa is an aromatic amino acid (Phe, Trp, Tyr)
<400> 2
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Cys Xaa
1 5 10 15
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3
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Glu Xaa Xaa Xaa
20 25 30
Xaa Gly Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa
35 40 45
Xaa Xaa Cys
<210> 3
<211> 51
<212> PRT
<213> Raphanus sativus
<400> 3
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly
1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg
20 25 30
His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys Tyr
35 40 45
Phe Pro Cys
<210> 4
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Reference
peptide
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
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4
<220>
<221> MOD RES
<222> (12)
<223> AMIDATION
<400> 4
Arg His Gly Ser Cys Asn Tyr Val Phe Pro Ala His
1 5 10
<210> 5
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 5
Lys Ala Arg His Gly Ser Xaa Asn Tyr Val Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
<210> 6
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 6
Lys Ala Arg His Gly Arg Xaa Asn Tyr Val Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 7
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
6
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 7
Lys Ala Arg His Gly Ser Xaa Arg Tyr Val Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 8
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
7
<400> 8
Lys Ala Arg His Gly Ser Xaa Trp Tyr Val Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 9
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 9
Lys Ala Arg His Gly Ser Xaa Asn Phe Val Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
8
<210> 10
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 10
Lys Ala Arg His Gly Ser Xaa Asn Leu Val Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 11
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
9
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 11
Lys Ala Arg His Gly Ser Xaa Asn Tyr Arg Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 12
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 12
Lys Ala Arg His Gly Ser Xaa Asn Tyr Val Trp Pro Ala His Lys Xaa
1 5 10 15
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
Ile Xaa Tyr Phe
<210> 13
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 13
Lys Ala Arg His Gly Ser Xaa Asn Tyr Val Phe Pro Tyr His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 14
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
11
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 14
Lys Ala Arg His Gly Ser Xaa Asn Tyr Val Phe Pro Lys His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 15
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
12
<400> 15
Lys Ala Arg His Gly Ser Xaa Arg Leu Val Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 16
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Desor~.ptio_n_ of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 16
Lys Ala Arg His Gly Ser Xaa Arg Tyr Val Trp Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
13
<210> 17
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 17
Lys Ala Arg His Gly Ser Xaa Arg Tyr Arg Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 18
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
14
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 18
Lys Ala Arg His Gly Ser Xaa Asn Leu Val Phe Pro Lys His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 19
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 19
Lys Ala Arg His Gly Ser Xaa Trp Tyr Arg Phe Pro Ala His Lys Xaa
1 5 10 15
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
Ile Xaa Tyr Phe
<210> 20
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 20
Lys Ala Arg His Gly Ser Xaa Asn Phe Val Phe Pro Tyr His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 21
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
16
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 21
Lys Ala Arg His Gly Ser Xaa Arg Phe Arg Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 22
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
17
<400> 22
Lys Ala Arg His Gly Ser Xaa Arg Lys Arg Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 23
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 23
Lys Ala Arg His Gly Ser Xaa Arg Tyr Arg Phe Pro Lys His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
18
<210> 24
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 24
Lys Ala Arg His Gly Ser Xaa Arg Leu Arg Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 25
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
19
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 25
Lys Ala Arg His Gly Arg Xaa Arg Tyr Arg Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 26
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 26
Lys Ala Arg His Gly Arg Ile Trp Tyr Arg Phe Pro Ala His Lys Xaa
1 5 10 15
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
Ile Xaa Tyr Phe
<210> 27
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
<400> 27
Lys Ala Arg His Gly Ser Xaa Trp Leu Arg Phe Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 28
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
21
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 28
Lys Ala Arg His Gly Ser Xaa Arg Leu Arg Phe Pro Tyr His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 29
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution peptide of Rs-AFP2
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
22
<400> 29
Lys Ala Arg His Gly Ser Xaa Trp Leu Arg Phe Pro Lys His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
<210> 30
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 30
Lys Ala Arg His Gly Ser Xaa Arg Leu Arg Trp Pro Ala His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
CA 02378432 2001-12-19
WO 01/09174 PCT/GB00/02941
23
<210> 31
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD RES
<222> (1)
<223> ACETYLATION
<220>
<221> MOD RES
<222> (20)
<223> AMIDATION
<220>
<221> MOD RES
<222> (7, 16, 18)
<223> Abu
<220>
<223> Description of Artificial Sequence: Substitution
peptide of Rs-AFP2
<400> 31
Lys Ala Arg His Gly Arg Xaa Arg Phe Arg Trp Pro Tyr His Lys Xaa
1 5 10 15
Ile Xaa Tyr Phe
