Note: Descriptions are shown in the official language in which they were submitted.
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COMPREND PLUS D'UN TOME.
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NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
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SYNTHETIC ANTIMICROBIAL POLYPEPTIDES
BACKGROUND
Antimicrobial peptides (AMPs) are a relatively newly discovered group of
antimicrobial
agents with new modes of action.
AMPs are widely distributed in animals, plants and microbes and are among the
most
ancient host defense factors. Most of the peptides are cationic and amphipatic
in nature; a
feature that allows interaction with the negatively charged bacterial or
fungal membrane.
The peptides range in size from 6-7 amino acids up to 60. More than 500
different
AMPs have been isolated to date. They can be divided into several classes
based on
structure or amino acid composition. The simplest structures are small alpha-
helical peptides.
Other AMPs folds into beta-sheet structures, while others again form rigid,
disulfide bridged
tertiary structures.
The AMPs are normally microbicidal (as opposed to static) and are extremely
fast
acting. Usually, the target organism is killed within minutes. They work by
interfering with the
membrane function of the target organisms. Several different mechanisms of
actions have
been shown to exist, but for most AMPs, the overall result is membrane
disruption and/or cell
lysis.
The selectivity of microbial membranes is mediated by membrane composition,
membrane charge and trans-membrane potential. Microbial membranes have a
higher
negative charge than the membrane of higher organisms, contains different
types of
phospholipids, and no cholesterol.
It has proven extremely difficult to induce resistance to AMPs in target
organisms. This
is a reflection of the target; multiple genomic alterations would have to
occur to significantly
alter the membrane composition or charge.
SUMMARY
In a first aspect the present invention relates to polypeptides having
antimicrobial
activity, comprising the amino acid sequence:
X1-X2-X3-X4-X5; wherein each of X1, X2, X3, X4 and X5 are selected from the
group of amino
acid sequences:
P-P-R-F, P-R-F-P, R-F-P-P, F-P-P-R, P-R-P-F, R-P-F-P, P-F-P-R, F-P-R-P, P-P-F-
R,
P-F-R-P, F-R-P-P, R-P-P-F, P-P-R-L, P-R-L-P, R-L-P-P, L-P-P-R, P-R-P-L, R-P-L-
P,
P-L-P-R, L-P-R-P, P-P-L-R, P-L-R-P, L-R-P-P, R-P-P-L, P-P-R-V, P-R-V-P, R-V-P-
P,
V-P-P-R, P-R-P-V, R-P-V-P, P-V-P-R, V-P-R-P, P-P-V-R, P-V-R-P, V-R-P-P, R-P-P-
V,
P-P-R-I, P-R-I-P, R-I-P-P, I-P-P-R, P-R-P-I, R-P-I-P, P-I-P-R, I-P-R-P, P-P-I-
R, P-I-R-P,
I-R-P-P and R-P-P-I;
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and wherein at the most four of X, - X5 are identical.
In a second aspect the present invention relates to polynucleotides having a
nucleotide sequence which encodes for the polypeptide of the invention.
In a third aspect the present invention relates to a nucleic acid construct
comprising
the nucleotide sequence, which encodes for the polypeptide of the invention,
operably linked
to one or more control sequences that direct the production of the polypeptide
in a suitable
host.
In a fourth aspect the present invention relates to a recombinant expression
vector
comprising the nucleic acid construct of the invention.
In a fifth aspect the present invention relates to a recombinant host cell
comprising the
nucleic acid construct of the invention.
In a sixth aspect the present invention relates to a method for producing a
polypeptide
of the invention, the method comprising:
(a) cultivating a recombinant host cell of the invention under conditions
conducive for
production of the polypeptide; and
(b) recovering the polypeptide.
Other aspects of the present invention will be apparent from the below
description and
from the appended claims.
DEFINITIONS
Before discussing the present invention in further details, the following
terms and
conventions will first be defined:
Antimicrobial activity: The term "antimicrobial activity" is defined herein as
an activity
which is capable of killing or inhibiting growth of microbial cells. In the
context of the present
invention the term "antimicrobial" is intended to mean that there is a
bactericidal and/or a
bacteriostatic and/or fungicidal and/or fungistatic effect and/or a virucidal
effect, wherein the
term "bactericidal" is to be understood as capable of killing bacterial cells.
The term
"bacteriostatic" is to be understood as capable of inhibiting bacterial
growth, i.e. inhibiting
growing bacterial cells. The term "fungicidal" is to be understood as capable
of killing fungal
cells. The term "fungistatic" is to be understood as capable of inhibiting
fungal growth, i.e.
inhibiting growing fungal cells. The term "virucidal" is to be understood as
capable of
inactivating virus. The term "microbial cells" denotes bacterial or fungal
cells (including
yeasts).
In the context of the present invention the term "inhibiting growth of
microbial cells" is
intended to mean that the cells are in the non-growing state, i.e., that they
are not able to
propagate.
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For purposes of the present invention, antimicrobial activity may be
determined
according to the procedure described by Lehrer et al., Journal of
Immunological Methods, Vol.
137 (2) pp. 167-174 (1991).
Polypeptides having antimicrobial activity may be capable of reducing the
number of
living cells of Escherichia coli (DSM 1576) to 1/100 after 8 hours (preferably
after 4 hours,
more preferably after 2 hours, most preferably after 1 hour, and in particular
after 30 minutes)
incubation at 20 C in an aqueous solution of 25%(w/w); preferably in an
aqueous solution of
10%(w/w); more preferably in an aqueous solution of 5%(w/w); even more
preferably in an
aqueous solution of 1 %(w/w); most preferably in an aqueous solution of
0.5%(w/w); and in
particular in an aqueous solution of 0.1%(w/w) of the polypeptides having
antimicrobial
activity.
Polypeptides having antimicrobial activity may also be capable of inhibiting
the
outgrowth of Escherichia coli (DSM 1576) for 24 hours at 25 C in a microbial
growth
substrate, when added in a concentration of 1000 ppm; preferably when added in
a
concentration of 500 ppm; more preferably when added in a concentration of 250
ppm; even
more preferably when added in a concentration of 100 ppm; most preferably when
added in a
concentration of 50 ppm; and in particular when added in a concentration of 25
ppm.
Polypeptides having antimicrobial activity may be capable of reducing the
number of
living cells of Bacillus subtilis (ATCC 6633) to 1/100 after 8 hours
(preferably after 4 hours,
more preferably after 2 hours, most preferably after 1 hour, and in particular
after 30 minutes)
incubation at 20 C in an aqueous solution of 25%(w/w); preferably in an
aqueous solution of
10%(w/w); more preferably in an aqueous solution of 5%(w/w); even more
preferably in an
aqueous solution of 1%(w/w); most preferably in an aqueous solution of
0.5%(w/w); and in
particular in an aqueous solution of 0.1 %(w/w) of the polypeptides having
antimicrobial
activity.
Polypeptides having antimicrobial activity may also be capable of inhibiting
the
outgrowth of Bacillus subtilis (ATCC 6633) for 24 hours at 25 C in a microbial
growth
substrate, when added in a concentration of 1000 ppm; preferably when added in
a
concentration of 500 ppm; more preferably when added in a concentration of 250
ppm; even
more preferably when added in a concentration of 100 ppm; most preferably when
added in a
concentration of 50 ppm; and in particular when added in a concentration of 25
ppm.
The polypeptides of the present invention should preferably have at least 20%
of the
antimicrobial activity of the polypeptide consisting of the amino acid
sequence of anyone of
SEQ ID NO:1 to SEQ ID NO:319. In a particular preferred embodiment, the
polypeptides
should have at least 40%, such as at least 50%, preferably at least 60%, such
as at least
70%, more preferably at least 80%, such as at least 90%, most preferably at
least 95%, such
as about or at least 100% of the antimicrobial activity of the polypeptide
consisting of the
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amino acid sequence of anyone of SEQ ID NO:1 to SEQ ID NO:319.
Fragment: When used herein, a "fragment" of the amino acid sequences of the
invention is a subsequence of the polypeptides wherein one or more amino acids
have been
deleted from the amino and/or carboxyl terminus. Preferably the one or more
amino acids
have been deleted from the carboxyl terminus. Preferably a fragment consists
of at least 20
amino acids.
Allelic variant: In the present context, the term "allelic variant" denotes
any of two or
more alternative forms of a gene occupying the same chromosomal locus. Allelic
variation
arises naturally through mutation, and may result in polymorphism within
populations. Gene
mutations can be silent (no change in the encoded polypeptide) or may encode
polypeptides
having altered amino acid sequences. An allelic variant of a polypeptide is a
polypeptide
encoded by an allelic variant of a gene.
Substantially pure polynucleotide: The term "substantially pure
polynucleotide" as used
herein refers to a polynucleotide preparation, wherein the polynucleotide has
been removed
from its natural genetic milieu, and is thus free of other extraneous or
unwanted coding
sequences and is in a form suitable for use within genetically engineered
protein production
systems. Thus, a substantially pure polynucleotide contains at the most 10% by
weight of
other polynucleotide material with which it is natively associated (lower
percentages of other
polynucleotide material are preferred, e.g. at the most 8% by weight, at the
most 6% by
weight, at the most 5% by weight, at the most 4% at the most 3% by weight, at
the most 2%
by weight, at the most I% by weight, and at the most 1/2% by weight). A
substantially pure
polynucleotide may, however, include naturally occurring 5' and 3'
untranslated regions, such
as promoters and terminators. It is preferred that the substantially pure
polynucleotide is at
least 92% pure, i.e. that the polynucleotide constitutes at least 92% by
weight of the total
polynucleotide material present in the preparation, and higher percentages are
preferred such
as at least 94% pure, at least 95% pure, at least 96% pure, at least 96% pure,
at least 97%
pure, at least 98% pure, at least 99%, and at the most 99.5% pure. The
polynucleotides
disclosed herein are preferably in a substantially pure form. In particular,
it is preferred that
the polynucleotides disclosed herein are in "essentially pure form", i.e. that
the polynucleotide
preparation is essentially free of other polynucleotide material with which it
is natively
associated. Herein, the term "substantially pure polynucleotide" is synonymous
with the terms
"isolated polynucleotide" and "polynucleotide in isolated form".
Modification(s) In the context of the present invention the term
"modification(s)" is
intended to mean any chemical modification of the polypeptide consisting of
the amino acid
sequence X,-X2-X3-X4-X5i wherein each of X1, X2, X3, X4 and X5 are selected
from the group
of amino acid sequences:
P-P-R-F, P-R-F-P, R-F-P-P, F-P-P-R, P-R-P-F, R-P-F-P, P-F-P-R, F-P-R-P, P-P-F-
R,
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P-F-R-P, F-R-P-P, R-P-P-F, P-P-R-L, P-R-L-P, R-L-P-P, L-P-P-R, P-R-P-L, R-P-L-
P,
P-L-P-R, L-P-R-P, P-P-L-R, P-L-R-P, L-R-P-P, R-P-P-L, P-P-R-V, P-R-V-P, R-V-P-
P,
V-P-P-R, P-R-P-V, R-P-V-P, P-V-P-R, V-P-R-P, P-P-V-R, P-V-R-P, V-R-P-P, R-P-P-
V,
P-P-R-I, P-R-I-P, R-I-P-P, I-P-P-R, P-R-P-I, R-P-1-P, P-I-P-R, 1-P-R-P, P-P-I-
R, P-I-R-P,
1-R-P-P and R-P-P-I; and wherein at the most four of X1 - X5 are identical; or
the amino acid
sequence of anyone of SEQ ID NO:1 to SEQ ID NO:319 as well as genetic
manipulation of
the DNA encoding the polypeptides. The modification(s) can be replacement(s)
of the amino
acid side chain(s), substitution(s), deletion(s) and/or insertions(s) in or at
the amino acid(s) of
interest; or use of unnatural amino acids with similar characteristics in the
amino acid
sequence. In particular the modification(s) can be amidations, such as
amidation of the C-
terminus.
cDNA: The term "cDNA" when used in the present context, is intended to cover a
DNA
molecule which can be prepared by reverse transcription from a mature,
spliced, mRNA
molecule derived from a eukaryotic cell. cDNA lacks the intron sequences that
are usually
present in the corresponding genomic DNA. The initial, primary RNA transcript
is a precursor
to mRNA and it goes through a series of processing events before appearing as
mature
spliced mRNA. These events include the removal of intron sequences by a
process called
splicing. When cDNA is derived from mRNA it therefore lacks intron sequences.
Nucleic acid construct: When used herein, the term "nucleic acid construct"
means a
nucleic acid molecule, either single- or double-stranded, which is isolated
from a naturally
occurring gene or which has been modified to contain segments of nucleic acids
in a manner
that would not otherwise exist in nature. The term nucleic acid construct is
synonymous with
the term "expression cassette" when the nucleic acid construct contains the
control
sequences required for expression of a coding sequence of the present
invention.
Control sequence: The term "control sequences" is defined herein to include
all
components, which are necessary or advantageous for the expression of a
polypeptide of the
present invention. Each control sequence may be native or foreign to the
nucleotide sequence
encoding the polypeptide. Such control sequences include, but are not limited
to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal peptide
sequence, and
transcription terminator. At a minimum, the control sequences include a
promoter, and
transcriptional and translational stop signals. The control sequences may be
provided with
linkers for the purpose of introducing specific restriction sites facilitating
ligation of the control
sequences with the coding region of the nucleotide sequence encoding a
polypeptide.
Operably linked: The term "operably linked" is defined herein as a
configuration in
which a control sequence is appropriately placed at a position relative to the
coding sequence
of the DNA sequence such that the control sequence directs the expression of a
polypeptide.
Coding sequence: When used herein the term "coding sequence" is intended to
cover
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a nucleotide sequence, which directly specifies the amino acid sequence of its
protein
product. The boundaries of the coding sequence are generally determined by an
open
reading frame, which usually begins with the ATG start codon. The coding
sequence typically
include DNA, cDNA, and recombinant nucleotide sequences.
Expression: In the present context, the term "expression" includes any step
involved in
the production of the polypeptide including, but not limited to,
transcription, post-
transcriptional modification, translation, and post-translational
modification. Preferably
expression also comprise secretion of the polypeptide.
Expression vector: In the present context, the term "expression vector" covers
a DNA
molecule, linear or circular, that comprises a segment encoding a polypeptide
of the invention,
and which is operably linked to additional segments that provide for its
transcription.
Host cell. The term "host cell", as used herein, includes any cell type which
is
susceptible to transformation with a nucleic acid construct.
The terms "polynucleotide probe", "hybridization" as well as the various
stringency
conditions are defined in the section titled "Polypeptides Having
Antimicrobial Activity".
DETAILED DESCRIPTION
Polypeptides Having Antimicrobial Activity
In a first aspect, the present invention relates to polypeptides having
antimicrobial
activity and where the polypeptides comprise, preferably consist of the amino
acid sequence
X1-X2-X3-X4-X5; wherein X1, X2, X3, X4 and X5 independently are P-P-R-Z1, P-R-
Z2-P, R-Z3-P-
P, Z4-P-P-R, P-R-P-Z5, R-P-Z6-P, P-Z7-P-R, Z$-P-R-P, P-P-Z9-R, P-Z10-R-P, Z11-
R-P-P or R-P-
P-Z12; wherein Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z3, Z9, Z1o, Z11 and Z12
independently are F, L, V or I.
The invention also relates to polypeptides having antimicrobial activity and
where the
polypeptides comprise, preferably consist of the amino acid sequence X1-X2-X3-
X4-X5;
wherein each of X1, X2, X3, X4 and X5 is selected from the group of amino acid
sequences:
P-P-R-F, P-R-F-P, R-F-P-P, F-P-P-R, P-R-P-F, R-P-F-P, P-F-P-R, F-P-R-P, P-P-F-
R,
P-F-R-P, F-R-P-P, R-P-P-F, P-P-R-L, P-R-L-P, R-L-P-P, L-P-P-R, P-R-P-L, R-P-L-
P,
P-L-P-R, L-P-R-P, P-P-L-R, P-L-R-P, L-R-P-P, R-P-P-L, P-P-R-V, P-R-V-P, R-V-P-
P,
V-P-P-R, P-R-P-V, R-P-V-P, P-V-P-R, V-P-R-P, P-P-V-R, P-V-R-P, V-R-P-P, R-P-P-
V,
P-P-R-I, P-R-I-P, R-I-P-P, I-P-P-R, P-R-P-I, R-P-I-P, P-I-P-R, I-P-R-P, P-P-I-
R, P-I-R-P,
I-R-P-P and R-P-P-I.
In an embodiment X1, X2, X3, X4 and X5 are selected from the group of amino
acid
sequences consisting of:
P-P-R-Z1i P-R-Z2-P, R-Z3-P-P, Z4-P-P-R, P-R-P-Z5, R-P-Z6-P, P-Z7-P-R, Z8-P-R-
P, P-P-Z9-R,
P-Z1o-R-P, Z11-R-P-P and R-P-P-Z12; wherein X1 - X5 are identical, except for
the Z1 - Z12
amino acids; wherein Z1 - Z12 are selected from the group of amino acids
consisting of F, L, V
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and I.
Preferably at the most four of X1 - X5 are identical; more preferably at the
most three,
even more preferably at the most two of X1 - X5 are identical; most preferably
none of X1 - X5
are identical.
In another embodiment X1 = X2 = X3 = X4 = X5 are P-P-R-Z1, P-R-Z2-P, R-Z3-P-P,
Z4-P-
P-R, P-R-P-Z5, R-P-Z6-P, P-Z7-P-R, Z8-P-R-P, P-P-Z9-R, P-Z10-R-P, Z11-R-P-P or
R-P-P-Z12;
except that Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11 and Z12 independently
of X1, X2, X3, X4 and
X5 are F, L, V or I.
In another embodiment the present invention relates to polypeptides having
antimicrobial activity and where the polypeptides comprises, preferably
consists of the amino
acid sequence X1-X2-X3-X4-X5-X6i wherein X1, X2, X3, X4, X5 and X6
independently are P-P-R-
Z1, P-R-Z2-P, R-Z3-P-P, Z4-P-P-R, P-R-P-Z5, R-P-Z6-P, P-Z7-P-R, Z8-P-R-P, P-P-
Z9-R, P-Z1o-
R-P, Z11-R-P-P or R-P-P-Z12; wherein Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z1o,
Z11 and Z12
independently are F, L, V or I.
In another embodiment the invention relates to polypeptides having
antimicrobial
activity and where the polypeptides comprise, preferably consist of the amino
acid sequence
X1-X2-X3-X4-X5-X6; wherein each of X1, X2, X3, X4, X5 and X6 is selected from
the group of
amino acid sequences:
P-P-R-F, P-R-F-P, R-F-P-P, F-P-P-R, P-R-P-F, R-P-F-P, P-F-P-R, F-P-R-P, P-P-F-
R,
P-F-R-P, F-R-P-P, R-P-P-F, P-P-R-L, P-R-L-P, R-L-P-P, L-P-P-R, P-R-P-L, R-P-L-
P,
P-L-P-R, L-P-R-P, P-P-L-R, P-L-R-P, L-R-P-P, R-P-P-L, P-P-R-V, P-R-V-P, R-V-P-
P,
V-P-P-R, P-R-P-V, R-P-V-P, P-V-P-R, V-P-R-P, P-P-V-R, P-V-R-P, V-R-P-P, R-P-P-
V,
P-P-R-I, P-R-l-P, R-l-P-P, I-P-P-R, P-R-P-I, R-P-l-P, P-l-P-R, I-P-R-P, P-P-l-
R, P-l-R-P,
I-R-P-P and R-P-P-I.
In another embodiment XI, X2, X3, X4, X5 and X6 are selected from the group of
amino
acid sequences consisting of:
P-P-R-Z1a P-R-Z2-P, R-Z3-P-P, Z4-P-P-R, P-R-P-Z5, R-P-Z6-P, P-Z7-P-R, Z6-P-R-
P, P-P-Z9-R,
P-Z10-R-P, Z11-R-P-P and R-P-P-Z12; wherein X1 - X6 are identical, except for
the Z1 - Z12
amino acids; wherein Z1 - Z12 are selected from the group of amino acids
consisting of F, L, V
and I.
Preferably at the most four of X1 - X6 are identical; more preferably at the
most three,
even more preferably at the most two of X1 - X6 are identical; most preferably
none of X1 - X6
are identical.
In another embodiment X1 = X2 = X3 = X4 = X5 = X6 are P-P-R-Z1, P-R-Z2-P, R-Z3-
P-P,
Z4-P-P-R, P-R-P-Z5, R-P-Z6-P, P-Z7-P-R, Z5-P-R-P, P-P-Z9-R, P-Z10-R-P, Z11-R-P-
P or R-P-P-
Z12; except that Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z1o, Z11 and Z12
independently of X1, X2, X3, X4,
X5 and X6 are F, L,VorI.
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In a preferred embodiment, the present invention relates to polypeptides
having
antimicrobial activity and where the polypeptides comprise, preferably consist
of the amino
acid sequence of anyone of SEQ ID NO:1 to SEQ ID NO:319.
In yet another embodiment, the amino acid sequence differs by at the most five
amino
acids (e.g. by five amino acids), such as by at the most four amino acids
(e.g. by four amino
acids), e.g. by at the most three amino acids (e.g. by three amino acids),
particularly by at the
most two amino acids (e.g. by two amino acids), such as by one amino acid from
the amino
acid sequence X1-X2-X3-X4-X5; wherein each of X1, X2, X3, X4 and X5 are
selected from:
P-P-R-F, P-R-F-P, R-F-P-P, F-P-P-R, P-R-P-F, R-P-F-P, P-F-P-R, F-P-R-P, P-P-F-
R,
P-F-R-P, F-R-P-P, R-P-P-F, P-P-R-L, P-R-L-P, R-L-P-P, L-P-P-R, P-R-P-L, R-P-L-
P,
P-L-P-R, L-P-R-P, P-P-L-R, P-L-R-P, L-R-P-P, R-P-P-L, P-P-R-V, P-R-V-P, R-V-P-
P,
V-P-P-R, P-R-P-V, R-P-V-P, P-V-P-R, V-P-R-P, P-P-V-R, P-V-R-P, V-R-P-P, R-P-P-
V,
P-P-R-I, P-R-I-P, R-I-P-P, I-P-P-R, P-R-P-I, R-P-l-P, P-I-P-R, I-P-R-P, P-P-I-
R, P-l-R-P,
I-R-P-P and R-P-P-I;
or the amino acid sequence X1-X2-X3-X4-X5-X6; wherein each of X1, X2, X3, X4,
X5 and X6 are
selected from:
P-P-R-F, P-R-F-P, R-F-P-P, F-P-P-R, P-R-P-F, R-P-F-P, P-F-P-R, F-P-R-P, P-P-F-
R,
P-F-R-P, F-R-P-P, R-P-P-F, P-P-R-L, P-R-L-P, R-L-P-P, L-P-P-R, P-R-P-L, R-P-L-
P,
P-L-P-R, L-P-R-P, P-P-L-R, P-L-R-P, L-R-P-P, R-P-P-L, P-P-R-V, P-R-V-P, R-V-P-
P,
V-P-P-R, P-R-P-V, R-P-V-P, P-V-P-R, V-P-R-P, P-P-V-R, P-V-R-P, V-R-P-P, R-P-P-
V,
P-P-R-I, P-R-I-P, R-I-P-P, I-P-P-R, P-R-P-I, R-P-I-P, P-I-P-R, I-P-R-P, P-P-I-
R, P-I-R-P,
I-R-P-P and R-P-P-I;
or the amino acid sequence of anyone of SEQ ID NO:1 to SEQ ID NO:319.
Preferably, the polypeptides of the present invention comprise the amino acid
sequence X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6 or the amino acid sequence of
anyone of SEQ
ID NO:1 to SEQ ID NO:319; or a fragment thereof that has antimicrobial
activity. In another
preferred embodiment, the polypeptides consist of the amino acid sequence X1-
X2-X3-X4-X5 or
X1-X2-X3-X4-X5-X6 or the amino acid sequence of anyone of SEQ ID NO:1 to SEQ
ID NO:319.
The amino acids making up the polypeptides of the invention may independently
be
selected from D or L forms.
The polypeptides of the invention may consist of from 20 to 500 amino acids,
preferably from 20 to 400 amino acids, more preferably from 20 to 300 amino
acids, even
more preferably from 20 to 200 amino acids (such as from 20 to 150 amino
acids), most
preferably from 20 to 100 amino acids and in particular from 20 to 50 amino
acids.
The amino acid sequence X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6 may be directly
preceded by an amino acid sequence of from 3 to 10 amino acids (such as from 3
to 5 amino
acids) of which at least two amino acids are positively charged at neutral pH
(such as
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arginine, lysine or histidine). Preferably the amino acid sequence X1-X2-X3-X4-
X5 or X1-X2-X3-
X4-X5-X6 may be directly preceded by the amino acid sequence R-X7-R; wherein
X7 is R, F, L,
V or I. The term "directly preceded by" means that the amino acid sequence is
attached to the
N-terminal of the amino acid sequence X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6.
The polypeptide of the invention may be an artificial variant which comprises,
preferably consists of, an amino acid sequence that has at the most three,
e.g. at the most
two, such as at the most one, substitutions, deletions and/or insertions of
amino acids as
compared to the amino acid sequences X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6; or
the amino
acid sequence of anyone of SEQ ID NO:1 to SEQ ID NO:319.
Such artificial variants may be constructed by standard techniques known in
the art,
such as by site-directed/random mutagenesis of the polypeptide comprising the
amino acid
sequence X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6 or the amino acid sequence of
anyone of SEQ
ID NO:1 to SEQ ID NO:319. In one embodiment of the invention, amino acid
changes are of a
minor nature, that is conservative amino acid substitutions that do not
significantly affect the
folding and/or activity of the protein; small deletions, typically of one to
about 5 amino acids;
small amino- or carboxyl-terminal extensions, such as an amino-terminal
methionine residue;
a small linker peptide of up to about 10-25 residues; or a small extension
that facilitates
purification by changing net charge or another function, such as a poly-
histidine tract, an
antigenic epitope or a binding domain.
Examples of conservative substitutions are within the group of basic amino
acids
(arginine, lysine and histidine), acidic amino acids (glutamic acid and
aspartic acid), polar
amino acids (glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine, valine
and methionine), aromatic amino acids (phenylalanine, tryptophan and
tyrosine), and small
amino acids (glycine, alanine, serine and threonine). Amino acid substitutions
which do not
generally alter the specific activity are known in the art and are described,
for example, by H.
Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The
most
commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,
Ala/Thr,
Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, LeuNal,
Ala/Glu, and
Asp/Gly as well as these in reverse.
In an interesting embodiment of the invention, the amino acid changes are of
such a
nature that the physico-chemical properties of the polypeptides are altered.
For example,
amino acid changes may be performed, which improve the thermal stability of
the polypeptide,
which alter the substrate specificity, which changes the pH optimum, and the
like.
N-terminal extension
An N-terminal extension of the polypeptides of the invention may suitably
consist of
from I to 50 amino acids, preferably 2-20 amino acids, especially 3-15 amino
acids. In one
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embodiment N-terminal peptide extension does not contain an Arg (R). In
another
embodiment the N-terminal extension comprises a kex2 or kex2-like cleavage
site as will be
defined further below. In a preferred embodiment the N-terminal extension is a
peptide,
comprising at least two Glu (E) and/or Asp (D) amino acid residues, such as an
N-terminal
extension comprising one of the following sequences: EAE, EE, DE and DD.
Kex2 sites
Kex2 sites (see, e.g., Methods in Enzymology Vol 185, ed. D. Goeddel, Academic
Press Inc. (1990), San Diego, CA, "Gene Expression Technology") and kex2-like
sites are di-
basic recognition sites (i.e., cleavage sites) found between the pro-peptide
encoding region
and the mature region of some proteins.
Insertion of a kex2 site or a kex2-like site have in certain cases been shown
to
improve correct endopeptidase processing at the pro-peptide cleavage site
resulting in
increased protein secretion levels.
In the context of the invention insertion of a kex2 or kex2-like site result
in the
possibility to obtain cleavage at a certain position in the N-terminal
extension resulting in an
antimicrobial polypeptide being extended in comparison to the mature
polypeptide shown as
the amino acid sequences X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6; or the amino
acid sequence
of anyone of SEQ ID NO:1 to SEQ ID NO:319.
Fused polypeptides
The polypeptides of the present invention also include fused polypeptides or
cleavable
fusion polypeptides in which another polypeptide is fused at the N-terminus or
the C-terminus
of the polypeptide of the invention or a fragment thereof. A fused polypeptide
is produced by
fusing a nucleotide sequence (or a portion thereof) encoding another
polypeptide to a
nucleotide sequence (or a portion thereof) of the present invention.
Techniques for producing
fusion polypeptides are known in the art, and include ligating the coding
sequences encoding
the polypeptides so that they are in frame and that expression of the fused
polypeptide is
under control of the same promoter(s) and terminator.
Polynucleotides and Nucleotide Sequences
The present invention also relates to polynucleotides having a nucleotide
sequence
which encodes for a polypeptide of the invention. In particular, the present
invention relates to
polynucleotides consisting of a nucleotide sequence which encodes for a
polypeptide of the
invention. Due to the degeneracy of the genetic code, the skilled person will
easily recognize
that several nucleotide sequences encoding each of the polypeptides of the
invention may be
prepared. It is well known in the art which nucleotides make up codons
encoding the amino
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acids of the polypeptides of the invention.
The present invention also relates to polynucleotides which encode fragments
of the
amino acid sequences X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6; or the amino acid
sequence of
anyone of SEQ ID NO:1 to SEQ ID NO:319 that have antimicrobial activity. A
subsequence of
the polynucleotides is a nucleotide sequence wherein one or more nucleotides
from the 5'
and/or 3' end have been deleted.
The nucleotide sequence may be obtained by standard cloning procedures used in
genetic engineering to relocate the nucleotide sequence from one location to a
different site
where it will be reproduced. The cloning procedures may involve excision and
isolation of a
desired fragment comprising the nucleotide sequence encoding the polypeptide,
insertion of
the fragment into a vector molecule, and incorporation of the recombinant
vector into a host
cell where multiple copies or clones of the nucleotide sequence will be
replicated. The
nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic
origin, or any
combinations thereof.
Modification of a nucleotide sequence encoding a polypeptide of the present
invention
may be necessary for the synthesis of a polypeptide, which comprises an amino
acid
sequence that has at least one substitution, deletion and/or insertion as
compared to the
amino acid sequences X1-X2-X3-X4-X5 or X1-X2-X3-X4-X5-X6; or the amino acid
sequence of
anyone of SEQ ID NO:1 to SEQ ID NO:319. These artificial variants may differ
in some
engineered way from the polypeptide isolated from its native source, e.g.,
variants that differ
in specific activity, thermostability, pH optimum, or the like.
It will be apparent to those skilled in the art that such modifications can be
made
outside the regions critical to the function of the molecule and still result
in an active
polypeptide. Amino acid residues essential to the activity of the polypeptide
encoded by the
nucleotide sequence of the invention, and therefore preferably not subject to
modification,
such as substitution, may be identified according to procedures known in the
art, such as site-
directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham
and Wells,
1989, Science 244: 1081-1085). In the latter technique, mutations are
introduced at every
positively charged residue in the molecule, and the resultant mutant molecules
are tested for
antimicrobial activity to identify amino acid residues that are critical to
the activity of the
molecule. Sites of substrate-enzyme interaction can also be determined by
analysis of the
three-dimensional structure as determined by such techniques as nuclear
magnetic
resonance analysis, crystallography or photoaffinity labelling (see, e.g., de
Vos et al., 1992,
Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224:
899-904;
Wlodaver at al., 1992, FEBS Letters 309: 59-64).
Moreover, a nucleotide sequence encoding a polypeptide of the present
invention may
be modified by introduction of nucleotide substitutions which do not give rise
to another amino
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acid sequence of the polypeptide encoded by the nucleotide sequence, but which
correspond
to the codon usage of the host organism intended for production of the enzyme.
The introduction of a mutation into the nucleotide sequence to exchange one
nucleotide for another nucleotide may be accomplished by site-directed
mutagenesis using
any of the methods known in the art. Particularly useful is the procedure,
which utilizes a
supercoiled, double stranded DNA vector with an insert of interest and two
synthetic primers
containing the desired mutation. The oligonucleotide primers, each
complementary to
opposite strands of the vector, extend during temperature cycling by means of
Pfu DNA
polymerase. On incorporation of the primers, a mutated plasmid containing
staggered nicks is
generated. Following temperature cycling, the product is treated with Dpnl
which is specific for
methylated and hemimethylated DNA to digest the parental DNA template and to
select for
mutation-containing synthesized DNA. Other procedures known in the art may
also be used.
For a general description of nucleotide substitution, see, e.g., Ford et al.,
1991, Protein
Expression and Purification 2: 95-107.
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising a
nucleotide
sequence of the present invention operably linked to one or more control
sequences that
direct the expression of the coding sequence in a suitable host cell under
conditions
compatible with the control sequences.
A nucleotide sequence encoding a polypeptide of the present invention may be
manipulated in a variety of ways to provide for expression of the polypeptide.
Manipulation of
the nucleotide sequence prior to its insertion into a vector may be desirable
or necessary
depending on the expression vector. The techniques for modifying nucleotide
sequences
utilizing recombinant DNA methods are well known in the art.
The control sequence may be an appropriate promoter sequence, a nucleotide
sequence which is recognized by a host cell for expression of the nucleotide
sequence. The
promoter sequence contains transcriptional control sequences, which mediate
the expression
of the polypeptide. The promoter may be any nucleotide sequence which shows
transcriptional activity in the host cell of choice including mutant,
truncated, and hybrid
promoters, and may be obtained from genes encoding extracellular or
intracellular
polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the nucleic
acid
constructs of the present invention, especially in a bacterial host cell, are
the promoters
obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene
(dagA), Bacillus
subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene
(amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliguefaciens
alpha-
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amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP),
Bacillus subtilis xylA
and xy1B genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al.,
1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as
the tac
promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences
USA 80:
21-25). Further promoters are described in "Useful proteins from recombinant
bacteria" in
Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.
Examples of suitable promoters for directing the transcription of the nucleic
acid
constructs of the present invention in a filamentous fungal host cell are
promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic
proteinase,
Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-
amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor
miehei lipase,
Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase,
Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease
(WO
96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the
genes for
Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose
phosphate isomerase),
and mutant, truncated, and hybrid promoters thereof.
In a yeast host, useful promoters are obtained from the genes for
Saccharomyces
cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1),
Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate
dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate
kinase.
Other useful promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8:
423-488.
The control sequence may also be a suitable transcription terminator sequence,
a
sequence recognized by a host cell to terminate transcription. The terminator
sequence is
operably linked to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any
terminator which is functional in the host cell of choice may be used in the
present invention.
Preferred terminators for filamentous fungal host cells are obtained from the
genes for
Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus
nidulans
anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-
like protease.
Preferred terminators for yeast host cells are obtained from the genes for
Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CM),
and
Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other
useful
terminators for yeast host cells are described by Romanos et al., 1992, supra.
The control sequence may also be a suitable leader sequence, a nontranslated
region
of an mRNA which is important for translation by the host cell. The leader
sequence is
operably linked to the 5' terminus of the nucleotide sequence encoding the
polypeptide. Any
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leader sequence that is functional in the host cell of choice may be used in
the present
invention.
Preferred leaders for filamentous fungal host cells are obtained from the
genes for
Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate
isomerase.
Suitable leaders for yeast host cells are obtained from the genes for
Saccharomyces
cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate
kinase,
Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence
operably
linked to the 3' terminus of the nucleotide sequence and which, when
transcribed, is
recognized by the host cell as a signal to add polyadenosine residues to
transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell of choice
may be used in the
present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are
obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger
glucoamylase,
Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like
protease, and
Aspergillus niger alpha-glucosidase.
Useful polyadenylation sequences for yeast host cells are described by Guo and
Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.
The control sequence may also be a signal peptide coding region that codes for
an
amino acid sequence linked to the amino terminus of a polypeptide and directs
the encoded
polypeptide into the cell's secretory pathway. The 5' end of the coding
sequence of the
nucleotide sequence may inherently contain a signal peptide coding region
naturally linked in
translation reading frame with the segment of the coding region which encodes
the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may contain a
signal peptide
coding region which is foreign to the coding sequence. The foreign signal
peptide coding
region may be required where the coding sequence does not naturally contain a
signal
peptide coding region. Alternatively, the foreign signal peptide coding region
may simply
replace the natural signal peptide coding region in order to enhance secretion
of the
polypeptide. However, any signal peptide coding region which directs the
expressed
polypeptide into the secretory pathway of a host cell of choice may be used in
the present
invention.
Effective signal peptide coding regions for bacterial host cells are the
signal peptide
coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic
amylase, Bacillus
stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus
licheniformis beta-
lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM),
and Bacillus
subtilis prsA. Further signal peptides are described by Simonen and Palva,
1993,
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Microbiological Reviews 57: 109-137.
Effective signal peptide coding regions for filamentous fungal host cells are
the signal
peptide coding regions obtained from the genes for Aspergillus oryzae TAKA
amylase,
Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei
aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa
lipase.
Useful signal peptides for yeast host cells are obtained from the genes for
Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
Other
useful signal peptide coding regions are described by Romanos et al., 1992,
supra.
The control sequence may also be a propeptide coding region that codes for an
amino
acid sequence positioned at the amino terminus of a polypeptide. The resultant
polypeptide is
known as a proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is
generally inactive and can be converted to a mature active polypeptide by
catalytic or
autocatalytic cleavage of the propeptide from the propolypeptide. The
propeptide coding
region may be obtained from the genes for Bacillus subtilis alkaline protease
(aprE), Bacillus
subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor,
Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).
Where both signal peptide and propeptide regions are present at the amino
terminus
of a polypeptide, the propeptide region is positioned next to the amino
terminus of a
polypeptide and the signal peptide region is positioned next to the amino
terminus of the
propeptide region.
It may also be desirable to add regulatory sequences which allow the
regulation of the
expression of the polypeptide relative to the growth of the host cell.
Examples of regulatory
systems are those which cause the expression of the gene to be turned on or
off in response
to a chemical or physical stimulus, including the presence of a regulatory
compound.
Regulatory systems in prokaryotic systems include the lac, tac, and trp
operator systems. In
yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the
TAKA alpha-
amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus
oryzae
glucoamylase promoter may be used as regulatory sequences. Other examples of
regulatory
sequences are those which allow for gene amplification. In eukaryotic systems,
these include
the dihydrofolate reductase gene which is amplified in the presence of
methotrexate, and the
metallothionein genes which are amplified with heavy metals. In these cases,
the nucleotide
sequence encoding the polypeptide would be operably linked with the regulatory
sequence.
Expression Vectors
The present invention also relates to recombinant expression vectors
comprising the
nucleic acid construct of the invention. The various nucleotide and control
sequences
described above may be joined together to produce a recombinant expression
vector which
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may include one or more convenient restriction sites to allow for insertion or
substitution of the
nucleotide sequence encoding the polypeptide at such sites. Alternatively, the
nucleotide
sequence of the present invention may be expressed by inserting the nucleotide
sequence or
a nucleic acid construct comprising the sequence into an appropriate vector
for expression. In
creating the expression vector, the coding sequence is located in the vector
so that the coding
sequence is operably linked with the appropriate control sequences for
expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus)
which
can be conveniently subjected to recombinant DNA procedures and can bring
about the
expression of the nucleotide sequence. The choice of the vector will typically
depend on the
compatibility of the vector with the host cell into which the vector is to be
introduced. The
vectors may be linear or closed circular plasmids.
The vector may be an autonomously replicating vector, i.e., a vector which
exists as
an extrachromosomal entity, the replication of which is independent of
chromosomal
replication, e.g., a plasmid, an extrachromosomal element, a minichromosome,
or an artificial
chromosome.
The vector may contain any means for assuring self-replication. Alternatively,
the
vector may be one which, when introduced into the host cell, is integrated
into the genome
and replicated together with the chromosome(s) into which it has been
integrated.
Furthermore, a single vector or plasmid or two or more vectors or plasmids
which together
contain the total DNA to be introduced into the genome of the host cell, or a
transposon may
be used.
The vectors of the present invention preferably contain one or more selectable
markers which permit easy selection of transformed cells. A selectable marker
is a gene the
product of which provides for biocide or viral resistance, resistance to heavy
metals,
prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are the dal genes from Bacillus
subtilis or
Bacillus licheniformis, or markers which confer antibiotic resistance such as
ampicillin,
kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for
yeast host cells
are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use
in a
filamentous fungal host cell include, but are not limited to, amdS
(acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase),
hygB (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate
decarboxylase),
sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as
equivalents thereof.
Preferred for use in an Aspergillus cell are the amdS and pyrG genes of
Aspergillus
nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
The vectors of the present invention preferably contain an element(s) that
permits
stable integration of the vector into the host cell's genome or autonomous
replication of the
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vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the
nucleotide
sequence encoding the polypeptide or any other element of the vector for
stable integration of
the vector into the genome by homologous or nonhomologous recombination.
Alternatively,
the vector may contain additional nucleotide sequences for directing
integration by
homologous recombination into the genome of the host cell. The additional
nucleotide
sequences enable the vector to be integrated into the host cell genome at a
precise
location(s) in the chromosome(s). To increase the likelihood of integration at
a precise
location, the integrational elements should preferably contain a sufficient
number of
nucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base
pairs, and most
preferably 800 to 1,500 base pairs, which are highly homologous with the
corresponding
target sequence to enhance the probability of homologous recombination. The
integrational
elements may be any sequence that is homologous with the target sequence in
the genome
of the host cell. Furthermore, the integrational elements may be non-encoding
or encoding
nucleotide sequences. On the other hand, the vector may be integrated into the
genome of
the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of
replication
enabling the vector to replicate autonomously in the host cell in question.
Examples of
bacterial origins of replication are the origins of replication of plasmids
pBR322, pUC19,
pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194,
pTA1060,
and pAM131 permitting replication in Bacillus. Examples of origins of
replication for use in a
yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1
and CEN3, and the combination of ARS4 and CEN6. The origin of replication may
be one
having a mutation which makes its functioning temperature-sensitive in the
host cell (see,
e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:
1433).
More than one copy of a nucleotide sequence of the present invention may be
inserted
into the host cell to increase production of the gene product. An increase in
the copy number
of the nucleotide sequence can be obtained by integrating at least one
additional copy of the
sequence into the host cell genome or by including an amplifiable selectable
marker gene
with the nucleotide sequence where cells containing amplified copies of the
selectable marker
gene, and thereby additional copies of the nucleotide sequence, can be
selected for by
cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the
recombinant expression vectors of the present invention are well known to one
skilled in the
art (see, e.g., Sambrook et al., 1989, supra).
Host Cells
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The present invention also relates to recombinant a host cell comprising the
nucleic
acid construct of the invention, which are advantageously used in the
recombinant production
of the polypeptides. A vector comprising a nucleotide sequence of the present
invention is
introduced into a host cell so that the vector is maintained as a chromosomal
integrant or as a
self-replicating extra-chromosomal vector as described earlier.
The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-
unicellular microorganism, e.g., a eukaryote.
Useful unicellular cells are bacterial cells such as gram positive bacteria
including, but
not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus
brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus
lautus, Bacillus lentus,
Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Bacillus subtilis, and
Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or
Streptomyces
murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a
preferred
embodiment, the bacterial host cell is a Bacillus lentus, Bacillus
licheniformis, Bacillus
stearothermophilus, or Bacillus subtilis cell. In another preferred
embodiment, the Bacillus cell
is an alkalophilic Bacillus.
The introduction of a vector into a bacterial host cell may, for instance, be
effected by
protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General
Genetics
168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961,
Journal of
Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of
Molecular
Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988,
Biotechniques
6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169:
5771-5278).
The host cell may be a eukaryote, such as a mammalian, insect, plant, or
fungal cell.
In a preferred embodiment, the host cell is a fungal cell. "Fungi" as used
herein
includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota
(as
defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition,
1995, CAB International, University Press, Cambridge, UK) as well as the
Oomycota (as cited
in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi
(Hawksworth et al.,
1995, supra).
In a more preferred embodiment, the fungal host cell is a yeast cell. "Yeast"
as used
herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous
yeast, and
yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the
classification of yeast may
change in the future, for the purposes of this invention, yeast shall be
defined as described in
Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport,
R.R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
In an even more preferred embodiment, the yeast host cell is a Candida,
Hansenula,
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Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
In a most preferred embodiment, the yeast host cell is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,
Saccharomyces
douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces
oviformis
cell. In another most preferred embodiment, the yeast host cell is a
Kluyveromyces lactis cell.
In another most preferred embodiment, the yeast host cell is a Yarrowia
lipolytica cell.
In another more preferred embodiment, the fungal host cell is a filamentous
fungal
cell. "Filamentous fungi" include all filamentous forms of the subdivision
Eumycota and
Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi
are
characterized by a mycelial wall composed of chitin, cellulose, glucan,
chitosan, mannan, and
other complex polysaccharides. Vegetative growth is by hyphal elongation and
carbon
catabolism is obligately aerobic. In contrast, vegetative growth by yeasts
such as
Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon
catabolism may
be fermentative.
In an even more preferred embodiment, the filamentous fungal host cell is a
cell of a
species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola,
Mucor,
Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or
Trichoderma.
In a most preferred embodiment, the filamentous fungal host cell is an
Aspergillus
awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger or
Aspergillus oryzae cell. In another most preferred embodiment, the filamentous
fungal host
cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium
culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium
negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium
sulphureum,
Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In
an even most
preferred embodiment, the filamentous fungal parent cell is a Fusarium
venenatum
(Nirenberg sp. nov.) cell. In another most preferred embodiment, the
filamentous fungal host
cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora
thermophila,
Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma
harzianum,
3o Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma
viride cell.
Fungal cells may be transformed by a process involving protoplast formation,
transformation of the protoplasts, and regeneration of the cell wall in a
manner known per se.
Suitable procedures for transformation of Aspergillus host cells are described
in EP 238 023
and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA
81: 1470-
1474. Suitable methods for transforming Fusarium species are described by
Malardier at aL,
1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the
procedures
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WO 2005/026204 PCT/DK2004/000606
described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors,
Guide to Yeast
Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187,
Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:
163; and
Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:
1920.
Methods of Production
The present invention also relates to methods for producing a polypeptide of
the
present invention comprising (a) cultivating a host cell under conditions
conducive for
production of the polypeptide; and (b) recovering the polypeptide.
In the production methods of the present invention, the cells are cultivated
in a nutrient
medium suitable for production of the polypeptide using methods known in the
art. For
example, the cell may be cultivated by shake flask cultivation, small-scale or
large-scale
fermentation (including continuous, batch, fed-batch, or solid state
fermentations) in
laboratory or industrial fermentors performed in a suitable medium and under
conditions
allowing the polypeptide to be expressed and/or isolated. The cultivation
takes place in a
suitable nutrient medium comprising carbon and nitrogen sources and inorganic
salts, using
procedures known in the art. Suitable media are available from commercial
suppliers or may
be prepared according to published compositions (e.g., in catalogues of the
American Type
Culture Collection). If the polypeptide is secreted into the nutrient medium,
the polypeptide
can be recovered directly from the medium. If the polypeptide is not secreted,
it can be
recovered from cell lysates.
The polypeptides may be detected using methods known in the art that are
specific for
the polypeptides. These detection methods may include use of specific
antibodies, formation
of an enzyme product, or disappearance of an enzyme substrate. For example, an
enzyme
assay may be used to determine the activity of the polypeptide as described
herein.
The resulting polypeptide may be recovered by methods known in the art. For
example, the polypeptide may be recovered from the nutrient medium by
conventional
procedures including, but not limited to, centrifugation, filtration,
extraction, spray-drying,
evaporation, or precipitation.
The polypeptides of the present invention may be purified by a variety of
procedures
known in the art including, but not limited to, chromatography (e.g., ion
exchange, affinity,
hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures
(e.g.,
preparative isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation),
SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and
Lars Ryden,
editors, VCH Publishers, New York, 1989).
Plants
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The present invention also relates to a transgenic plant, plant part, or plant
cell which
has been transformed with a nucleotide sequence encoding a polypeptide having
antimicrobial activity of the present invention so as to express and produce
the polypeptide in
recoverable quantities. The polypeptide may be recovered from the plant or
plant part.
Alternatively, the plant or plant part containing the recombinant polypeptide
may be used as
such for improving the quality of a food or feed, e.g., improving nutritional
value, palatability,
and rheological properties, or to destroy an antinutritive factor. The
recovered polypeptide,
plant or plant part may also be used to improve or alter digestive flora in
animals and
livestock.
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a
monocot). Examples of monocot plants are grasses, such as meadow grass (blue
grass,
Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis,
and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
Examples of dicot plants are tobacco, potato, sugar beet, legumes, such as
lupins,
pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as
cauliflower,
rape seed, and the closely related model organism Arabidopsis thaliana.
Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and
tubers. Also
specific plant tissues, such as chioroplast, apoplast, mitochondria, vacuole,
peroxisomes, and
cytoplasm are considered to be a plant part. Furthermore, any plant cell,
whatever the tissue
origin, is considered to be a plant part.
Also included within the scope of the present invention are the progeny of
such plants,
plant parts and plant cells.
The transgenic plant or plant cell expressing a polypeptide of the present
invention
may be constructed in accordance with methods known in the art. Briefly, the
plant or plant
cell is constructed by incorporating one or more expression constructs
encoding a polypeptide
of the present invention into the plant host genome and propagating the
resulting modified
plant or plant cell into a transgenic plant or plant cell.
Conveniently, the expression construct is a nucleic acid construct which
comprises a
nucleotide sequence encoding a polypeptide of the present invention operably
linked with
appropriate regulatory sequences required for expression of the nucleotide
sequence in the
plant or plant part of choice. Furthermore, the expression construct may
comprise a
selectable marker useful for identifying host cells into which the expression
construct has
been integrated and DNA sequences necessary for introduction of the construct
into the plant
in question (the latter depends on the DNA introduction method to be used).
The choice of regulatory sequences, such as promoter and terminator sequences
and
optionally signal or transit sequences is determined, for example, on the
basis of when,
where, and how the polypeptide is desired to be expressed. For instance, the
expression of
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the gene encoding a polypeptide of the present invention may be constitutive
or inducible, or
may be developmental, stage or tissue specific, and the gene product may be
targeted to a
specific tissue or plant part such as seeds or leaves. Regulatory sequences
are, for example,
described by Tague et al., 1988, Plant Physiology 86: 506.
For constitutive expression, the 35S-CaMV promoter may be used (Franck et al.,
1980, Cell 21: 285-294). Organ-specific promoters may be, for example, a
promoter from
storage sink tissues such as seeds, potato tubers, and fruits (Edwards &
Coruzzi, 1990, Ann.
Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems
(Ito et al., 1994,
Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin,
prolamin, globulin,
or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39:
885-889), a
Vicia faba promoter from the legumin B4 and the unknown seed protein gene from
Vicia faba
(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), a promoter
from a seed oil
body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941), the
storage protein
napA promoter from Brassica napus, or any other seed specific promoter known
in the art,
e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf
specific
promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993,
Plant
Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene
promoter (Mitra
and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene
promoter from rice
(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound
inducible
promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular
Biology 22: 573-
588).
A promoter enhancer element may also be used to achieve higher expression of
the
enzyme in the plant. For instance, the promoter enhancer element may be an
intron which is
placed between the promoter and the nucleotide sequence encoding a polypeptide
of the
present invention. For instance, Xu et al., 1993, supra disclose the use of
the first intron of the
rice actin 1 gene to enhance expression.
The selectable marker gene and any other parts of the expression construct may
be
chosen from those available in the art.
The nucleic acid construct is incorporated into the plant genome according to
conventional techniques known in the art, including Agrobacterium-mediated
transformation,
virus-mediated transformation, microinjection, particle bombardment, biolistic
transformation,
and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,
Bio/Technology
8: 535; Shimamoto et al., 1989, Nature 338: 274).
Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of
choice
for generating transgenic dicots (for a review, see Hooykas and Schilperoort,
1992, Plant
Molecular Biology 19: 15-38). However it can also be used for transforming
monocots,
although other transformation methods are generally preferred for these
plants. Presently, the
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method of choice for generating transgenic monocots is particle bombardment
(microscopic
gold or tungsten particles coated with the transforming DNA) of embryonic
calli or developing
embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current
Opinion
Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An
alternative
method for transformation of monocots is based on protoplast transformation as
described by
Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.
Following transformation, the transformants having incorporated therein the
expression construct are selected and regenerated into whole plants according
to methods
well-known in the art.
The present invention also relates to methods for producing a polypeptide of
the
present invention comprising (a) cultivating a transgenic plant or a plant
cell comprising a
nucleotide sequence encoding a polypeptide having antimicrobial activity of
the present
invention under conditions conducive for production of the polypeptide; and
(b) recovering the
polypeptide.
Compositions
In a still further aspect, the present invention relates to compositions, such
as
pharmaceutical compositions, comprising an antimicrobial polypeptide of the
invention.
The composition may comprise a polypeptide of the invention as the major
polypeptide
component, e.g., a mono-component composition. Alternatively, the composition
may
comprise multiple enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase,
carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin
glycosyltransferase,
deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,
glucoamylase, alpha-
glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase,
mannosidase,
oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,
polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The compositions may further comprise another pharmaceutically active agent,
such
as an additional biocidal agent, such as another antimicrobial polypeptide
exhibiting
antimicrobial activity as defined above. The biocidal agent may be an
antibiotic, as known in
the art. Classes of antibiotics include penicillins, e.g. penicillin G,
penicillin V, methicillin,
oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in
combination with beta-lactamase
inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam,
etc.;
carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides;
lincomycins;
polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole;
spectinomycin;
trimethoprim; vancomycin; etc. The biocidal agent may also be an anti-mycotic
agent,
including polyenes, e.g. amphotericin B, nystatin; 5-flucosyn; and azoles,
e.g. miconazol,
ketoconazol, itraconazol and fluconazol.
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In an embodiment the biocidal agent is a non-enzymatic chemical agent. In
another
embodiment the biocidal agent is a non-polypeptide chemical agent.
The biocidal agent may be capable of reducing the number of living cells of
Escherichia coli (DSM 1576) or Bacillus subtilis (ATCC 6633) to 1/100 after 8
hours
(preferably after 4 hours, more preferably after 2 hours, most preferably
after 1 hour, and in
particular after 30 minutes) incubation at 20 C in an aqueous solution of
25%(w/w); preferably
in an aqueous solution of 10%(w/w); more preferably in an aqueous solution of
5%(w/w); even
more preferably in an aqueous solution of 1%(w/w); most preferably in an
aqueous solution of
0.5%(w/w); and in particular in an aqueous solution of 0.1 %(w/w) of the
biocidal agent.
The biocidal agent may also be capable of inhibiting the outgrowth of
Escherichia coli
(DSM 1576) or Bacillus subtilis (ATCC 6633) for 24 hours at 25 C in a
microbial growth
substrate, when added in a concentration of 1000 ppm; preferably when added in
a
concentration of 500 ppm; more preferably when added in a concentration of 250
ppm; even
more preferably when added in a concentration of 100 ppm; most preferably when
added in a
concentration of 50 ppm; and in particular when added in a concentration of 25
ppm.
The antimicrobial polypeptide of the invention and the biocidal agent of the
composition may be selected so that a synergistic antimicrobial effect is
obtained.
The compositions may comprise a suitable carrier material. The compositions
may
also comprise a suitable delivery vehicle capable of delivering the
antimicrobial polypeptides
of the invention to the desired locus when the compositions are used as a
medicament.
The compositions may be prepared in accordance with methods known in the art
and
may be in the form of a liquid or a dry composition. For instance, the
polypeptide composition
may be in the form of a granulate or a microgranulate. The polypeptide to be
included in the
composition may be stabilized in accordance with methods known in the art.
Examples are given below of preferred uses of the polypeptide compositions of
the
invention. The dosage of the polypeptide composition of the invention and
other conditions
under which the composition is used may be determined on the basis of methods
known in
the art.
Methods and Uses
The present invention also encompasses various uses of the antimicrobial
polypeptides of the invention. The antimicrobial polypeptides are typically
useful at any locus
subject to contamination by bacteria, fungi, yeast or algae. Typically, loci
are in aqueous
systems such as cooling water systems, laundry rinse water, oil systems such
as cutting oils,
lubricants, oil fields and the like, where microorganisms need to be killed or
where their
growth needs to be controlled. However, the present invention may also be used
in all
applications for which known antimicrobial compositions are useful, such as
protection of
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wood, latex, adhesive, glue, paper, cardboard, textile, leather, plastics,
caulking, and feed.
Other uses include preservation of foods, beverages, cosmetics such as
lotions,
creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants,
deodorants, mouth
wash, contact lens products, enzyme formulations, or food ingredients.
Thus, the antimicrobial polypeptides of the invention may by useful as a
disinfectant,
e.g., in the treatment of acne, infections in the eye or the mouth, skin
infections; in
antiperspirants or deodorants; in foot bath salts; for cleaning and
disinfection of contact
lenses, hard surfaces, teeth (oral care), wounds, bruises and the like.
In general it is contemplated that the antimicrobial polypeptides of the
present
invention are useful for cleaning, disinfecting or inhibiting microbial growth
on any hard
surface. Examples of surfaces, which may advantageously be contacted with the
antimicrobial
polypeptides of the invention are surfaces of process equipment used e.g.
dairies, chemical
or pharmaceutical process plants, water sanitation systems, oil processing
plants, paper pulp
processing plants, water treatment plants, and cooling towers. The
antimicrobial polypeptides
of the invention should be used in an amount, which is effective for cleaning,
disinfecting or
inhibiting microbial growth on the surface in question.
Further, it is contemplated that the antimicrobial polypeptides of the
invention can
advantageously be used in a cleaning-in-place (C.I.P.) system for cleaning of
process
equipment of any kind.
The antimicrobial polypeptides of the invention may additionally be used for
cleaning
surfaces and cooking utensils in food processing plants and in any area in
which food is
prepared or served such as hospitals, nursing homes, restaurants, especially
fast food
restaurants, delicatessens and the like. It may also be used as an
antimicrobial in food
products and would be especially useful as a surface antimicrobial in cheeses,
fruits and
vegetables and food on salad bars.
It may also be used as a preservation agent or a disinfection agent in water
based
paints.
The antimicrobial polypeptides of the present invention are also useful for
microbial
control of water lines, and for disinfection of water, in particular for
disinfection of industrial
water.
The invention also relates to the use of an antimicrobial polypeptide or
composition of
the invention as a medicament. Further, an antimicrobial polypeptide or
composition of the
invention may also be used for the manufacture of a medicament for controlling
or combating
microorganisms, such as fungal organisms or bacteria, preferably gram positive
bacteria.
The composition and antimicrobial polypeptide of the invention may be used as
an
antimicrobial veterinarian or human therapeutic or prophylactic agent. Thus,
the composition
and antimicrobial polypeptide of the invention may be used in the preparation
of veterinarian
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or human therapeutic agents or prophylactic agents for the treatment of
microbial infections,
such as bacterial or fungal infections, preferably gram positive bacterial
infections. In
particular the microbial infections may be associated with lung diseases
including, but not
limited to, tuberculosis, pneumonia and cystic fibrosis; and sexual
transmitted diseases
including, but not limited to, gonorrhea and chlamydia.
The composition of the invention comprises an effective amount of the
antimicrobial
polypeptide of the invention.
The term "effective amount" when used herein is intended to mean an amount of
the
antimicrobial polypeptides of the invention, which is sufficient to inhibit
growth of the
microorganisms in question.
The invention also relates to wound healing compositions or products such as
bandages, medical devices such as, e.g., catheters and further to anti-
dandruff hair products,
such as shampoos.
Formulations of the antimicrobial polypeptides of the invention are
administered to a
host suffering from or predisposed to a microbial infection. Administration
may be topical,
localized or systemic, depending on the specific microorganism, preferably it
will be localized.
Generally the dose of the antimicrobial polypeptides of the invention will be
sufficient to
decrease the microbial population by at least about 50%, usually by at least 1
log, and may be
by 2 or more logs of killing. The compounds of the present invention are
administered at a
dosage that reduces the microbial population while minimizing any side-
effects. It is
contemplated that the composition will be obtained and used under the guidance
of a
physician for in vivo use. The antimicrobial polypeptides of the invention are
particularly useful
for killing gram negative bacteria, including Pseudomonas aeruginosa, and
Chlamydia
trachomatis; and gram-positive bacteria, including various staphylococci and
streptococci.
The antimicrobial polypeptides of the invention are also useful for in vitro
formulations
to kill microbes, particularly where one does not wish to introduce quantities
of conventional
antibiotics. For example, the antimicrobial polypeptides of the invention may
be added to
animal and/or human food preparations; or they may be included as an additive
for in vitro
cultures of cells, to prevent the overgrowth of microbes in tissue culture.
The susceptibility of a particular microbe to killing with the antimicrobial
polypeptides of
the invention may be determined by in vitro testing, as detailed in the
experimental section.
Typically a culture of the microbe is combined with the antimicrobial
polypeptide at varying
concentrations for a period of time sufficient to allow the protein to act,
usually between about
one hour and one day. The viable microbes are then counted, and the level of
killing
determined.
Microbes of interest include, but are not limited to, Gram-negative bacteria,
for
example: Citrobacter sp.; Enterobacter sp.; Escherichia sp., e.g. E. coli;
Klebsiella sp.;
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Morganella sp.; Proteus sp.; Providencia sp.; Salmonella sp., e.g. S. typhi,
S. typhimurium;
Serratia sp.; Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa; Yersinia sp.,
e.g. Y. pestis,
Y. pseudotuberculosis, Y. enterocolitica; Franciscella sp.; Pasturella sp.;
Vibrio sp., e.g. V.
cholerae, V. parahemolyticus; Campylobacter sp., e.g. C. jejuni; Haemophilus
sp., e.g. H.
influenzae, H. ducreyi; Bordetella sp., e.g. B. pertussis, B. bronchiseptica,
B. parapertussis;
Brucella sp., Neisseria sp., e.g. N. gonorrhoeae, N. meningitidis, etc. Other
bacteria of
interest include Legionella sp., e.g. L. pneumophila; Listeria sp., e.g. L.
monocytogenes;
Mycoplasma sp., e.g. M. hominis, M. pneumoniae; Mycobacterium sp., e.g. M.
tuberculosis,
M. leprae; Treponema sp., e.g. T. pallidum; Borrelia sp., e.g. B. burgdorferi;
Leptospirae sp.;
Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C.
trachomatis, C. pneumoniae,
C. psittaci; Helicobacter sp., e.g. H. pylon, etc.
Non bacterial pathogens of interest include fungal and protozoan pathogens,
e.g.
Plasmodia sp., e.g. P. falciparum, Trypanosoma sp., e.g. T. brucei;
shistosomes;
Entaemoeba sp., Cryptococcus sp., Candida sp., e.g. C. albicans; etc.
Various methods for administration may be employed. The polypeptide
formulation
may be given orally, or may be injected intravascularly, subcutaneously,
peritoneally, by
aerosol, opthalmically, intra-bladder, topically, etc. For example, methods of
administration by
inhalation are well-known in the art. The dosage of the therapeutic
formulation will vary widely,
depending on the specific antimicrobial polypeptide to be administered, the
nature of the
disease, the frequency of administration, the manner of administration, the
clearance of the
agent from the host, and the like. The initial dose may be larger, followed by
smaller
maintenance doses. The dose may be administered as infrequently as weekly or
biweekly, or
fractionated into smaller doses and administered once or several times daily,
semi-weekly,
etc. to maintain an effective dosage level. In many cases, oral administration
will require a
higher dose than if administered intravenously. The amide bonds, as well as
the amino and
carboxy termini, may be modified for greater stability on oral administration.
For example, the
carboxy terminus may be amidated.
Formulations
The compounds of this invention can be incorporated into a variety of
formulations for
therapeutic administration. More particularly, the compounds of the present
invention can be
formulated into pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents, and may be formulated into
preparations in
solid, semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules,
ointments, creams, foams, solutions, suppositories, injections, inhalants,
gels, microspheres,
lotions, and aerosols. As such, administration of the compounds can be
achieved in various
ways, including oral, buccal, rectal, parenteral, intraperitoneal,
intradermal, transdermal,
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intracheal, etc., administration. The antimicrobial polypeptides of the
invention may be
systemic after administration or may be localized by the use of an implant or
other formulation
that acts to retain the active dose at the site of implantation.
In one embodiment, a formulation for topical use comprises a chelating agent
that
decreases the effective concentration of divalent cations, particularly
calcium and magnesium.
For example, agents such as citrate, EGTA or EDTA may be included, where
citrate is
preferred. The concentration of citrate will usually be from about 1 to 10 mM.
The compounds of the present invention can be administered alone, in
combination
with each other, or they can be used in combination with other known compounds
(e.g.,
perforin, anti-inflammatory agents, antibiotics, etc.) In pharmaceutical
dosage forms, the
compounds may be administered in the form of their pharmaceutically acceptable
salts. The
following methods and excipients are merely exemplary and are in no way
limiting.
For oral preparations, the compounds can be used alone or in combination with
appropriate additives to make tablets, powders, granules or capsules, for
example, with
conventional additives, such as lactose, mannitol, corn starch or potato
starch; with binders,
such as crystalline cellulose, cellulose derivatives, acacia, corn starch or
gelatins; with
disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired, with diluents,
buffering agents,
moistening agents, preservatives and flavoring agents.
The compounds can be formulated into preparations for injections by
dissolving,
suspending or emulsifying them in an aqueous or nonaqueous solvent, such as
vegetable or
other similar oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or
propylene glycol; and if desired, with conventional additives such as
solubilizers, isotonic
agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The compounds can be utilized in aerosol formulation to be administered via
inhalation. The compounds of the present invention can be formulated into
pressurized
acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and
the like.
The compounds can be used as lotions, for example to prevent infection of
burns, by
formulation with conventional additives such as solubilizers, isotonic agents,
suspending
agents, emulsifying agents, stabilizers and preservatives.
Furthermore, the compounds can be made into suppositories by mixing with a
variety
of bases such as emulsifying bases or water-soluble bases. The compounds of
the present
invention can be administered rectally via a suppository. The suppository can
include vehicles
such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body
temperature,
yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs,
and
suspensions may be provided wherein each dosage unit, for example,
teaspoonful,
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tablespoonful, tablet or suppository, contains a predetermined amount of the
composition
containing one or more compounds of the present invention. Similarly, unit
dosage forms for
injection or intravenous administration may comprise the compound of the
present invention
in a composition as a solution in sterile water, normal saline or another
pharmaceutically
acceptable carrier.
Implants for sustained release formulations are well-known in the art.
Implants are
formulated as microspheres, slabs, etc. with biodegradable or non-
biodegradable polymers.
For example, polymers of lactic acid and/or glycolic acid form an erodible
polymer that is well-
tolerated by the host. The implant containing the antimicrobial polypeptides
of the invention is
placed in proximity to the site of infection, so that the local concentration
of active agent is
increased relative to the rest of the body.
The term "unit dosage form", as used herein, refers to physically discrete
units suitable
as unitary dosages for human and animal subjects, each unit containing a
predetermined
quantity of compounds of the present invention calculated in an amount
sufficient to produce
the desired effect in association with a pharmaceutically acceptable diluent,
carrier or vehicle.
The specifications for the unit dosage forms of the present invention depend
on the particular
compound employed and the effect to be achieved, and the pharmacodynamics
associated
with the compound in the host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants,
carriers or
diluents, are readily available to the public. Moreover, pharmaceutically
acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity adjusting
agents, stabilizers,
wetting agents and the like, are readily available to the public.
Typical dosages for systemic administration range from 0.1 pg to 100
milligrams per
kg weight of subject per administration. A typical dosage may be one tablet
taken from two to
six times daily, or one time-release capsule or tablet taken once a day and
containing a
proportionally higher content of active ingredient. The time-release effect
may be obtained by
capsule materials that dissolve at different pH values, by capsules that
release slowly by
osmotic pressure, or by any other known means of controlled release.
Those of skill will readily appreciate that dose levels can vary as a function
of the
specific compound, the severity of the symptoms and the susceptibility of the
subject to side
effects. Some of the specific compounds are more potent than others. Preferred
dosages for
a given compound are readily determinable by those of skill in the art by a
variety of means. A
preferred means is to measure the physiological potency of a given compound.
The use of liposomes as a delivery vehicle is one method of interest. The
liposomes
fuse with the cells of the target site and deliver the contents of the lumen
intracellularly. The
liposomes are maintained in contact with the cells for sufficient time for
fusion, using various
means to maintain contact, such as isolation, binding agents, and the like. In
one aspect of
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the invention, liposomes are designed to be aerosolized for pulmonary
administration.
Liposomes may be prepared with purified proteins or peptides that mediate
fusion of
membranes, such as Sendai virus or influenza virus, etc. The lipids may be any
useful
combination of known liposome forming lipids, including cationic or
zwitterionic lipids, such as
phosphatidylcholine. The remaining lipid will be normally be neutral or acidic
lipids, such as
cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
For preparing the liposomes, the procedure described by Kato et al. (1991) J.
Biol.
Chem. 266:3361 may be used. Briefly, the lipids and lumen composition
containing peptides
are combined in an appropriate aqueous medium, conveniently a saline medium
where the
total solids will be in the range of about 1-10 weight percent. After intense
agitation for short
periods of time, from about 5-60 sec., the tube is placed in a warm water
bath, from about 25-
40 C and this cycle repeated from about 5-10 times. The composition is then
sonicated for a
convenient period of time, generally from about 1-10 sec. and may be further
agitated by
vortexing. The volume is then expanded by adding aqueous medium, generally
increasing the
volume by about from 1-2 fold, followed by shaking and cooling. This method
allows for the
incorporation into the lumen of high molecular weight molecules.
Formulations with Other Active Agents
For use in the subject methods, the antimicrobial polypeptides of the
invention may be
formulated with other pharmaceutically active agents, particularly other
antimicrobial agents.
Other agents of interest include a wide variety of antibiotics, as known in
the art. Classes of
antibiotics include penicillins, e.g. penicillin G, penicillin V, methicillin,
oxacillin, carbenicillin,
nafcillin, ampicillin, etc.; penicillins in combination with beta-lactamase
inhibitors,
cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.;
carbapenems;
rnonobactams; aminoglycosides; tetracyclines; macrolides; lincomycins;
polymyxins;
sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin;
trimethoprim;
vancomycin; etc.
Anti-mycotic agents are also useful, including polyenes, e.g. amphotericin B,
nystatin;
5-flucosyn; and azoles, e.g. miconazol, ketoconazol, itraconazol and
fluconazol.
3o Antituberculotic drugs include isoniazid, ethambutol, streptomycin and
rifampin. Cytokines
may also be included in a formulation of the antimicrobial polypeptides of the
invention, e.g.
interferon gamma, tumor necrosis factor alpha, interleukin 12, etc.
in vitro synthesis
The antimicrobial peptides of the invention may be prepared by in vitro
synthesis,
using conventional methods as known in the art. Various commercial synthetic
apparatuses
are available, for example automated synthesizers by Applied Biosystems Inc.,
Beckman, etc.
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By using synthesizers, naturally occurring amino acids may be substituted with
unnatural
amino acids, particularly D-isomers (or D-forms) e.g. D-alanine and D-
isoleucine,
diastereoisomers, side chains having different lengths or functionalities, and
the like. The
particular sequence and the manner of preparation will be determined by
convenience,
economics, purity required, and the like.
Chemical linking may be provided to various peptides or proteins comprising
convenient functionalities for bonding, such as amino groups for amide or
substituted amine
formation, e.g. reductive amination, thiol groups for thioether or disulfide
formation, carboxyl
groups for amide formation, and the like.
If desired, various groups may be introduced into the peptide during synthesis
or
during expression, which allow for linking to other molecules or to a surface.
Thus cysteines
can be used to make thioethers, histidines for linking to a metal ion complex,
carboxyl groups
for forming amides or esters, amino groups for forming amides, and the like.
The polypeptides may also be isolated and purified in accordance with
conventional
methods of recombinant synthesis. A lysate may be prepared of the expression
host and the
lysate purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity
chromatography, or other purification technique. For the most part, the
compositions which
are used will comprise at least 20% by weight of the desired product, more
usually at least
about 75% by weight, preferably at least about 95% by weight, and for
therapeutic purposes,
usually at least about 99.5% by weight, in relation to contaminants related to
the method of
preparation of the product and its purification. Usually, the percentages will
be based upon
total protein
Animal Feed
The present invention is also directed to methods for using the polypeptides
having
antimicrobial activity in animal feed, as well as to feed compositions and
feed additives
comprising the antimicrobial polypeptides of the invention.
The term animal includes all animals, including human beings. Examples of
animals
are non-ruminants, and ruminants, such as cows, sheep and horses. In a
particular
embodiment, the animal is a non-ruminant animal. Non-ruminant animals include
mono-
gastric animals, e.g. pigs or swine (including, but not limited to, piglets,
growing pigs, and
sows); poultry such as turkeys and chicken (including but not limited to
broiler chicks, layers);
young calves; and fish (including but not limited to salmon).
The term feed or feed composition means any compound, preparation, mixture, or
composition suitable for, or intended for intake by an animal.
In the use according to the invention the antimicrobial polypeptide can be fed
to the
animal before, after, or simultaneously with the diet. The latter is
preferred.
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In a particular embodiment, the antimicrobial polypeptide, in the form in
which it is
added to the feed, or when being included in a feed additive, is well defined.
Well-defined
means that the antimicrobial polypeptide preparation is at least 50% pure as
determined by
Size-exclusion chromatography (see Example 12 of WO 01/58275). In other
particular
embodiments the antimicrobial polypeptide preparation is at least 60, 70, 80,
85, 88, 90, 92,
94, or at least 95% pure as determined by this method.
A well-defined antimicrobial polypeptide preparation is advantageous. For
instance, it
is much easier to dose correctly to the feed an antimicrobial polypeptide that
is essentially
free from interfering or contaminating other antimicrobial polypeptides. The
term dose
correctly refers in particular to the objective of obtaining consistent and
constant results, and
the capability of optimising dosage based upon the desired effect.
For the use in animal feed, however, the antimicrobial polypeptide need not be
that
pure; it may e.g. include other enzymes, in which case it could be termed an
antimicrobial
polypeptide preparation.
The antimicrobial polypeptide preparation can be (a) added directly to the
feed (or
used directly in a treatment process of vegetable proteins), or (b) it can be
used in the
production of one or more intermediate compositions such as feed additives or
premixes that
is subsequently added to the feed (or used in a treatment process). The degree
of purity
described above refers to the purity of the original antimicrobial polypeptide
preparation,
whether used according to (a) or (b) above.
Antimicrobial polypeptide preparations with purities of this order of
magnitude are in
particular obtainable using recombinant methods of production, whereas they
are not so
easily obtained and also subject to a much higher batch-to-batch variation
when the
antimicrobial polypeptide is produced by traditional fermentation methods.
Such antimicrobial polypeptide preparation may of course be mixed with other
enzymes.
The term vegetable proteins as used herein refers to any compound,
composition,
preparation or mixture that includes at least one protein derived from or
originating from a
vegetable, including modified proteins and protein-derivatives. In particular
embodiments, the
protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, or
60% (w/w).
Vegetable proteins may be derived from vegetable protein sources, such as
legumes
and cereals, for example materials from plants of the families Fabaceae
(Leguminosae),
Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal
and
rapeseed meal.
In a particular embodiment, the vegetable protein source is material from one
or more
plants of the family Fabaceae, e.g. soybean, lupine, pea, or bean.
In another particular embodiment, the vegetable protein source is material
from one or
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more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or
quinoa.
Other examples of vegetable protein sources are rapeseed, and cabbage.
Soybean is a preferred vegetable protein source.
Other examples of vegetable protein sources are cereals such as barley, wheat,
rye,
oat, maize (corn), rice, and sorghum.
The antimicrobial polypeptide can be added to the feed in any form, be it as a
relatively pure antimicrobial polypeptide, or in admixture with other
components intended for
addition to animal feed, i.e. in the form of animal feed additives, such as
the so-called pre-
mixes for animal feed.
In a further aspect the present invention relates to compositions for use in
animal feed,
such as animal feed, and animal feed additives, e.g. premixes.
Apart from the antimicrobial polypeptide of the invention, the animal feed
additives of
the invention contain at least one fat soluble vitamin, and/or at least one
water soluble
vitamin, and/or at least one trace mineral, and/or at least one macro mineral.
Further, optional, feed-additive ingredients are colouring agents, aroma
compounds,
stabilisers, and/or at least one other enzyme selected from amongst phytases
EC 3.1.3.8 or
3.1.3.26; xylanases EC 3.2.1.8; galactanases EC 3.2.1.89; and/or beta-
glucanases EC
3.2.1.4.
In a particular embodiment these other enzymes are well defined (as defined
above
for antimicrobial polypeptide preparations).
Examples of other antimicrobial peptides (AMP's) are CAP18, Leucocin A,
Tritrpticin,
Protegrin-1, Thanatin, Defensin, Ovispirin such as Novispirin (Robert Lehrer,
2000), and
variants, or fragments thereof which retain antimicrobial activity.
Examples of other antifungal polypeptides (AFP's) are the Aspergillus
giganteus, and
Aspergillus niger peptides, as well as variants and fragments thereof which
retain antifungal
activity, as disclosed in WO 94/01459 and PCT/DK02/00289 [replace with WO
number once
published].
Usually fat and water soluble vitamins, as well as trace minerals form part of
a so-
called premix intended for addition to the feed, whereas macro minerals are
usually
separately added to the feed. Either of these composition types, when enriched
with an
,antimicrobial polypeptide of the invention, is an animal feed additive of the
invention.
In a particular embodiment, the animal feed additive of the invention is
intended for
being included (or prescribed as having to be included) in animal diets or
feed at levels of
0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g
additive per 100 g
feed). This is so in particular for premixes.
The following are non-exclusive lists of examples of these components:
Examples of fat soluble vitamins are vitamin A, vitamin D3, vitamin E, and
vitamin K,
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e.g. vitamin K3.
Examples of water soluble vitamins are vitamin B12, biotin and choline,
vitamin 131,
vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-
panthothenate.
Examples of trace minerals are manganese, zinc, iron, copper, iodine,
selenium, and
cobalt.
Examples of macro minerals are calcium, phosphorus and sodium.
The nutritional requirements of these components (exemplified with poultry and
piglets/pigs)
are listed in Table A of WO 01/58275. Nutritional requirement means that these
components
should be provided in the diet in the concentrations indicated.
In the alternative, the animal feed additive of the invention comprises at
least one of
the individual components specified in Table A of WO 01/58275. At least one
means either of,
one or more of, one, or two, or three, or four and so forth up to all
thirteen, or up to all fifteen
individual components. More specifically, this at least one individual
component is included in
the additive of the invention in such an amount as to provide an in-feed-
concentration within
the range indicated in column four, or column five, or column six of Table A.
The present invention also relates to animal feed compositions. Animal feed
compositions or diets have a relatively high content of protein. Poultry and
pig diets can be
characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets
can be
characterised as indicated in column 4 of this Table B. Furthermore such fish
diets usually
have a crude fat content of 200-310 g/kg.
An animal feed composition according to the invention has a crude protein
content of
50-800 g/kg, and furthermore comprises at least one antimicrobial polypeptide
as claimed
herein.
Furthermore, or in the alternative (to the crude protein content indicated
above), the
animal feed composition of the invention has a content of metabolisable energy
of 10-30
MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of
available phosphorus
of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a
content of
methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50
g/kg.
In particular embodiments, the content of metabolisable energy, crude protein,
calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is
within any one of
ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).
Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e.
Crude
protein (g/kg)= N (g/kg) x 6.25. The nitrogen content is determined by the
Kjeldahl method
(A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of
Official Analytical
Chemists, Washington DC).
Metabolisable energy can be calculated on the basis of the NRC publication
Nutrient
requirements in swine, ninth revised edition 1988, subcommittee on swine
nutrition,
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committee on animal nutrition, board of agriculture, national research
council. National
Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy
Values for
Poultry Feed-stuffs, Spelderholt centre for poultry research and extension,
7361 DA
Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen by, Wageningen.
ISBN 90-
71463-12-5.
The dietary content of calcium, available phosphorus and amino acids in
complete
animal diets is calculated on the basis of feed tables such as Veevoedertabel
1997, gegevens
over chemische samenstelling, verteerbaarheid en voederwaarde van
voedermiddelen,
Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.
In a particular embodiment, the animal feed composition of the invention
contains at
least one vegetable protein or protein source as defined above.
In still further particular embodiments, the animal feed composition of the
invention
contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70%
Barley;
and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0-
20% whey.
Animal diets can e.g. be manufactured as mash feed (non pelleted) or pelleted
feed.
Typically, the milled feed-stuffs are mixed and sufficient amounts of
essential vitamins and
minerals are added according to the specifications for the species in
question. Enzymes can
be added as solid or liquid enzyme formulations. For example, a solid enzyme
formulation is
typically added before or during the mixing step; and a liquid enzyme
preparation is typically
added after the pelleting step. The enzyme may also be incorporated in a feed
additive or
premix.
The final enzyme concentration in the diet is within the range of 0.01-200 mg
enzyme
protein per kg diet, for example in the range of 5-30 mg enzyme protein per kg
animal diet.
The antimicrobial polypeptide may be administered in one or more of the
following
amounts (dosage ranges): 0.01-200; or 0.01-100; or 0.05-100; or 0.05-50; or
0.10-10 - all
these ranges being in mg antimicrobial polypeptide protein per kg feed (ppm).
For determining mg antimicrobial polypeptide protein per kg feed, the
antimicrobial
polypeptide is purified from the feed composition, and the specific activity
of the purified
antimicrobial polypeptide is determined using a relevant assay (see under
antimicrobial
activity, substrates, and assays). The antimicrobial activity of the feed
composition as such is
also determined using the same assay, and on the basis of these two
determinations, the
dosage in mg antimicrobial polypeptide protein per kg feed is calculated.
The same principles apply for determining mg antimicrobial polypeptide protein
in feed
additives. Of course, if a sample is available of the antimicrobial
polypeptide used for
preparing the feed additive or the feed, the specific activity is determined
from this sample (no
need to purify the antimicrobial polypeptide from the feed composition or the
additive).
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Detergent composition
The antimicrobial polypeptides of the invention may be added to and thus
become a
component of a detergent composition.
The detergent composition of the invention may for example be formulated as a
hand
or machine laundry detergent composition including a laundry additive
composition suitable
for pre-treatment of stained fabrics and a rinse added fabric softener
composition, or be
formulated as a detergent composition for use in general household hard
surface cleaning
operations, or be formulated for hand or machine dishwashing operations.
In a specific aspect, the invention provides a detergent additive comprising
the
antimicrobial polypeptides of the invention and a surfactant. The detergent
additive as well as
the detergent composition may comprise one or more other enzymes such as a
protease, a
lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a
mannanase, an
arabinase, a galactanase, a xylanase, an oxidase (such as a laccase), and/or a
peroxidase
(such as a haloperoxidase).
In general the properties of the chosen enzyme(s) should be compatible with
the
selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and
non-enzymatic
ingredients, etc.), and the enzyme(s) should be present in effective amounts.
Proteases: Suitable proteases include those of animal, vegetable or microbial
origin.
Microbial origin is preferred. Chemically modified or protein engineered
mutants are included.
The protease may be a serine protease or a metallo protease, preferably an
alkaline microbial
protease or a trypsin-like protease. Examples of alkaline proteases are
subtilisins, especially
those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg,
subtilisin 309, subtilisin
147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like
proteases are
trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described
in WO
89/06270 and WO 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO
98/20115, WO 98/20116, and WO 98/34946, especially the variants with
substitutions in one
or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120,
123, 167, 170, 194,
206, 218, 222, 224, 235 and 274.
Lipases: Suitable lipases include those of bacterial or fungal origin.
Chemically
modified or protein engineered mutants are included. Examples of useful
lipases include
lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T.
lanuginosus) as
described in EP 258 068 and EP 305 216 or from H. insolens as described in WO
96/13580,
a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218
272), P.
cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas
sp. strain SD
705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus
lipase,
e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,
1131, 253-360), B.
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stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO
94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO
94/25578, WO 95114783, WO 95/22615, WO 97/04079 and WO 97/07202.
Amylases: Suitable amylases (alpha and/or beta) include those of bacterial or
fungal
origin. Chemically modified or protein engineered mutants are included.
Amylases include, for
example, alpha-amylases obtained from Bacillus, e.g. a special strain of B.
licheniformis,
described in more detail in GB 1,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO
94/18314, WO 96/23873, and WO 97/43424, especially the variants with
substitutions in one
or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156,
181, 188, 190,
197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
Cellulases: Suitable cellulases include those of bacterial or fungal origin.
Chemically
modified or protein engineered mutants are included. Suitable cellulases
include cellulases
from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
Acremonium, e.g.
the fungal cellulases produced from Humicola insolens, Myceliophthora
thermophila and
Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US
5,776,757
and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having
colour care
benefits. Examples of such cellulases are cellulases described in EP 0 495
257, EP 0 531
372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants
such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US
5,686,593, US
5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant,
bacterial
or fungal origin. Chemically modified or protein engineered mutants are
included. Examples of
useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus,
and variants
thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
The detergent enzyme(s) may be included in a detergent composition by adding
separate additives containing one or more enzymes, or by adding a combined
additive
comprising all of these enzymes. A detergent additive of the invention, i.e. a
separate additive
or a combined additive, can be formulated e.g. as a granulate, a liquid, a
slurry, etc. Preferred
detergent additive formulations are granulates, in particular non-dusting
granulates, liquids, in
particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and
4,661,452 and may optionally be coated by methods known in the art. Examples
of waxy
coating materials are polyethylene oxide) products (polyethyleneglycol, PEG)
with mean
molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50
ethylene
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oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12
to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols;
fatty acids; and
mono- and di- and triglycerides of fatty acids. Examples of film-forming
coating materials
suitable for application by fluid bed techniques are given in GB 1483591.
Liquid enzyme
preparations may, for instance, be stabilized by adding a polyol such as
propylene glycol, a
sugar or sugar alcohol, lactic acid or boric acid according to established
methods. Protected
enzymes may be prepared according to the method disclosed in EP 238,216.
The detergent composition of the invention may be in any convenient form,
e.g., a bar,
a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be
aqueous, typically
containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
The detergent composition comprises one or more surfactants, which may be non-
ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
The surfactants
are typically present at a level of from 0.1 % to 60% by weight.
When included therein the detergent will usually contain from about 1% to
about 40%
of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-
olefinsulfonate, alkyl
sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary
alkanesulfonate, alpha-sulfo
fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
When included therein the detergent will usually contain from about 0.2% to
about
40% of a non-ionic surfactant such as alcohol ethoxylate, nonyiphenol
ethoxylate,
alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid
monoethanolamide, fatty
acid monoethanolarnide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl
derivatives of
glucosamine ("glucamides").
The detergent may contain 0-65 % of a detergent builder or complexing agent
such as
zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate,
nitrilotriacetic acid,
ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or
alkenylsuccinic
acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Examples are
carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),
poly(vinyl alcohol),
poly(vinylpyridine-N -oxide), poly(vinylimidazole), polycarboxylates such as
polyacrylates,
maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid
copolymers.
The detergent may contain a bleaching system which may comprise a H202 source
such as perborate or percarbonate which may be combined with a peracid-forming
bleach
activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively,
the bleaching system may comprise peroxyacids of e.g. the amide, imide, or
sulfone type.
The enzyme(s) of the detergent composition of the invention may be stabilized
using
conventional stabilizing agents, e.g., a polyol such as propylene glycol or
glycerol, a sugar or
sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an
aromatic borate ester,
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or a phenyl boronic acid derivative such as 4-formyiphenyl boronic acid, and
the composition
may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
The detergent may also contain other conventional detergent ingredients such
as e.g.
fabric conditioners including clays, foam boosters, suds suppressors, anti-
corrosion agents,
soil-suspending agents, anti-soil redeposition agents, dyes, bactericides,
optical brighteners,
hydrotropes, tarnish inhibitors, or perfumes.
It is at present contemplated that in the detergent compositions any enzyme,
and the
antimicrobial polypeptides of the invention, may be added in an amount
corresponding to
0.01-100 mg of enzyme protein per liter of wash ligour, preferably 0.05-10 mg
of enzyme
protein per liter of wash liqour, more preferably 0.1-5 mg of enzyme protein
per liter of wash
ligour, and most preferably 0.1-1 mg of enzyme protein per liter of wash
liqour.
The antimicrobial polypeptides of the invention may additionally be
incorporated in the
detergent formulations disclosed in WO 97/07202.
The present invention is further described by the following examples which
should not
be construed as limiting the scope of the invention.
EXAMPLES
Chemicals used as buffers and substrates were commercial products of at least
reagent grade.
EXAMPLE 1
Design, construction and evaluation of synthetic antimicrobial peptides
To investigate and design new AMPs with potent antimicrobial activity, a
series of
putative AMPs were constructed. Three proximal arginines were chosen to
precede 6
consecutive 4 amino acid repeats. Based on its membrane-interacting ability
and abundance
in natural PR-rich peptides, phenylalanine was chosen as the hydrophobic
residue (Z,-Z,2) for
the Initial series. This resulted in 12 different derivatives designated SEQ
ID NO:1 to SEQ ID
NO:12 (see Table 1).
The 12 genes encoding SEQ ID NO:1 to SEQ ID NO:12 were synthesized from 12
specific oligonucleotides (Primerl to Primerl2) each encoding one of the
peptides. The
single-stranded oligonucleotide was turned into double-stranded DNA by
polymerization with a
complementary primer (Primerl3) by 5 cycles in a standard PCR reaction. A Nool
(underlined) DNA restriction site Is located in the proximal part while a Xbal
(underlined)
restriction site Is located In the distal part of the primer - these sites are
used for cloning into
the corresponding expression vector, pBAD/glllA (invitrogen, USA).
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Primerl - SEQ ID NO:1 forward (SEQ ID NO:320):
CCATAGCACC ATGGCGCGTC GCCGTCCGCC ACGTTTTCCA CCTCGTTTTC CACCTCGTTT
CCCTCCACGT TTCCCTCCAC GCTTCCCACC TCGTTTCTAA TTGCTCTAGA ACAAAAACTC
Primer2 - SEQ ID NO:2 forward (SEQ ID NO:321):
CCATAGCACC ATGGCGCGTC GCCGTCCACG TTTTCCACCT CGTTTTCCAC CTCGTTTCCC
TCCACGTTTC CCTCCACGCT TCCCACCTCG TTTCCCGTAA TTGCTCTAGA ACAAAAACTC
Primer3 - SEQ ID NO:3 forward (SEQ ID NO:322):
CCATAGCACC ATGGCGCGTC GCCGTCGTTT TCCACCTCGT TTTCCACCTC GTTTCCCTCC
ACGTTTCCCT CCACGCTTCC CACCTCGTTT CCCGCCATAA TTGCTCTAGA ACAAAAACTC
Primer4 - SEQ ID NO:4 forward (SEQ ID NO:323):
CCATAGCACC ATGGCGCGTC GCCGTTTTCC ACCTCGTTTT CCACCTCGTT TCCCTCCACG
TTTCCCTCCA CGCTTCCCAC CTCGTTTCCC GCCACGTTAA TTGCTCTAGA ACAAAAACTC
Primer5 - SEQ ID NO:5 forward (SEQ ID NO:324):
CCATAGCACC ATGGCGCGTC GCCGTCCACG TCCTTTTCCG CGCCCTTTTC CACGTCCATT
CCCTCGTCCT TTCCCACGCC CTTTTCCACG CCCATTCTAA TTGCTCTAGA ACAAAAACTC
Primer6 - SEQ ID NO:6 forward (SEQ ID NO:325):
CCATAGCACC ATGGCGCGTC GCCGTCGTCC TTTTCCGCGC CCTTTTCCAC GTCCATTCCC
TCGTCCTTTC CCACGCCCTT TTCCACGCCC ATTCCCATAA TTGCTCTAGA ACAAAAACTC
Primer? - SEQ ID NO:7 forward (SEQ ID NO:326):
CCATAGCACC ATGGCGCGTC GCCGTCCTTT TCCGCGCCCT TTTCCACGTC CATTCCCTCG
TCCTTTCCCA CGCCCTTTTC CACGCCCATT CCCACGTTAA TTGCTCTAGA ACAAAAACTC
Primer8 - SEQ ID NO:8 forward (SEQ ID NO:327):
CCATAGCACC ATGGCGCGTC GCCGTTTTCC GCGCCCTTTT CCACGTCCAT TCCCTCGTCC
TTTCCCACGC CCTTTTCCAC GCCCATTCCC ACGTCCTTAA TTGCTCTAGA ACAAAAACTC
Primer9 - SEQ ID NO:9 forward (SEQ ID NO:328):
CCATAGCACC ATGGCGCGTC GCCGTCCACC ATTTCGTCCA CCTTTCCGTC CTCCATTTCG
CCCGCCGTTT CGGCCACCGT TTCGACCTCC TTTCCGTTAA TTGCTCTAGA ACAAAAACTC
PrimerlO - SEQ ID NO:10 forward (SEQ ID NO:329):
CCATAGCACC ATGGCGCGTC GCCGTCCATT TCGTCCACCT TTCCGTCCTC CATTTCGCCC
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GCCGTTTCGG CCACCGTTTC GACCTCCTTT CCGTCCATAA TTGCTCTAGA ACAAAAACTC
Primed 1 - SEQ ID NO: 11 forward (SEQ ID NO:330):
CCATAGCACC ATGGCGCGTC GCCGTTTTCG TCCACCTTTC CGTCCTCCAT TTCGCCCGCC
GTTTCGGCCA CCGTTTCGAC CTCCTTTCCG TCCACCATAA TTGCTCTAGA ACAAAAACTC
Primerl2 - SEQ ID NO:12 forward (SEQ ID NO:331):
CCATAGCACC ATGGCGCGTC GCCGTCGTCC ACCTTTCCGT CCTCCATTTC GCCCGCCGTT
TCGGCCACCG TTTCGACCTC CTTTCCGTCC ACCATTTTAA TTGCTCTAGA ACAAAAACTC
Primerl3 - reverse (SEQ ID NO:332):
GAGTTTTTGT TCTAGAGCAA TTA
The antibiotic activity of AMPs can be tested in a suicide expression system
(SES), as
disclosed in Example 1 of International patent application WO 00/73433. This
system employs
a sensitive host (here E. cols) and a plasmid with an inducible promoter
(pBAD/gIIIA). If the
peptide has antibacterial activity, induction of peptide synthesis will result
in growth inhibition
and/or cell death of the producing cell - hence suicide expression system.
Generally, the
stronger the antimicrobial activity, the stronger the inhibition observed.
The 12 double-stranded PCR fragments were purified, restricted with Xbal and
Ncol,
and cloned into pBAD/g I I IA, which were transformed into E. coli TOP10
(Invitrogen), and re-
streaked on LB agar plates with 100 pg/ml Ampicillin. Individual clones of SEQ
ID NO:1 to
SEQ ID NO:12 was grown in a 100-microwell plate (Honeycomb plate) in 150 pl RM
+ 0.2%
glucose + 100 pg/ml arnpicillin at 37 degrees Celsius at medium shaking in a
Bioscreen C
(Thermosystems, Finland).
The overnight cultures were diluted 50-fold in 150 pl RM + 0.2% glycerol + 100
ug/ml
ampicillin with either 0 0o or 0.1% inducer (arabinose) in a 100-well
honeycomb plate. Cell
density was monitored every 30 minutes by measuring at OD450 in a Bioscreen C
at 37
degrees Celsius. The percentage of growth inhibition was calculated as the end
point OD
measurement of each sample divided by the end point OD measurement obtained
from cells
containing the control vector and multiplied by 100. The formula is the
following:
Growth inhibition sample OD - blank OD X100
( control vector OD - blank OD
where "blank OD" corresponds to the OD of only the growth medium.
The amino acid sequences of the synthetic AMPs and the corresponding growth
inhibitions are shown in Table 1.
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Table 1.
SEQ ID NO Amino acid sequence Growth
inhibition
( lo)
Control TMELEICSWYHMGIRSFLEQKLISEEDLNSAVDHHHHHH 0
1 RRRPPRFPPRFPPRFPPRFPPRFPPRF 48
2 RRRPRFPPRFPPRFPPRFPPRFPPRFP 45
3 RRRRFPPRFPPRFPPRF PPRFPPRFPP 43
4 RRRFPPRFPPRFPPRFPPRFPPRFPPR 46
RRRPRPFPRPFPRPFPRPFPRPFPRPF 57
6 RRRRPFPRPFPRPFPRPFPRPFPRPFP 57
7 RRRPFPRPFPRPFPRPFPRPFPRPFPR 39
8 RRRFPRPFPRPFPRPFPRPFPRPFPRP 38
9 RRRPPFRPPFRPPFRPPFRPPFRPPFR 67
RRRPFRPPFRPPFRPPFRPPFRPPFRP 78
11 RRRFRPPFRPPFRPPFRPPFRPPFRPP 73
12 RRRRPPFRPPFRPPFRPPFRPPFRPPF 76
As expected, no or very little inhibition was observed in the absence of
inducer (data
not shown). In the presence of 0.1% inducer all synthetic AMPs resulted in
growth inhibition.
5
EXAMPLE 2
Expression of synthetic AMPs in yeast
The AMPs designated SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:10 were cloned
10 into the yeast expression vector pHH3885. Briefly, pHH3885 is a yeast/E.
coli shuttle vector
based on pYES (Invitrogen). This plasmid contains the 2p yeast replication
origin and the
Ura3 gene for selection in yeast. For propagation in E. coli the pUC origin
and bla gene is
used. Expression is controlled by the Gal promoter, and the S. cerevisiae
alpha-leader is used
to mediate secretion of the peptides.
As expression of a potent AMP will kill the producing organism, the
antimicrobial
activity has to be shielded. This can be done using a stretch of anionic amino
acids that will
shield the proximal charges that are crucial for antimicrobial activity.
Correspondingly, all
three AMPs are preceded by a stretch of aspartates and glutamates (DDDDE). As
some
proteases specifically cleave after glutamic acid residues (E), the anionic
stretch can be
liberated from the AMP allowing for monitoring of the antimicrobial activity
of the AMP.
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The PR-rich AMPs designated SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:10 were
amplified using the specific Forward primer and the general Reverse primer in
a standard
PCR reaction. No template is needed as the forward oligo contains all the
coding information.
Primer14 - SEQ ID NO:1 forward (SEQ ID NO:333):
GTATCGATGG CCAAGAGAGA AGCCGACGAT GACGATGAAC GTCGCCGTCC GCCACGTTTT
CCACCTCGTT TTCCACCTCG TTTCCCTCCA CGTTTCCCTC CACGCTTCCC ACCTCGTTTC
TAGATGGCTC TAGAGGGCCG
Primerl5 - SEQ ID NO:5 forward (SEQ ID NO:334):
GTATCGATGG CCAAGAGAGA AGCCGACGAT GACGATGAAC GTCGCCGTCC ACGTCCTTTT
CCGCGCCCTT TTCCACGTCC ATTCCCTCGT CCTTTCCCAC GCCCTTTTCC ACGCCCATTC
TAGATGGCTC TAGAGGGCCG
Primerl6 - SEQ ID NO:10 forward (SEQ ID NO:335):
GTATCGATGG CCAAGAGAGA AGCCGACGAT GACGATGAAC GTCGCCGTCC ATTTCGTCCA
CCTTTCCGTC CTCCATTTCG CCCGCCGTTT CGGCCACCGT TTCGACCTCC TTTCCGTCCA
TAGATGGCTC TAGAGGGCCG
Primer17 - reverse (SEQ ID NO:336):
CGGCCCTCTA GAGCCATCTA
The double-stranded PCR fragments were restricted with Ball and Xbal and
cloned
into pHH3885 restricted with the same two enzymes. The corresponding plasmids
were
transformed into E. coli and sequenced. After sequence-verification, the
shuttle-vectors were
electro-transformed to S. cerevisiae (JG169) and plated on SC + 2% glucose +
100 pg/ml
Ampicillin agar plates.
The yeast colonies were re-streaked and grown in 5 ml of liquid SC ground
media
(supplemented with 2% glucose) at 30 degrees Celsius for 3 days.
Cells were pelleted by centrifugation at 3500 rpm for 5 minutes. 500 pl were
transferred to microcon YM-3 centrifugational filters (Millipore Corp., USA)
and the volume
reduced to approximately 20 pl by centrifugation. The concentrated samples
were mixed with
Tricine loading buffer and analyzed in 16% Tricine gels (Novex, Invitrogen,
USA).
Distinct bands with the expected molecular weight were observed in each of the
three
cultures indicating expression of the three peptides. No bands were observed
in supernatants
of yeast cells harboring the control plasmid.
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EXAMPLE 3
Evaluation of variants of SEQ ID NO:1, SEQ ID NOS and SEQ ID NO:10
SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:10 were used as a starting point in a
mutational high throughput screening for obtaining AMPs with improved
activity. For simplicity
SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:10 all contained phenylalanine as the
hydrophobic residue. Employing only one type of hydrophobic amino acid is
probably not
optimal for antimicrobial activity; this is emphasized by natural peptides,
that all contain a mix
of hydrophobic residues. Degenerate DNA oligos were designed that codes for
each of the 4
hydrophobic amino acids instead of being restricted to phenylalanine. In
principle, as SEQ ID
NO:1, SEQ ID NO:5 and SEQ ID NO:10 each contains 6 phenylalanine residues, the
library
theoretically can encode 46 or 4096 different variants.
The libraries were constructed as above with one long forward primer encoding
the
entire peptide (SEQ ID NO:1 degenerate primer, SEQ ID NO:5 degenerate primer
and SEQ
ID NO:10 degenerate primer) and a smaller reverse primer for synthesis of the
reverse strand
(Primer13). N represent 25% of each of the 4 nucleotides (G, A, T and C)
allowing for
incorporation of the four hydrophobic amino acids in the designated positions.
SEQ ID NO:1 degenerate primer (SEQ ID NO:337)
CCATAGCACC ATGGCGCGTC GCCGTCCGCC ACGTNTTCCA CCTCGTNTTC CACCTCGTNT
CCCTCCACGT NTCCCTCCAC GCNTCCCACC TCGTNTCTAA TTGCTCTAGA ACAAAAACTC
SEQ ID NO:5 degenerate primer (SEQ ID NO:338)
CCATAGCACC ATGGCGCGTC GCCGTCCACG TCCTNTTCCG CGCCCTNTTC CACGTCCANT
CCCTCGTCCT NTCCCACGCC CTNTTCCACG CCCANTCTAA TTGCTCTAGA ACAAAAACTC
SEQ ID NO:10 degenerate primer (SEQ ID NO:339)
CCATAGCACC ATGGCGCGTC GCCGTCCANT TCGTCCACCT NTCCGTCCTC CANTTCGCCC
GCCGNTTCGG CCACCGNTTC GACCTCCTNT CCGTCCATAA TTGCTCTAGA ACAAAAACTC
The DNA fragments were cloned into the SES vector pBAD/gIIIA and transformed
into
E. coli to give three independent libraries of approximately a million
colonies each.
The libraries were plated and around 10,000 individual colonies from each
library were
picked into 96-well microtiter plates containing 20 x diluted RM + 200 pg/ml
Ampicillin by a
colony picker. These master plates were replica-plated into a new set of 96-
well plates
containing 20 x diluted RM media and either 0.1% or 0% inducer and incubated
overnight at
37 degrees Celsius. A number of the most growth inhibited clones from each
library were
isolated, sequenced and re-tested for growth inhibition in the Bioscreen C.
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The amino acid sequences of the variants of SEQ ID NO:1 and the corresponding
growth inhibitions are shown in Table 2; variants of SEQ ID NO:5 are shown in
Table 3; and
variants of SEQ ID NO:10 are shown in Table 4.
Table 2. Variants of SEQ ID NO:1 and the corresponding growth inhibitions.
SEQ ID NO Amino acid sequence Growth
inhibition
M)
13 RRRPPRFPPRFPPRFPPRFPPRFPPRF 51
14 RRRPPRFPPRIPPRFPPRLPPRIPPRL 90
RRRPPRFPPRIPPRLPPRVPPRVPPRL 57
16 RRRPPRIPPRFPPRVPPRFPPRVPPRV 98
17 RRRPPRIPPRLPPRIPPRFPPRFPPRL 95
18 RRRPPRLPPRFPPRFPPRIPPRFPPRI 99
19 RRRPPRLPPRFPPRFPPRIPPRFPPRI 78
RRRPPRLPPRFPPRFPPRVPPRFPPRV 86
21 RRRPPRLPPRFPPRFPPRVPPRFPPRV 86
22 RRRPPRLPPRFPPRFPPRVPPRLPPRF 81
23 RRRPPRLPPRFPPRFPPRVPPRVPPRF 82
24 RRRPPRLPPRFPPRFPPRVPPRVPPRF 90
RRRPPRLPPRFPPRFPPRVPPRVPPRI 81
26 RRRPPRVPPRFPPRVPPRFPPRVPPRL 86
27 RRRPPRLPPRLPPRLPPRVPPRVPLVSNCSRTKTHLRRGSE 90
Table 3. Variants of SEQ ID NO:5 and the corresponding growth inhibitions.
SEQ ID NO Amino acid sequence Growth
inhibition
28 RRRPRPFPRPFPRPFPRPFPRPFPRPF 57
29 RRRPRLPRPFPRPVPRPLPRPVPRPI 63
RRRPRPFPPPFPRPIPRPFPRPFPR PL 61
31 RRRPRPFPRPFPHPFPRPFPRPIPRPI 73
32 RRRPRPFPRPFPHPIPRPLPRPFPR PI 77
33 RRRPRPFPRPFPRPFPRPFPRPFPRPF 79
34 RRRPRPFPRPFPRPFPRPFPRPFPRPL 74
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35 RRRPRPFPRPFPRPFPRPFPRPIPRPF 78
36 RRRPRPFPRPFPRPFPRPFPRPIPRPI 77
37 RRRPRPFPRPFPRPFPRPIPRPFPRPV 68
38 RRRPRPFPRPFPRPFPRPIPRPVPRPI 64
39 RRRPRPFPRPFPRPFPRPLPRPFPRPF 56
40 RRRPRPFPRPFPRPFPRPVPRPLPRPI 69
41 RRRPRPFPRPFPRPFPRPVPRPLPRPV 71
42 RRRPRPFPRPFPRPIPRPFPRPLPRPI 66
43 RRRPRPFPRPFPRPIPRPFPRPVPRPV 68
44 RRRPRPFPRPFPRPLPRPFPRPVPRPF 95
45 RRRPRPFPRPFPRPLPRPIPRPFPRPV 69
46 RRRPRPFPRPFPRPVPRPFPRPFPRPI 84
47 RRRPRPFPRPFPRPVPRPIPRPFPRPF 92
48 RRRPRPFPRPFPRPVPRPVPRPVPRPF 59
49 RRRPRPFPRPLPRPVPRPFPRPVPRPL 51
50 RRRPRPFPRPLPRPVPRPFPRPVPRPV 69
51 RRRPRPFPRPVPRPFPRPFPRPIPRPF 77
52 RRRPRPFPRPVPRPIPRPFPRPVPRPF 53
53 RRRPRPFPRPVPRPIPRPFPRPVPRPV 62
54 RRRPRPFPRPVPRPVPRPFPRPIPRPL 65
55 RRRPRPFPRPVPRPVPRPFPRPLPRPI 63
56 RRRPRPIPRPFPRPFPRPFPRPFPRPI 51
57 RRRPRPIPRPFPRPIPRPVPRPFPRPF 73
58 RRRPRPIPRPFPRPIPRPVPRPFPRPF 77
59 RRRPRPIPRPFPRPIPRPVPRPFPRPF 75
60 RRRPRPIPRPFPRPVPRPVPRPFPRPF 70
61 RRRPRPVPRPFPRPVPRPLPRPFPRPF 97
62 RRRPRPIPRPIPRPFPRPFPRPFHAQSNCSRTKTHLRRGSE 77
63 RRRPRPFPRPFPRPVPRPFPRPFPRQSNCSRTKTHLRRGSE 80
Table 4. Variants of SEQ ID NO:10 and the corresponding growth inhibitions.
SEQ ID NO Amino acid sequence Growth
inhibition
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64 RRRPFRPPFRPPFRPPFRPPFRPPFRP 82
65 RRRPLRPPLRPPVRPPVRPPFRPPFRP 89
66 RRRPFRPPFRPPFRPPIRPPLRPPIRP 76
67 RRRPLRPPLRPPIRPPFRPPVRPPFRP 80
68 RRRPIRPPFRPPFRPPLRPPVRPPFRP 78
69 RRRPIRPPFRPPFRPPLRPPFRPPIRP 75
70 RRRPLRPPLRPPFRPPIRPPLRPPFRP 76
71 RRRPLRPPIRPPFRPPIRPPFRPPFRP 82
72 RRRPFRPPFRPPFRPPFRPPFRPPFRP 49
73 RRRPFRPPVRPPFRPPVRPPIRPPVRP 65
74 RRRPFRPPVRPPFRPPFRPPIRPPIRP 76
75 RRRPFRPPIRPPVRPPFRPPFRPPFRP 79
76 RRRPIRPPFRPPIRPPVRPPFRPPIRP 86
77 RRRPFRPPFRPPIRPPVRPPFRPPFRP 72
78 RRRPFRPPFRPPFRPPFRPPFRPPFRP 83
79 RRRPFRPPFRPPFRPPVRPPVRPPFRP 82
80 RRRPFRPPFRPPIRPPVRPPVRPPFRP 81
81 RRRPFRPPIRPPFRPPFRPPVRPPIRP 80
82 RRRPIRPPFRPPVRPPFRPPFRPPFRP 71
83 RRRPLRPPFRPPIRPPVRPPFRPPVRP 72
84 RRRPIRPPLRPPIRPPFRPPFRPPLRP 80
85 RRRPFRPPIRPPIRPPFRPPFRPPIRP 67
86 RRRPIRPPFRPPLRPPLRPPVRPPFRP 74
87 RRRPFRPPFRPPIRPPFRPPVRPPVRP 80
88 RRRPFRPPLRPPLRPPVRPPVRPPFRP 82
89 RRRPFRPPFRPPFRPPVRPPVRPPLRP 70
90 RRRPFRPPIRPPFRPPFRPPVRPPFRP 74
91 RRRPFRPPVRPPIRPPVRPPFRPPVRP 52
92 RRRPVRPPIRPPFRPPFRPPFRPPFRP 85
93 RRRPFRPPFRPPFRPPVRPPLRPPIRP 61
94 RRRPFRPPVRPPIRPPVRPPFRPPFRP 77
95 RRRPFRPPLRPPLRPPIRPPVRPPFRP 80
96 RRRPFRPPIRPPFRPPIRPPLRPPIRP 71
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97 RRRPLRPPFRPPFRPPLRPPFRPPIRP 69
98 RRRPFRPPFRPPVRPPIRPPLRPPFRP 63
99 RRRPFRPPIRPPIRPPFRPPIRPPFRP 79
100 RRRPIRPPFRPPFRPPFRPPFRPPFRP 84
101 RRRPIRPPFRPPLRPPFRPPVRPPVRP 81
102 RRRPIRPPFRPPIRPPFRPPLRPPFRP 77
103 RRRPLRPPIRPPIRPPVRPPFRPPIRP 73
104 RRRPFRPPFRPPIRPPFRPPFRPPLRP 76
105 RRRPIRPPLRPPLRPPFRPPFRPPFRP 73
106 RRRPFRPPFRPPFRPPIRPPIRPPLRP 75
107 RRRPFRPPFRPPVRPPFRPPIRPPFRP 69
108 RRRPLRPPFRPPIRPPVRPPVRPPFRP 76
109 RRRPFRPPIRPPVRPPLRPPVRPPFRP 71
110 RRRPFRPPLRPPFRPPIRPPIRPPVRP 73
111 RRRPLRPPVRPPFRPPVRPPLRPPFRP 59
112 RRRPFRPPIRPPFRPPVRPPVRPPVRP 69
113 RRRPLRPPVRPPFRPPVRPPFRPPFRP 75
114 RRRPLRPPLRPPFRPPLRPPFRPPFRP 74
115 RRRPLRPPFRPPIRPPFRPPFRPPFRP 79
116 RRRPIRPPFRPPFRPPVRPPIRPPFRP 69
117 RRRPIRPPIRPPFRPPFRPPIRPPFRP 77
118 RRRPFRPPFRPPLRPPFRPPVRPPLRP 73
119 RRRPIRPPFRPPIRPPFRPPVRPPVRP 69
120 RRRPLRPPFRPPFRPPFRPPVRPPIRP 68
121 RRRPIRPPLRPPVRPPIRPPFRPPFRP 65
122 RRRPFRPPFRPPFRPPVRPPFRPPLRP 64
123 RRRPFRPPLRPPIRPPIRPPFRPPFRP 65
124 RRRPFRPPIRPPLRPPVRPPLRPPFRP 73
125 RRRPIRPPFRPPLRPPFRPPVRPPVRP 73
126 RRRPIRPPIRPPLRPPFRPPFRPPFRP 77
127 RRRPIRPPFRPPLRPPFRPPFRPPLRP 67
128 RRRPFRPPFRPPLRPPFRPPVRPPLRP 69
129 RRRPFRPPFRPPVRPPFRPPIRPPLRP 69
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130 RRRPFRPPIRPPLRPPFRPPVRPPLRP 78
131 RRRPIRPPFRPPFRPPIRPPIRPPVRP 75
132 RRRPFRPPFRPPLRPPVRPPVRPPIRP 72
133 RRRPFRPPIRPPFRPPVRPPIRPPVRP 62
134 RRRPFRPPFRPPFRPPVRPPLRPPVRP 68
135 RRRPIRPPFRPPIRPPIRPPIRPPVRP 38
136 RRRPFRPPFRPPIRPPFRPPIRPPLRP 77
137 RRRPLRPPFRPPFRPPIRPPFRPPVRP 58
138 RRRPLRPPFRPPVRPPFRPPFRPPFRP 78
139 RRRPFRPPFRPPIRPPFRPPIRPPVRP 67
140 RRRPIRPPIRPPFRPPVRPPVRPPFRP 70
141 RRRPIRPPIRPPFRPPFRPPLRPPIRP 65
142 RRRPLRPPFRPPVRPPFRPPVRPPFRP 70
143 RRRPFRPPVRPPVRPPFRPPLRPPVRP 43
144 RRRPIRPPLRPPVRPPVRPPFRPPFRP 64
145 RRRPLRPPVRPPFRPPFRPPLRPPFRP 59
146 RRRPLRPPFRPPIRPPFRPPFRPPLRP 68
147 RRRPFRPPFRPPIRPPVRPPVRPPFRP 79
148 RRRPIRPPFRPPFRPPFRPPFRPPLRP 76
149 RRRPIRPPIRPPFRPPFRPPVRPPVRP 62
150 RRRPIRPPIRPPFRPPIRPPFRPPVRP 68
151 RRRPLRPPVRPPLRPPVRPPFRPPFRP 68
152 RRRPFRPPFRPPFRPPFRPPVRPPIRP 75
153 RRRPIRPPFRPPIRPPVRPPIRPPFRP 68
154 RRRPFRPPFRPPVRPPFRPPFRPPFRP 75
155 RRRPFRPPFRPPFRPPFRPPFRPPLRP 83
156 RRRPFRPPLRPPFRPPFRPPIRPPFRP 67
157 RRRPIRPPFRPPIRPPFRPPVRPPLRP 64
158 RRRPIRPPFRPPIRPPIRPPIRPPVRP 67
159 RRRPVRPPIRPPIRPPVRPPLRPPVRP 35
160 RRRPFRPPVRPPFRPPVRPPFRPPFRP 77
161 RRRPFRPPVRPPIRPPVRPPFRPPFRP 78
162 RRRPFRPPIRPPFRPPFRPPLRPPLRP 68
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163 RRRPIRPPFRPPFRPPFRPPIRPPIRP 79
164 RRRPFRPPIRPPLRPPFRPPFRPPFRP 78
165 RRRPFRPPLRPPFRPPVRPPLRPPVRP 65
166 RRRPLRPPVRPPFRPPVRPPVRPPLRP 55
167 RRRPFRPPLRPPFRPPLRPPLRPPVRP 58
168 RRRPLRPPFRPPIRPPVRPPLRPPFRP 71
169 RRRPIRPPLRPPVRPPFRPPVRPPFRP 67
170 RRRPIRPPFRPPFRPPFRPPVRPPFRP 80
171 RRRPIRPPIRPPIRPPIRPPFRPPFRP 77
172 RRRPFRPPIRPPFRPPIRPPVRPPFRP 82
173 RRRPLRPPIRPPLRPPVRPPFRPPFRP 81
174 RRRPIRPPIRPPFRPPLRPPFRPPFRP 80
175 RRRPFRPPVRPPFRPPLRPPFRPPFRP 77
176 RRRPLRPPVRPPIRPPIRPPFRPPFRP 81
177 RRRPFRPPIRPPFRPPIRPPVRPPIRP 75
178 RRRPLRPPFRPPVRPPFRPPFRPPFRP 81
179 RRRPFRPPIRPPIRPPFRPPFRPPFRP 87
180 RRRPLRPPIRPPIRPPIRPPFRPPFRP 76
181 RRRPIRPPIRPPFRPPFRPPFRPPFRP 83
182 RRRPFRPPIRPPFRPPIRPPVRPPFRP 83
183 RRRPFRPPVRPPLRPPFRPPFRPPFRP 80
184 RRRPIRPPIRPPFRPPLRPPFRPPFRP 78
185 RRRPLRPPIRPPIRPPFRPPFRPPFRP 79
186 RRRPIRPPIRPPIRPPFRPPFRPPFRP 77
187 RRRPIRPPFRPPFRPPVRPPFRPPFRP 85
188 RRRPFRPPFRPPIRPPVRPPVRPPFRP 83
189 RRRPFRPPFRPPIRPPVRPPIRPPFRP 79
190 RRRPFRPPIRPPFRPPIRPPFRPPFRP 83
191 RRRPFRPPFRPPIRPPFRPPVRPPFRP 77
192 RRRPFRPPFRPPFRPPVRPPVRPPFRP 92
193 RRRPFRPPIRPPFRPPIRPPFRPPFRP 81
194 RRRPLRPPFRPPFRPPIRPPFRPPFRP 81
195 RRRPFRPPIRPPFRPPVRPPVRPPVRP 76
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196 RRRPFRPPLRPPIRPPIRPPFRPPFRP 77
197 RRRPFRPPFRPPFRPPFRPPLRPPFRP 86
198 RRRPFRPPFRPPFRPPFRPPFRPPFRP 91
199 RRRPLRPPIRPPFRPPVRPPFRPPFRP 80
200 RRRPFRPPFRPPLRPPFRPPVRPPFRP 87
201 RRRPFRPPIRPPIRPPFRPPIRPPFRP 78
202 RRRPFRPPFRPPFRPPLRPPFRPPFRP 84
203 RRRPFRPPIRPPFRPPIRPPFRPPFRP 79
204 RRRPLRPPLRPPFRPPIRPPFRPPFRP 76
205 RRRPFRPPFRPPIRPPVRPPFRPPFRP 77
206 RRRPFRPPFRPPFRPPFRPPFRPPFRP 77
EXAMPLE 4
Evaluation of synthetic PR-rich AMPs from a mixed-motif library
The AMPs tested in Example 3 consisted of degenerate but otherwise identical
repeats. To test whether the 12 different "4 amino acid sequence motifs" when
mixed in one
sequence would give rise to potent AMPs, a new library was constructed. The
parent
backbone in this series was composed of the following sequence motifs: PPRX,
PRPX,
PXRP, PPRX, PRPX and PXRP; where X denotes any of the hydrophobic amino acids
valine,
phenylalamine, isoleucine or leucine. Again, an equal ratio of hydrophobic
amino acids was
used.
The library was constructed as in Example 3 above with one long forward primer
encoding the entire peptide (Mixed-motif degenerate primer) and a smaller
reverse primer for
synthesis of the reverse strand (Primerl3). N represent 25% of each of the 4
nucleotides (G,
A, T and C).
Mixed-motif degenerate primer (SEQ ID NO:340)
CCATAGCACC ATGGCGCGTC GCCGTCCGCC ACGTNTTCCA CGTCCTNTTC CANTTCGTCC
ACCGCCACGT NTTCCACGTC CTNTTCCANT TCGTCCATAA TTGCTCTAGA ACAAAAACTC
The PCR synthesized DNA fragments were cloned into the SES vector pBAD/gIIIA
and transformed into E. co/i to a library of approximately a million colonies
each.
The library was plated and around 10,000 individual colonies were picked into
96-well
microtiter plates containing 20 x diluted RM + 200 pg/ml Ampicillin by a
colony picker. These
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master plates were replica-plated into a new set of 96-well plates containing
20 x diluted RM
media and either 0.1% or 0% inducer and incubated overnight at 37 degrees
Celsius. A
number of the most growth inhibited clones were isolated, sequenced and re-
tested for
growth inhibition in the Bioscreen C.
The amino acid sequences of the variants from the mixed-motif library and the
corresponding growth inhibitions are shown in Table 5.
Table 5. Synthetic PR-rich AMPs with a mixed-motif.
SEQ ID NO Amino acid sequence Growth
inhibition
207 RRRPPRFPRPFPFRPPPRFPRPIPFRP 93
208 RRRPPRFPRPFPVRPPPRFPRPIPVRP 95
209 RRRPPRLPRPFPFRPPPRFPRPIPFRP 78
210 RRRPPRIPRPIPVRPPPRIPRPIPFRP 94
211 RRRPPRLPRPFPFRPPPRFPRPIPFRP 96
212 RRRPPRLPRPFPFRPPPRFPRPVPIRP 96
213 RRRPPRLPRPFPFRPPPRLPRPFPIRP 94
214 RRRPPRLPRPFPFRPPPRFPRPIPFRP 95
215 RRRPPRLPRPFPFRPPPRPRPFPFRP 94
216 RRRPPRLPRPFPFRPPPRPRPLPVRP 95
217 RRRPPRLPRPFPFRPPPRVPRPFPIRP 93
218 RRRPPRLPRPFPFRPPPRVPRPIPVRP 93
219 RRRPPRLPRPFPFRPPPRVPRPLPFRP 93
220 RRRPPRLPRPFPIRPPPRFPRPFPVRP 90
221 RRRPPRLPRPFPIRPPPRFPRPLPFRP 93
222 RRRPPRLPRPFPIRPPPRFPRPVPLRP 93
223 RRRPPRLPRPFPIRPPPRLPRPLPFRP 90
224 RRRPPRLPRPFPIRPPPRVPRPFPVRP 93
225 RRRPPRLPRPFPFRPPPRVPRPIPVRP 97
226 RRRPPRLPRPFPFRPPPRVPRPIPVRP 94
227 RRRPPRLPRPFPFRPPPRVPRPVPIRP 94
228 RRRPPRLPRPFPLRPPPRFPRPFPIRP 96
229 RRRPPRLPRPFPLRPPPRFPRPFPLRP 95
230 RRRPPRLPRPFPLRPPPRFPRPLPLRP 94
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231 RRRPPRLPRPFPLRPPPRLPRPFPFRP 98
232 RRRPPRLPRPFPLRPPPRLPRPFPVRP 92
233 RRRPPRLPRPFPLRPPPRLPRPFPFRP 91
234 RRRPPRLPRPFPLRPPPRVPRPLPIRP 93
235 RRRPPRLPRPFPLRPPPRVPRPVPFRP 94
236 RRRPPRLPRPFPLRPPPRVPRPVPFRP 91
237 RRRPPRLPRPFPLRPPPRVPRPVPVRP 96
238 RRRPPRLPRPFPVRPPPRFPRPFPIRP 95
239 RRRPPRLPRPFPVRPPPRFPRPFPLRP 99
240 RRRPPRLPRPFPVRPPPRFPRPVPFRP 91
241 RRRPPRLPRPFPVRPPPRFPRPVPFRP 96
242 RRRPPRLPRPFPVRPPPRLPRPVPFRP 90
243 RRRPPRLPRPFPVRPPPRLPRPVPFRP 91
244 RRRPPRLPRPFPVRPPPRLPRPVPFRP 90
245 RRRPPRLPRPFPVRPPPRVPRPFPVRP 95
246 RRRPPRLPRPFPVRPPPRVPRPFPVRP 95
247 RRRPPRLPRPFPVRPPPRVPRPLPFRP 93
248 RRRPPRLPRPFPVRPPPRVPRPVPIRP 94
249 RRRPPRLPRPFPVRPPPRVPRPVPIRP 93
250 RRRPPRLPRPIPFRPPPRIPRPIPVRP 96
251 RRRPPRLPRPIPFRPPPRIPRPLPFRP 96
252 RRRPPRLPRPIPFRPPPRIPRPVPVRP 92
253 RRRPPRLPRPIPFRPPPRLPRPIPIRP 91
254 RRRPPRLPRPIPFRPPPRVPRPFPIRP 77
255 RRRPPRLPRPFPVRPPPRFPRPVPFRP 93
256 RRRPPRLPRPIPLRPPPRVPRPIPIRP 91
257 RRRPPRLPRPIPVRPPPRFPRPIPFRP 92
258 RRRPPRLPRPIPVRPPPRFPRPIPFRP 92
259 RRRPPRLPRPIPVRPPPRFPRPIPVRP 98
260 RRRPPRLPRPIPVRPPPRIPRPIPVRP 90
261 RRRPPRLPRPIPVRPPPRIPRPVPFRP 92
262 RRRPPRLPRPIPVRPPPRLPRPVPLRP 93
263 RRRPPRLPRPIPVRPPPRVPRPIPLRP 94
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264 RRRPPRLPRPLPFRPPPRFPRPVPVRP 92
265 RRRPPRLPRPLPFRPPPRLPRPFPVRP 94
266 RRRPPRLPRPLPFRPPPRLPRPVPVRP 94
267 RRRPPRLPRPLPFRPPPRVPRPFPIRP 94
268 RRRPPRLPRPLPFRPPPRVPRPVPLRP 93
269 RRRPPRLPRPLPFRPPPRVPRPVPVRP 90
270 RRRPPRLPRPLPFRPPPRVPRPVPVRP 90
271 RRRPPRLPRPLPFRPPPRVPRPVPVRP 97
272 RRRPPRLPRPLPIRPPPRFPRPIPVRP 91
273 RRRPPRLPRPLPIRPPPRFPRPVPLRP 92
274 RRRPPRLPRPLPIRPPPRFPRPVPVRP 93
275 RRRPPRLPRPLPIRPPPRLPRPFPVRP 92
276 RRRPPRLPRPLPIRPPPRLPRPVPVRP 94
277 RRRPPRLPRPLPIRPPPRVPRPFPIRP 91
278 RRRPPRLPRPLPIRPPPRVPRPLPFRP 98
279 RRRPPRLPRPLPIRPPPRVPRPLPLRP 91
280 RRRPPRLPRPLPFRPPPRFPRPFPVRP 97
281 RRRPPRLPRPLPLRPPPRFPRPVPLRP 91
282 RRRPPRLPRPLPLRPPPRLPHPIPFRP 95
283 RRRPPRLPRPLPVRPPPRFPRPVPFRP 95
284 RRRPPRLPRPLPVRPPPRFPRPVPVRP 89
285 RRRPPRLPRPLPVRPPPRVPRPFPVRP 91
286 RRRPPRLPRPLPVRPPPRVPRPLPLRP 95
287 RRRPPRLPRPLPFRPPPRLPRPFPIRP 94
288 RRRPPRLPRPLPVRPPPRVPRPFPLRP 96
289 RRRPPRLPRPLPVRPPPRVPRPFPVRP 95
290 RRRPPRLPRPLPVRPPPRVPRPLPLRP 94
291 RRRPPRLPRPLPVRPPPRVPRPVPFRP 90
292 RRRPPRLPRPLPVRPPPRVPRPVPFRP 93
293 RRRPPRLPRPVPFRPPPRFPRPFPIRP 93
294 RRRPPRLPRPVPFRPPPRFPRPVPLRP 95
295 RRRPPRLPRPVPFRPPPRIPRPFPLRP 59
296 RRRPPRLPRPVPFRPPPRVPRPIPFRP 88
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297 RRRPPRLPRPVPFRPPPRVPRPVPLRP 90
298 RRRPPRLPRPVPFRPPPRVPRPVPLRP 92
299 RRRPPRLPRPVPIRPPPRFPRPIPFRP 96
300 RRRPPRLPRPVPIRPPPRIPRPIPFRP 92
301 RRRPPRLPRPVPIRPPPRVPRPVPLRP 92
302 RRRPPRLPRPVPLRPPPRFPRPVPVRP 82
303 RRRPPRLPRPVPLRPPPRVPRPLPIRP 91
304 RRRPPRLPRPVPVRPPPRFPRPVPIRP 90
305 RRRPPRLPRPVPVRPPPRFPRPVPVRP 90
306 RRRPPRLPRPVPVRPPPRFPRPVPVRP 90
307 RRRPPRLPRPVPVRPPPRVPRPVPFRP 92
308 RRRPPRLPRPVPVRPPPRVPRPVPVRP 96
309 RRRPPRVPRPFPIRPPPRVPRPVPFRP 76
310 RRRPPRVPRPFPIRPPPRVPRPVPVRP 93
311 RRRPPRVPRPFPVRPPPRFPRPVPFRP 96
312 RRRPPRVPRPIPIRPPPRVPRPVPFRP 93
313 RRRPPRVPRPIPIRPPPRVPRPVPVRP 92
314 RRRPPRVPRPLPFRPPPRFPRPIPFRP 94
315 RRRPPRLPRPVPVRPPPRFPRPFPVRP 98
316 RRRTRHVFARPFPFRPPPRIPRPFPLRP 97
317 RRRTRHVFARPVPVRPPPRVPRPFPVRP 95
318 RRRTRHVVPRPLPVRPPPRVPRPFPLRP 93
319 RRRPPRVPRPFPVRPPPRVPRPIPFVHNCSRTKTHLRRGSE 93
EXAMPLE 5
Determination of the MIC, MBC and MEC of selected peptides
To verify the antimicrobial potency of the peptides identified and described
in
Examples 1-4, the Minimal Inhibitory Concentration (MIC), Minimal Bactericidal
Concentration
(MBC) and Minimal Effective Concentration (MEC) of three structurally
different peptides were
determined against a range of microorganisms.
The following three peptides were selected for chemical synthesis and
determination
of antimicrobial potency:
PR-1 (RRR-PRPV-PRPF-PRPV-PRPL-PRPF-PRPF) (SEQ ID NO:61)
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PR-2 (RRR-PFRP-PFRP-PFRP-PVRP-PVRP-PFRP) (SEQ ID NO:79)
PR-3 (RRR-PPRL-PRPF-PVRP-PPRF-PRPF-PLRP) (SEQ ID NO:239)
The tested microorganisms included E. coli (ATCC 10536), S. carnosus, S.
simulans,
M. luteus (ATCC 9341), B. subtilis (ATCC 6633) and S. cerevisiae (ATCC 9763).
The MIC and MBC determinations were carried out in accordance to the NCCLS
protocol with the minor modification that the peptide was dissolved in 0.01 %
Acetic acid and
0.1 % BSA to avoid precipitation at high concentrations and minimize peptide
binding to plastic
containers. The MEC determinations were done according to Steinberg and Lehrer
(Methods
1o in Molecular Biology, Vol. 78: Antibacterial Peptide Protocols) in a radial
diffusion assay setup.
The MEC protocol circumvents some of the problems associated testing
antimicrobial
peptides with the NCCLS protocol and provides a very sensitive test setup. The
results were
as shown in tables 6-8.
Table 6. MIC determination (all values are pg/mI)
Peptide E. coli S. carnosus S. simulans M. luteus B. subtilis S. cerevisiae
PR-1 128 4 16 64 16 >128
PR-2 64 16 64 64 64 >128
PR-3 64 4 8 64 4 >128
Table 7. MBC determination (all values are pg/ml)
Peptide E. coli S. carnosus S. simulans M. luteus B. subtilis S. cerevisiae
PR-1 128 64 64 >128 64 >128
PR-2 >128 >128 >128 64 64 >128
PR-3 128 64 32 >128 64 >128
Table 8. MEC determination (all values are pg/ml)
Peptide E. coli S. carnosus S. simulans M. luteus B. subtilis S. cerevisiae
PR-1 3.0 1.7 0.8 0.4 2.4 32
PR-2 3.0 2.0 1.1 0.5 3.6 32
PR-3 3.0 1.8 0.9 0.8 2.0 >128
It is evident that the three PR-rich peptides are broadly active against Gram-
positive
and Gram-negative organisms under defined conditions.
EXAMPLE 6
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Hemolytic activity of PR-1, PR-2 and PR-3
The ability of the antimicrobial peptides to lyse human red blood cells (hRBC)
was
analyzed. The Red Blood Cell Test assay provides a simple test system that
assesses the
membranolytic activity of test materials. It is suitable as a screening assay
for hemolytic
properties. Hemoglobin release is a useful endpoint of cell membrane
integrity. Released
hemoglobin is detected by measuring absorbance at 540 nm. Normally, the
concentration
resulting in 50% hemolysis relative to a totally lysed sample (HL50) would be
calculated;
however due to the low hemolytic activity of the tested peptides, this was not
done in this
Example. An 8% RBC suspension (12 days old) was used in the present test
setup.
In Table 9 is shown the individual hemolysis values relative to a 100 l0 lysed
sample.
Table 9. Hemolysis values.
Peptide Hemolysis (%)
concentration
(pg/mI) PR-1 PR-2 PR-3
0 15 15 15
47 16 16 19
94 16 16 16
188 16 17 16
375 17 17 18
Since the peptides were dissolved in 0.01% acetic acid, vehicle control of the
0.01%
acetic acid was included. PBS with 25%, 10%, 5% 0.01% acetic acid was included
on both
plates. No hemolytic reaction different from the background was observed at
any of the tested
concentrations.
To verify that a dose/response effect exists, the hemolytic activity of SDS
was also
tested.
Table 10. SDS dose/response values.
SDS concentration Hemolysis (%)
(pg/ml)
12.5 17
26
50 68
100 88
The three tested peptide variants, PR-1, PR-2 and PR-3, do not show hemolytic
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properties significantly different from the background level, under the
conditions of the assay.
The level of spontaneous hemolysis is high in this assay, probably due to the
age of
the red blood cells (12 days). The relatively high level of spontaneous
hernolysis does not
interfere with the conclusion that the three peptides are non-hemolytic at the
concentrations
tested.
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