Note: Descriptions are shown in the official language in which they were submitted.
CA 02485178 2004-11-22
LACTIC ACID BACTERIA-DERIVED BACTERIOCIN AND USES THEREOF FOR
PREVENTION OR TREATMENT OF CANCER
FIELD OF THE INVENTION
The invention relates to the use of bacteriocins
and compositions comprising such bacteriocins for the
treatment and/or prevention of cancer. The invention also
relates to the use of bacteriocins and compositions
comprising such bacteriocins for inhibiting proliferation of
cancerous cells. The invention particularly relates to
bacteriocins derived from lactic acid bacteria and
compositions comprising such bacteriocins.
BACKGROUND OF THE INVENTION
Conventional chemotherapeutic agents are usually
not specific towards cancerous cells and halt the progression
of any dividing cells. As such, these drugs are highly
cytotoxic and can cause serious side effects (loss of hair,
loss of intestine villosities, depletion of immune cells,
etc.) and can impair significantly the quality of life of the
patient (infertility, depression, etc.). Consequently, their
use if often limited by the amount one can tolerate.
In addition, the use of conventional
chemotherapeutic agents is also known to cause drug
resistance in cancerous cells, and often multi-drug
resistance. Hence, clinicians have to readjust, over the
course of the treatment, the dosage and/or the composition of
chemotherapeutic cocktails to take into account the newly
acquired drug resistance phenotype of the cancerous cells.
Further, to date, many cancers cannot be treated
and represent a significant percentage of mortality.
There is thus a need for improved chemotherapeutic
agents. These agents may be more effective, more selective
towards cancerous cells, may riot cause drug resistance and/or
CA 02485178 2004-11-22
2
may be used to halt the progression of multi-drug resistant
cancerous cells.
SUI~1ARY OF THE INVENTION
The present invention relates to the use of a
bacteriocin, and more particularly to the use of a
bacteriocin for the treatment and prevention of cancer.
In a first aspect, the present invention provides a
method of preventing or treating cancer in an animal, the
method comprising administering to the animal an agent
selected from the group consisting of (a) a bacteriocin
derived from a lactic-acid bacteria, and (b) a composition
comprising a bacteriocin derived from a lactic acid bacteria
and a carrier (or any combinations thereof).
In an embodiment, the agent is cytotoxic to the
cancerous cell of the cancer. In another embodiment, the
agent is administered through or adapted for administration
through a route selected from the group consisting of
intravenous, oral, transdermal, subcutaneous, mucosal,
intramuscular, intranasal, intrapulmonary, parenteral,
intrarectal, intratumoral and topical. In yet another
embodiment, the agent is the bacteriocin and, in a further
embodiment, the bacteriocin is substantially pure. In
another embodiment, the bacteriocin is a recombinant
bacteriocin and, in a further embodiment, the recombinant
bacteriocin is produced in a prokaryotic host. In yet
another embodiment, the bacter_iocin is selected from the
group consisting of bavaricin, helveticin, acidocin,
lactocin, lactacin, lacticin, nisin, leucocin, lactococcin,
pediocin, curvaticin, curvacin, mutacin, mesentericin,
plantaricin, streptin and sakacin. In another embodiment,
the bacteriocin is pediocin arid, in a further embodiment, the
bacteriocin is pediocin PA-1. In yet another embodiment, the
CA 02485178 2004-11-22
3
pediocin PA-1 comprises an amino acid sequence substantially
identical to the sequence set forth in SEQ ID NO: 2 or a
fragment thereof. In yet a further embodiment, the
bacteriocin is derived from a lactic acid bacteria species
selected from the group consisting of Streptococcus spp.,
Lactococcus spp., Lactobacillus spp., Pediococcus spp.,
Leuconostoc spp., Bifidobacterium spp. and Enterococcus spp.
In an embodiment, the bacteriocin is derived from Pediococcus
spp, in a further embodiment, the bacteriocin is derived from
Pediococcus acidilactici and, in yet a further embodiment,
the bacteriocin is derived from Pediococcus acidilactici PAC
1Ø
In an embodiment, the agent is the above-mentioned
composition. In another embodiment, the carrier is selected
from the group consisting of a pharmaceutically acceptable
carrier and a nutraceutically acceptable carrier and, in a
further embodiment, the carrier is a nutraceutically
acceptable carrier. In yet another embodiment, the agent is
administered through or adapted for administreation through
an oral route. In another embodiment, the composition is
substantially free of contaminants from the lactic acid
bacteria from which said bacteriocin is derived. In an
embodiment, the nutraceutically acceptable carrier is a food
product, in a further embodiment, the food product is a
fermented food product and, in yet a further embodiment, the
fermented food product is a fermented milk. In another
embodiment, the animal is a mammal and, in yet a further
embodiment, the mammal is a human. In an embodiment, the
cancer affects an organ, tissue, cell or system selected from
the group consisting of bone, soft tissue, brain, spinal
cord, breast, adrenal gland, pancreas, parathyroid,
pituitary, thyroid, anus, colon, rectum, esophagus,
gallbladder, stomach, liver, cervix, endometrium, uterus,
CA 02485178 2004-11-22
4
fallopian tube, ovaries, vagina, vulva, larynges,
oropharynges, immune cell, immune system, lung, lymph node
and plasma cell. In another embodiment, the
cancer is lung cancer and/or colon cancer.
In another aspect, the present invention provides
use of an agent selected from the group consisting of (a) a
bacteriocin derived from a lactic acid bacteria and (b) a
composition comprising a bacteriocin derived from a lactic
acid bacteria and a carrier for the prevention or treatment
of cancer in an animal. In a further aspect, the present
invention provides use of an agent selected from the group
consisting of (a) a bacteriocin derived from a lactic acid
bacteria and (b) a composition comprising a bacteriocin
derived from a lactic acid bacteria and a carrier for the
preparation of a medicament for the prevention or treatment
of cancer in an animal. In an embodiment, the agent used is
the agent described herein. In yet another embodiment, the
animal is the animal described herein. In another
embodiment, the cancer treated or prevented is the cancer
described herein.
In yet another aspect, the present invention
provides a bacteriocin derived from a lactic acid bacteria
for use in the prevention or treatment of cancer in an
animal.
In still a further aspect, the present invention
provides a composition comprising (i) a bacteriocin derived
from a lactic acid bacteria and (ii) a carrier for use in the
prevention or treatment of cancer in an animal.
In still another aspect, the present invention
provides a package comprising (a) an agent selected from the
group consisting of (i) a bacteriocin derived from a lactic
acid bacteria and (ii) a composition comprising a bacteriocin
derived from a lactic acid bacteria and a carrier and (b)
CA 02485178 2004-11-22
instructions for the use of said agent in the treatment or
prevention of cancer in an animal. In an embodiment, the
agent used is the agent described herein. In yet another
embodiment, the animal is the animal described herein. In
5 another embodiment, the instructions indicate the use of the
agent for the treatment or prevention of a cancer described
herein.
In still another aspect, the present invention
provides a method of inhibiting proliferation of a cancerous
cell in an animal, said method comprising administering to
said animal a bacteriocin derived from a lactic acid
bacteria. In an embodiment, the bacteriocin used is the
bacteriocin described herein. In yet another embodiment, the
animal is the animal described herein. In another
embodiment, the cancer treated or prevented is the cancer
described herein.
In another aspect, the present invention provides a
method of inhibiting proliferation of a cancerous cell, said
method comprising contacting said cell with a bacteriocin
derived from a lactic acid bacteria. In an embodiment, the
bacteriocin used is the bacteriocin described herein. In yet
another embodiment, the animal is the animal described
herein. In another embodiment, the cancer treated or
prevented is the cancer described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Pediococcus acidilactici bacteriocin complete
coding sequence (SEQ ID NO: 1, GenBank accession number
M83924)
Figure 2. Pediocin PA-1 polypeptide sequence (SEQ ID N0: 2,
GenBank accession number AAA25559)
CA 02485178 2004-11-22
6
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect, the invention relates to a
method of preventing and/or treating cancer. As used herein
the terms "preventing", "prevention", "treating" and
"treatment" relate to the retardation or inhibition of the
onset of a cancer and/or progression of a cancer (e. g.
formation and growth of a primary tumor, dissemination of
metastases, growth of metastases, involvement of lymph nodes
etc.). The invention also relates to the inhibition of
proliferation of cancerous cells. The term "cancer" as used
herein relates to the malignant growth of cells. The cancer
may be restricted to one organ, tissue or cell type or may be
disseminated throughout the body and affect several organs,
tissues or cell types. In an embodiment, the cancer may
affect bones (e. g. sarcoma, osteosarcoma, rhabdomyosarcoma),
brain or spinal cord (e. g. oligodendroglioma, ependymoma,
meningioma, lymphoma, schwannoma, medulloblastoma), breasts
(e.g. carcinoma in situ such as lobular carcinoma in situ and
ductal carcinoma in situ, stage I, II, II and IV carcinoma),
endocrine system (e. g. adrenal cancer or pheochromocytoma,
pancreatic cancer, parathyroid cancer, pituitary tumors,
thyroid cancer), gastrointestinal system (e. g. carcinoma,
adenocarcinoma, anal cancer, colon cancer, rectal cancer,
esophageal cancer, gallbladder cancer, gastric cancer, liver
cancer such as hepatocellular carcinoma, cholangiocarcinoma,
hemangiosarcoma and hepatoblastoma, pancreatic cancer, cancer
of the small intestine), reproductive system (e. g. cervical
cancer, endometrial cancer, uterine cancer, fallopian tube
cancer, gestational trophoblastic disease and
choriocarcinoma, ovarian cancer, vaginal cancer, vulvar
cancer), head and neck (e. g. laryngeal cancer, oropharyngeal
cancers, parathyroid cancer, t=hyroid cancer), immune system
(e.g acute lymphocytic leukemia (ALL), acute myelogenous
CA 02485178 2004-11-22
leukemia (AML), chronic lymphocytic leukemia (CLL), chronic
myelogenous leukemia (CML), hairy cell leukemia,
myeloproliferative disorders), lungs (e. g. mesothelioma, non-
small cell lung cancer, small-cell lung cancer), lymph nodes
(e. g. AIDS-related lymphoma, cutaneous T-cell
lymphoma/mucosis fungoides, Hodgkin's disease, non-Hodgkin's
disease), and plasma (e. g. multiple myeloma).
According to another aspect, the invention also
provides a method of preventing or treating cancer in an
animal. In an embodiment, the animal is a mammal, and, in a
further embodiment the mammal is a human. In another
embodiment, the methods described herein can be used to treat
other animals or other mammals (such as a cat, a dog or a
horse).
According to yet another aspect, the methods
described herein comprise administering an agent to said
animal. In an embodiment, the agent is cytotoxic to
cancerous cells. As used herein, the term "cytotoxic"
relates to the ability of the agent to halt or retard the
growth of cancerous cells or to induce the death of such
cells. In another embodiment, the agent may be administered
through a route selected from the group consisting of
intravenous, oral, transdermal, subcutaneous, mucosal,
intramuscular, intranasal, intrapulmonary, parenteral,
intrarectal, intratumoral and topical. In an embodiment, the
agent may be administered alone or in combination with other
chemotherapeutic agents. In another embodiment, the agent
may be administered in conjunction (before, simultaneously or
after) with a radiation therapy.
The invention also provides a method of treating or
preventing cancer, the method comprising administering an
agent selected from the group~consisting of (a) a bacteriocin
derived from a lactic acid bacteria and (b) a composition
CA 02485178 2004-11-22
8
comprising a bacteriocin derived from a lactic acid bacteria
and a carrier. As used herein the term "bacteriocin" relates
to a polypeptide produced by a microbial cell (e.g. a
bacteria), wherein such polypeptide possesses microbicidal
(e. g. bacteriocidal) activity. The microbicidal (e. g.
bactericiodal) activity of a bacteriocin can be measured
using several techniques known by those skilled in the art,
such as the agar spot test (see Example 4). The presence of
a bacteriocin can also be assessed with other standard
methods such as Western blotting, 2D electrophoresis,
capillary electrophoresis, imaging techniques (e. g. specific
antibodies coupled to immunof:luorescent compounds or an
enzyme that enables colorimet:ric visualisation), ELISA, RIA
and protein micro-array.
In an embodiment, the above-mentioned bacteriocin
is derived from a lactic acid bacteria (LAB). Such
bacteriocins include, but are not limited to, bavaricin,
helveticin, acidocin, lactocin, lactacin, lacticin, nisin,
leucocin, lactococcin, pediocin, curvaticin, curvacin,
mutacin, mesentericin, plantaricin, streptin and sakacin.
In an embodiment, the bacteriocin is pediocin and,
in a further embodiment, the bacteriocin is pediocin PA-1.
Pediocin AcH and pediocin PA-1 are used herein
interchangeably. In yet another embodiment, pediocin PA-1
has a sequence substantially identical to the sequence set
forth in Figure 2 (e.g. SEQ ID NO: 2) or is encoded by a
nucleotide sequence capable of encoding a polypeptide
substantially identical to the sequence set forth in Figure 2
(e. g. SEQ ID NO: 1). In another embodiment, amino acid
variants of pediocin (such as those described in Johnsen et
al., 2000 and Miller et al., 1998) can also be used in the
methods described herein. In another embodiment, the
bacteriocins and variants described herein may also retain
CA 02485178 2004-11-22
9
microbicidal (e.g. bacteriocidal) activity. In an
embodiment, the pediocin having a variation in its amino acid
sequence with respect to the natural pediocin sequence may
have enhanced stability, enhanced microbicidal activity
and/or enhanced anti-cancer activity.
Agents of the invention can be prepared, for
example, by replacing, deleting, or inserting an amino acid
residue of a bacteriocin derived from a lactic acid bacteria,
with other conservative amino acid residues, i.e., residues
having similar physical, biological, or chemical properties,
and screening for biological function. It is well known in
the art that some modifications and changes can be made in
the structure of a polypeptide without substantially altering
the biological function of that peptide, to obtain a
biologically equivalent polypeptide. The peptides, ligands
and domains of the present invention also extend to
biologically equivalent peptides, ligands and domains that
differ from a portion of the sequence of novel ligands of the
present invention by conservative amino acid substitutions.
As used herein, the term "conserved amino acid substitutions"
refers to the substitution of one amino acid for another at a
given location in the peptide, where the substitution can be
made without substantial loss of the relevant function. In
making such changes, substitutions of like amino acid
residues can be made on the basis of relative similarity of
side-chain substituents, for example, their size, charge,
hydrophobicity, hydrophilicity, and the like, and such
substitutions may be assayed for their effect on the function
of the peptide by routine testing. In some embodiments,
conserved amino acid substitutions may be made where an amino
acid residue is substituted for another having a similar
hydrophilicity value (e. g., within a value of plus or minus
2.0), where the following may be an amino acid having a
CA 02485178 2004-11-22
hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-
1.6)s are assigned to amino acid residues (as detailed in
United States Patent No. 4,554,101, incorporated herein by
reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0);
5 Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (-0.5); Thr
(-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val
(-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and
Trp (-3.4).
In alternative embodiments, conserved amino acid
10 substitutions may be made where an amino acid residue is
substituted for another having a similar hydropathic index
(e. g., within a value of plus or minus 2.0). In such
embodiments, each amino acid residue may be assigned a
hydropathic index on the basis of its hydrophobicity and
charge characteristics, as follows: Ile (+4.5); Val (+4.2);
Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8);
Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3);
Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5);
Asn (-3.5); Lys (-3.9); and Arg (-4.5).
In alternative embodiments, conserved amino acid
substitutions may be made where an amino acid residue is
substituted for another in the same class, where the amino
acids are divided into non-polar, acidic, basic and neutral
classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp,
Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral:
Gly, Ser, Thr, Cys, Asn, Gln, Tyr.
Conservative amino acid changes can include the
substitution of an L-amino acid by the corresponding D-amino
acid, by a conservative D-amino acid, or by a naturally-
occurring, non-genetically encoded form of amino acid, as
well as a conservative substit=ution of an L-amino acid.
Naturally-occurring non-genetically encoded amino acids
include beta-alanine, 3-amino-propionic acid, 2,3-diamino
CA 02485178 2004-11-22
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propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric
acid, N-methylglycine (sarcosine), hydroxyproline, ornithine,
citrulline, t-butylalanine, t-butylglycine, N-
methylisoleucine, phenylglycine, cyclohexylalanine,
norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-
benzothienyl alanine, 4-chlorophenylalanine, 2-
fluorophenylalanine, 3-fluorophenylalanine, 4-
fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-
isoquinoline-3-carboxylix acid, beta-2-thienylalanine,
methionine sulfoxide, homoarginine, N-acetyl lysine, 2-amino
butyric acid, 2-amino butyric acid, 2,4,-diamino butyric
acid, p-aminophenylalanine, N-methylvaline, homocysteine,
homoserine, cysteic acid, epsilon-amino hexanoic acid, delta-
amino valeric acid, or 2,3-diaminobutyric acid.
In alternative embodiments, conservative amino acid
changes include changes based on considerations of
hydrophilicity or hydrophobic.ity, size or volume, or charge.
Amino acids can be generally characterized as hydrophobic or
hydrophilic, depending primarily on the properties of the
amino acid side chain. A hydrophobic amino acid exhibits a
hydrophobicity of greater than zero, and a hydrophilic amino
acid exhibits a hydrophilicity of less than zero, based on
the normalized consensus hydrophobicity scale of Eisenberg et
al. (J. Mol. Bio. 179:125-142, 1984). Genetically encoded
hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, Ile,
Pro, Met and Trp, and genetically encoded hydrophilic amino
acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys.
Non-genetically encoded hydrophobic amino acids include t-
butylalanine, while non-genetically encoded hydrophilic amino
acids include citrulline and homocysteine.
Hydrophobic or hydrc>philic amino acids can be
further subdivided based on the characteristics of their side
chains. For example, an aromatic amino acid is a hydrophobic
CA 02485178 2004-11-22
12
amino acid with a side chain containing at least one aromatic
or heteroaromatic ring, which may contain one or more
substituents such as -OH, -SH, -CN, -F, -Cl, -Br, -I, -N02, -
N0, -NH2, -NHR, -NRR, -C (0) R, -C (O) OH, -C (0) OR, -C (0) NH2, -
C(0)NHR, -C(0)NRR, etc., where R is independently (Cl-C6)
alkyl, substituted (Cl-C6) alkyl, (C1-C6) alkenyl,
substituted (C1-C6) alkenyl, (Cl-C6) alkynyl, substituted
(Cl-C6) alkynyl, (C5-C20) aryl, substituted (C5-C20) aryl,
(C6-C26) alkaryl, substituted (C6-C26) alkaryl, 5-20 membered
heteroaryl, substituted 5-20 membered heteroaryl, 6-26
membered alkheteroaryl or substituted 6-26 membered
alkheteroaryl. Genetically encoded aromatic amino acids
include Phe, Tyr, and Tryp, while non-genetically encoded
aromatic amino acids include phenylglycine, 2-napthylalanine,
beta-2-thienylalanine, 1,2,3,4-tetrahydro-isoquinoline-3-
carboxylic acid, 4-chlorophenylalanine, 2-
fluorophenylalanine3-fluorophenylalanine, and 4-
fluorophenylalanine.
An apolar amino acid is a hydrophobic amino acid
with a side chain that is uncharged at physiological pH and
which has bonds in which a pair of electrons shared in common
by two atoms is generally held equally by each of the two
atoms (i.e., the side chain is not polar). Genetically
encoded apolar amino acids include Gly, Leu, Val, Ile, Ala,
and Met, while non-genetically encoded apolar amino acids
include cyclohexylalanine. Apolar amino acids can be further
subdivided to include aliphatic amino acids, which is a
hydrophobic amino acid having an aliphatic hydrocarbon side
chain. Genetically encoded aliphatic amino acids include
Ala, Leu, Val, and Ile, while non-genetically encoded
aliphatic amino acids include norleucine.
A polar amino acid is a hydrophilic amino acid with
a side chain that is uncharged at physiological pH, but which
CA 02485178 2004-11-22
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has one bond in which the pair of electrons shared in common
by two atoms is held more closely by one of the atoms.
Genetically encoded polar amino acids include Ser, Thr, Asn,
and Gln, while non-genetically encoded polar amino acids
include citrulline, N-acetyl lysine, and methionine
sulfoxide.
An acidic amino acid is a hydrophilic amino acid
with a side chain pKa value of. less than 7. Acidic amino
acids typically have negatively charged side chains at
physiological pH due to loss of a hydrogen ion. Genetically
encoded acidic amino acids include Asp and Glu. A basic
amino acid is a hydrophilic amino acid with a side chain pKa
value of greater than 7. Basic amino acids typically have
positively charged side chains at physiological pH due to
association with hydronium ion. Genetically encoded basic
amino acids include Arg, Lys, and His, while non-genetically
encoded basic amino acids include the non-cyclic amino acids
ornithine, 2,3,-diaminopropionic acid, 2,4-diaminobutyric
acid, and homoarginine.
The above classifications are not absolute and an
amino acid may be classified in more than one category. In
addition, amino acids can be r_lassified based on known
behaviour and or characteristic chemical, physical, or
biological properties based on specified assays or as
compared with previously identified amino acids. Amino acids
can also include bifunctional moieties having amino acid-like
side chains.
Conservative changes can also .include the
substitution of a chemically derivatised moiety for a non-
derivatised residue, by for example, reaction of a functional
side group of an amino acid. Thus, these substitutions can
include compounds whose free amino groups have been
derivatised to amine hydrochlorides, p-toluene sulfonyl
CA 02485178 2004-11-22
14
groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Similarly, free
carboxyl groups can be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides, and side
chains can be derivatized to form 0-acyl or 0-alkyl
derivatives for free hydroxyl groups or N-im-benzylhistidine
for the imidazole nitrogen of histidine. Peptide analogues
also include amino acids that have been chemically altered,
for example, by methylation, by amidation of the C-terminal
amino acid by an alkylamine such as ethylamine, ethanolamine,
or ethylene diamine, or acylation or methylation of an amino
acid side chain (such as acylation of the epsilon amino group
of lysine). Peptide analogues can also include replacement
of the amide linkage in the peptide with a substituted amide
(for example, groups of the formula -C(O)-NR, where R is (C1-
C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, substituted (C1-
C6) alkyl, substituted (C1-C6) alkenyl, or substituted (C1-
C6) alkynyl) or isostere of an amide linkage (for example, -
CH2NH-, -CH2S, -CH2CH2-, -CH=CH- (cis and trans), -C(O)CH2-,
-CH(OH)CH2-, or -CH2S0-).
"Homology" and "homologous" refers to sequence
similarity between two peptides or two nucleic acid
molecules. Homology can be determined by comparing each
position in the aligned sequences. A degree of homology
between nucleic acid or between amino acid sequences is a
function of the number of identical or matching nucleotides
or amino acids at positions shared by the sequences. As the
term is used herein, a nucleic acid sequence is "homologous"
to another sequence if the two sequences are substantially
identical and the functional activity of the sequences is
conserved (as used herein, the term "homologous" does not
infer evolutionary relatedness). Two nucleic acid sequences
are considered substantially identical if, when optimally
CA 02485178 2004-11-22
aligned (with gaps permitted), they share at least about 50%
sequence similarity or identity, or if the sequences share
defined functional motifs. In alternative embodiments,
sequence similarity in optimally aligned substantially
5 identical sequences may be at least 600, 70%, 750, 800, 850,
900 or 950. As used herein, a given percentage of homology
between sequences denotes the degree of sequence identity in
optimally aligned sequences. An "unrelated" or "non-
homologous" sequence shares less than 40% identity, though
10 preferably less than about 25 o identity, with any of SEQ ID
NOs. 1 and 2.
Substantially complementary nucleic acids are nucleic
acids in which the complement of one molecule is
substantially identical to the other molecule. Two nucleic
15 acid or protein sequences are considered substantially
identical if, when optimally aligned, they share at least
about 70o sequence identity. In alternative embodiments,
sequence identity may for example be at least 750, at least
800, at least 850, at least 900, or at least 95%. Optimal
alignment of sequences for comparisons of identity may be
conducted using a variety of algorithms, such as the local
homology algorithm of Smith arid Waterman, 1981, Adv. Appl.
Math 2: 482, the homology alignment algorithm of Needleman
and Wunsch, 1970, J. Mol. Biol. 48:443, the search for
similarity method of Pearson and Lipman, 1988, Proc. Natl.
Acad. Sci. USA 85: 2444, and the computerised implementations
of these algorithms (such as CAP, BESTFIT, FASTA and TFASTA
in the Wisconsin Genetics Software Package, Genetics Computer
Group, Madison, WI, U.S.A.). Sequence identity may also be
determined using the BLAST algorithm, described in Altschul
et al., 1990, J. Mol. Biol. 21.5:403-10 (using the published
default settings). Software for performing BLAST analysis may
be available through the National Center for Biotechnology
CA 02485178 2004-11-22
16
Information (through the Internet at
http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves
first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence
that either match or satisfy some positive-valued threshold
score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighbourhood word
score threshold. Initial neighbourhood word hits act as seeds
for initiating searches to find longer HSPs. The word hits
are extended in both directions along each sequence for as
far as the cumulative alignment score can be increased.
Extension of the word hits in each direction is halted when
the following parameters are met: the cumulative alignment
score falls off by the quantity X from its maximum achieved
value; the cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment. The BLAST program may
use as defaults a word length (W) of 11, the BLOSUM62 scoring
matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci.
USA 89: 10915-10919) alignments (B) of 50, expectation (B) of
10 (or 1 or 0.1 or 0.01 or 0.()01 or 0.0001), M=5, N=4, and a
comparison of both strands. One measure of the statistical
similarity between two sequences using the BLAST algorithm is
the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. In
alternative embodiments of the invention, nucleotide or amino
acid sequences are considered substantially identical if the
smallest sum probability in a comparison of the test
sequences is less than about 1, preferably less than about
CA 02485178 2004-11-22
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0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
An alternative indication that two nucleic acid
sequences are substantially complementary is that the two
sequences hybridize to each other under moderately stringent,
or preferably stringent, conditions. Hybridisation to filter-
bound sequences under moderately stringent conditions may,
for example, be performed in 0.5 M NaHP04, 7o sodium dodecyl
sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x
SSC/O.lo SDS at 42°C (see Ausubel, et al. (eds), 1989,
Current Protocols in Molecular Biology, Vol. 1, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., New
York, at p. 2.10.3). Alternatively, hybridization to filter-
bound sequences under stringent conditions may, for example,
be performed in 0.5 M NaHP04, 7o SDS, 1 mM EDTA at 65°C, and
washing in 0.1 x SSC/O.lo SDS at 68°C (see Ausubel, et al.
(eds), 1989, supra). Hybridization conditions may be modified
in accordance with known methods depending on the sequence of
interest (see Tijssen, 1993, Laboratory Techniques in
Biochemistry and Molecular Biology -- Hybridization with
Nucleic Acid Probes, Part I, Chapter 2 "Overview of
principles of hybridization and the strategy of nucleic acid
probe assays", Elsevier, New York). Generally, stringent
conditions are selected to be about 5°C lower than the
thermal melting point for the specific sequence at a defined
ionic strength and pH.
In another aspect, t=he invention provided methods
of using a bacteriocin derived from a lactic acid bacteria
(e.g. in the form of a composition or not) to treat or
prevent cancer. As used herein, the term "lactic acid
bacteria" (LAB) is a class of bacteria that are capable of
producing lactic acid. Usually, LAB are gram positive
bacteria. In embodiments, some LAB can be used in the
CA 02485178 2004-11-22
preparation (e. g. fermentation) of various food products
(such as milk, meat, vegetable and fruit). In embodiments,
the lactic acid bacteria species include, but are not limited
to Streptococcus spp., Lactococcus spp., Lactobacillus spp.,
Pediococcus spp., Bifidobacterium spp., Leuconostoc spp, and
Enterococcus spp. In an embodiment, the bacteriocin is
derived from Pediococcus acidilactici and, in a further
embodiment, the bacteriocin is derived from from Pediococcus
acidilactici PAC 1Ø
As used herein the term "derived from a LAB" is
defined as either produced by a LAB or being of LAB origin
but produced by other means (e. g. expressed in another
system; e.g. a prokaryotic system, such as E. coli).
In yet another embodiment, the bacteriocin
described herein is substantially pure. A compound or agent
that is "substantially pure" is separated from the components
that naturally accompany it. Typically, a compound is
substantially pure when it is at least 600, more generally
750 or over 900, by weight, of the total material in a
sample. Thus, for example, a polypeptide that is chemically
synthesised or produced by recombinant technology will
generally be substantially free from its naturally associated
components. A nucleic acid molecule is substantially pure
when it is not immediately contiguous with (i.e. covalently
linked to) the coding sequences with which it is normally
contiguous in the naturally occurring genome of the organism
from which the DNA of the invention is derived. A
substantially pure compound can be obtained, for example, by
extraction from a natural source; by expression of a
recombinant nucleic acid molecule encoding a polypeptide
compound; or by chemical synthesis. Purity can be measured
using any appropriate method such as column chromatography,
gel electrophoresis, HPLC, etc.
CA 02485178 2004-11-22
19
In a further embodiment, the agent described herein
is substantially free of bacterial contaminants from the
lactic acid bacteria (e.g. Pediococcus spp.) from which it is
derived. Such contaminants may be cell wall components,
organelles (such as the Golgi, endoplasmic reticulum,
ribosomes), nuclear components (such as the nuclear wall, the
nucleus or the nucleic acid it contains), nucleotides (such
as ribonucleotides and/or deoxyribonucleotides), lipids,
proteins and protein fragments, etc. In another embodiment,
the bacteriocin is isolated/purified from the lactic acid
bacteria in which it is produced (refer to the methods
described above and in the examples).
The invention also relates to a method of treating
or preventing cancer, said method comprising administering a
bacteriocin derived from a lactic acid bacteria and/or a
composition comprising such a bacteriocin and a suitable
carrier. In an embodiment, the bacteriocins (e. g. pediocin)
described herein may be modified/adapted to be more resistant
(e. g. less degraded) in the presence of gastro-intestinal
juices. In an embodiment, the agent may be administered in a
way such that the bacteriocin does not come into contact with
the gastro-instestinal (GI) tract (e. g. intravenous
administration). In another embodiment, the amino acid
sequence of the bacteriocin can be modified to increase its
stability in the GI tract. Such modifications include, but
are not limited to, replacing o-amino acids by z-amino acids.
In yet another embodiment, the agent can be coated with or
formulated with a material that enables the rapid and
specific delivery of the agent to a specific location in the
GI tract, thereby limiting the contact between the
bacteriocin and the GI juices. In another embodiment, the
bacteriocin can be provided in the form of a food product or
a nutraceutical comprising the bacteriocin, such as a
CA 02485178 2004-11-22
fermented food product or a fermented milk. The
incorporation of bacteriocins into a food product or
nutraceutical may prevent the degradation of the
bacteriocins.
5 In various embodiments, the agent described herein,
may be used therapeutically in formulations or medicaments to
prevent or treat cancer. The invention provides corresponding
methods of medical treatment, in which a therapeutic dose of
an agent is administered in a pharmacologically acceptable
10 formulation or nutraceutically acceptable formulation, e.g.
to a patient or subject in need thereof. Accordingly, the
invention also provides therapeutic compositions comprising a
bacteriocin derived from a lactic acid bacteria and a
carrier. In an embodiment, the carrier is a pharmaceutically
15 acceptable carrier or a nutraceutically acceptable carrier.
As used herein, the ~~nutraceutically acceptable carrier" is
defined as a carrier that is suitable for administration in a
nutraceutical. For those skilled in the art, a nutraceutical
is defined as any substance that is a food or a part of a
20 food and provides medical or health benefits, including the
prevention and treatment of disease. Such products may range
from isolated nutrients, dietary supplements and specific
diets to genetically engineered designer foods, herbal
products, and processed foods such as cereals, soups and
beverages. This definition also includes a bio-engineered
designer vegetable food, rich in antioxidant ingredients, and
a functional food or pharmafood. A nutraceutical is also
defined as a product isolated or purified from foods, and
generally sold in medicinal forms not usually associated with
food and demonstrated to have a physiological benefit or
provide protection against or improvement of a disease
condition. In an embodiment, the nutraceutically acceptable
carrier may be a food product (e. g. soy derivative, milk
CA 02485178 2004-11-22
21
derivative, meat product, juice, etc.) and, in a further
embodiment, it may be a fermented food product. Fermented
food products include, but are not limited to, fermented milk
products.
In one embodiment, such compositions include a
bacteriocin derived from a lactic acid bacteria in a
therapeutically or prophylactically effective amount
sufficient to treat or prevent cancer in an animal. The
composition may be soluble in an aqueous solution at a
physiologically acceptable pH.
A "therapeutically effective amount" refers to an
amount effective, at dosages and for periods of time
necessary, to achieve the desired therapeutic result, such as
the prevention or treatment of a cancer (e.g. inhibition or
reduction of growth of a primary tumor, inhibition or
reduction of implantation or growth of metastases, inhibition
or reduction of lymph nodes involvement). A therapeutically
effective amount of a bacteriocin derived from a lactic acid
bacteria may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the
ability of the agent to elicit a desired response in the
individual. Dosage regimens may be adjusted to provide the
optimum therapeutic response. A therapeutically effective
amount is also one in which any toxic or detrimental effects
of the compound are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount"
refers to an amount effective, at dosages and for periods of
time necessary, to achieve the desired prophylactic result,
such as preventing or inhibiting the rate of cancer onset or
progression. A prophylactical:ly effective amount can be
determined as described above for the therapeutically
effective amount. For any particular subject, specific dosage
regimens may be adjusted over time according to the
CA 02485178 2004-11-22
22
individual need and the professional judgement of the person
administering or supervising the administration of the
compositions.
As used herein ~~pharmaceutically acceptable
carrier" or ~~excipient" includes any and all solvents,
dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like
that are physiologically compatible. In one embodiment, the
carrier is suitable for parenteral administration.
Alternatively, the carrier can be suitable for intravenous,
intraperitoneal, intramuscular, sublingual, intratumoral or
oral administration. Pharmaceutically acceptable carriers
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media
and agents for pharmaceutically active substances is well
known in the art. Except insofar as any conventional media
or agent is incompatible with the active compound, use
thereof in the pharmaceutical compositions of the invention
is contemplated. Supplementary active compounds can also be
incorporated into the compositions.
Therapeutic compositions typically must be sterile
and stable under the conditions of manufacture and storage.
The composition can be formulated as a solution,
microemulsion, liposome, or other ordered structure suitable
to high drug concentration. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the :like), and suitable mixtures
thereof. The proper fluidity can be maintained, for example,
by the use of a coating such as lecithin, by the maintenance
of the required particle size in the case of dispersion and
by the use of surfactants. In many cases, it will be
CA 02485178 2004-11-22
23
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride
in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the
composition an agent which delays absorption, for example,
monostearate salts and gelatin. Moreover, a bacteriocin
derived from a lactic acid bacteria can be administered in a
time release formulation, for example in a composition which
includes a slow release polymer. The active compounds can be
prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation,
including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, polylactic acid and polylactic,
polyglycolic copolymers (PLG). Many methods for the
preparation of such formulations are patented or generally
known to those skilled in the art.
Sterile injectable solutions can be prepared by
incorporating the active compound (e. g. a bacteriocin derived
from a lactic acid bacteria) in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle
which contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum
drying and freeze-drying which yields a powder of the active
ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. In accordance
with an alternative aspect of the invention, a bacteriocin
CA 02485178 2004-11-22
24
derived from a lactic acid bacteria may be formulated with
one or more additional compounds that enhance the solubility
of the bacteriocin.
In accordance with another aspect of the invention,
therapeutic compositions of the present invention, comprising
an agent (e. g. a bacteriocin derived from a lactic acid
bacteria and/or a composition comprising a bacteriocin
derived from a lactic acid bacteria and a carrier), may be
provided in containers or packages (e. g. commercial packages)
which further comprise instructions for use of the agent for
preventing and/or treating of cancer.
Accordingly, the invention further provides a
package (e. g. commercial package) comprising a bacteriocin
derived from a lactic acid bacteria or the above-mentioned
composition together with instructions for the use of the
bacteriocin and/or composition for the prevention and/or
treatment of cancer.
The invention further provides use of a bacteriocin
derived from a lactic acid bacteria or the above-mentioned
composition for the prevention and/or treatment of cancer.
The invention further provides the use of a bacteriocin
derived from a lactic acid bacteria for the preparation of a
medicament for prevention and/or treatment of cancer.
In yet another aspect, the invention also provides
a gene therapy method for treating or preventing cancer.
Nucleic acids encoding a bacteriocin derived from a lactic
acid bacteria may be delivered to cells in vivo using methods
such as direct injection of DNA, receptor-mediated DNA
uptake, viral-mediated transfection or non-viral transfection
and lipid based transfection, all of which may involve the
use of gene therapy vectors. Direct injection has been used
to introduce naked DNA into cells in vivo (see e.g., Acsadi
et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science
CA 02485178 2004-11-22
247:1465-1468). A delivery apparatus (e.g., a Eugene gun") for
injecting DNA into cells in vivo may be used. Such an
apparatus may be commercially available (e. g., from BioRad).
Naked DNA may also be introduced into cells by complexing the
5 DNA to a can on, such as polylysine, which is coupled to a
ligand for a cell-surface receptor (see for example Wu, G.
and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson e1. al.
(1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.
5,166,320). Binding of the DNA-ligand complex to the
10 receptor may facilitate uptake of the DNA by receptor-
mediated endocytosis. A DNA-ligand complex linked to
adenovirus capsids which disrupt endosomes, thereby releasing
material into the cytoplasm, may be used to avoid degradation
of the complex by intracellular lysosomes (see for example
15 Curiel e1 al. (1991) Proc. Natl. Acad. Sci. USA 88:8850;
Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-
2126) .
Defective retroviruses are well characterized for
use as gene therapy vectors (for a review see Miller, A. D.
20 (1990) Blood 76:271). Protocols for producing recombinant
retroviruses and for infecting cells in vitro or in vivo with
such viruses can be found in Current Protocols in Molecular
Biology, Ausubel, F. M. et al. (eds.) Greene Publishing
Associates, (1989), Sections 9.10-9.14 and other standard
25 laboratory manuals. Examples of suitable retroviruses include
pLJ, pZIP, pWE and pEM which are well known to those skilled
in the art. Examples of suitable packaging virus lines
include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses
have been used to introduce a variety of genes into many
different cell types, including epithelial cells, endothelial
cells, lymphocytes, myoblasts, hepatocytes, bone marrow
cells, in vitro and/or in vivo (see for example Eglitis, et
al. (1985) Science 230:1395-1398; Danos and Mulligan (1988)
CA 02485178 2004-11-22
26
Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)
Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et
al. (1991) Science 254:1802-1805; van Beusechem et al. (1992)
Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992)
Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.
Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.
150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.
4,980,286; PCT Application WO 89/07136; PCT Application WO
89/02468; PCT Application WO 89/05345; and PCT Application WO
92/07573).
Adeno-associated virus (AAV) may be used as a gene
therapy vector for delivery of DNA for gene therapy purposes.
AAV is a naturally occurring defective virus that requires
another virus, such as an adenovirus or a herpes virus, as a
helper virus for efficient replication and a productive life
cycle (Muzyczka et al. Curr. Topics in Micro. and Immunol.
(1992) 158:97-129). AAV may be used to integrate DNA into
non-dividing cells (see for example Flotte et al. (1992) Am.
J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989)
J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J.
Virol. 62:1963-1973). An AAV vector such as that described in
Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 may be
used to introduce DNA into cells (see for example Hermonat et
al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin
et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.
(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J.
Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem.
268:3781-3790). Lentiviral gene therapy vectors may also be
adapted for use in the invention.
CA 02485178 2004-11-22
27
General methods for gene therapy are known in the
art. See for example, U.S. Pat. No. 5,399,346 by Anderson et
al. A biocompatible capsule for delivering genetic material
is described in PCT Publication WO 95/05452 by Baetge et al.
Methods of gene transfer into hematopoietic cells have also
previously been reported (see Clapp, D. W., et al., Blood 78:
1132-1139 (1991) Anderson, Science 288:627-9 (2000): and,
Cavazzana-Calvo et al., Science 288:669-72 (2000)).
In another embodiment, a vector encoding a
bacteriocin derived from a lactic acid bacteria can be
introduced into a cell in vitro. Such cell may, in an
embodiment, produce and/or secrete the bacteriocin. This
genetically modified cell can then be inserted into a host
having a cancer. In an embodiment, the genetically modified
cell is inserted into the host in proximity to the tumor or
the metastases. In yet another embodiment, the inserted
genetically modified cell produces or secretes the
bacteriocin into the host. In another embodiment, the
genetically modified cell is autologous or non-autologous to
the host.
Although various embodiments of the invention are
disclosed herein, many adaptations and modifications may be
made within the scope of the invention in accordance with the
common general knowledge of those skilled in this art. Such
modifications include the substitution of known equivalents
for any aspect of the invention in order to achieve the same
result in substantially the same way. Numeric ranges are
inclusive of the numbers defining the range. In the claims,
the word "comprising" is used as an open-ended term,
substantially equivalent to the phrase "including, but not
limited to". The following examples are illustrative of
various aspects of the invention, and do not limit the broad
aspects of the invention as disclosed herein.
CA 02485178 2004-11-22
28
EXAMPII~ES
Antimicrobial peptides, such as bacteriocins, possess
cationic and amphipathic properties which allow interactions
with the membrane of living cells. Many such cationic
peptides could have interesting and various biomedical and
health applications (Marshall and Arenas, 2003). Potential
uses may be as components of cosmetics (Reisch, 2002), as
antibiotics (Hancock and Lehrer, 1998), as analgesics
(Crescenzi et al., 2000), and as agents to prevent oral
mucositis (Chen et al., 2000) or to fight herpes viruses
(Wachsman et al., 1999; Wachsman et al., 2003). Some
bacteriocins have been reported to also possess antitumor
activity (Winder et al., 1998).
Bacteriocins produced by lactic acid bacteria (LAB) are
antimicrobial peptides, and they have recently gained
interest for their potential as food preservatives, as well
as for medical applications (Nes and Holo, 2000). An example
is nisin, which is produced by Lactococcus lactis. This
natural inhibitor is well known (Ross et al., 1999) and has
been used for a long time as a food preservative (Breukink
and Kruijff, 1999). Most recently, it has been considered to
be useful for the treatment of gastric Helicobacter and/or
ulcerous infections (Guder et al., 2000; Hancock, 1997; Ross
et al., 1999; Uteng et al., 2002). Other LAB bacteriocins,
belonging to the class IIa (pediocin, lactacine, etc.), are
very interesting and they are starting to be considered
seriously as a group of antimicrobial compounds worth
investigating for future medical use (Nes and Holo, 2000).
Pediocin PA-l, one of the known class IIa bacteriocins,
is produced by Pediococcus acidilactici PAC 1.0 and is
strongly active against Listeria monocytogenes (Bhunia et
al., 1988). The primary structure of pediocin PA-1 consists
CA 02485178 2004-11-22
29
of 44 amino acids and its theoretical molecular weight is
4624 Da (refer to figure 2, SEQ ID NO: 2), in the presence of
its two disulfide bonds, known to be very important for its
biological activity (Gaussier, 2003; Henderson et al., 1992;
Rodriguez et al., 2002). Advances in the understanding of
pediocin PA-1 structure, mode of action and biosynthesis will
greatly increase the potential usefulness of pediocin PA-1 in
different fields. Recent studies revealed that production of
recombinant pediocin PA-1 in the methylotrophic yeast Pichia
pastoris led to unexpected inhibition of its biological
activity (Example 2). This astonishing phenomenon raised
several questions and, especially, it has demonstrated a
particular feature of the function and/or structure of
pediocin PA-l, i.e., that pediocin PA-1 may possess a
somewhat "collagen-like" nature.
Recently, the role of collagen-derived proteolytic
fragments in angiogenesis was investigated (Marneros and
Olsen, 2001). Antitumoral effects have been demonstrated for
cecropin, melittin and magainine (Baker et al., 1993; Jaynes
et al., 1989; Moore et al., 1994; Ohsaki et al., 1992;
Sharma, 1992). In the case of melittin, the hyperactivation
of phospholipase A2 in ras-transformed cells was in part
responsible for the antitumoral activity (Winder et al.,
1998).
In the studies described herein, the potential of growth
inhibition of pediocin PA-1 on the A-549 human lung carcinoma
cell line and the DLD-1 human colon adenocarcinoma cell line
was evaluated.
Example I - Production of native/natural pediocin PA-I
Pediococcus acidilactici strain PAC 1.0 (Quest
International, Sarasota, Fla.) was cultivated in a 20 L
fermentor (Chemap, Switzerland) equipped with pH and p02
CA 02485178 2004-11-22
electrodes (Ingold), a foam sensor, and a mechanical foam
breaker. It was grown in 16 L of Lactobacilli MRS broth
(Oxoid); a 1% (v/v) inoculum was used and incubation was done
at 37°C for 18 h at an agitation rate of 200 rpm. No
5 additional aeration was provided and no chemical antifoam was
added during cultivation.
The cells were separated from the broth by
centrifugation (50008, 15 min, 20°C). The supernatant fluid
was concentrated 13-fold using a 0.5m2 1 kDa (cut-off)
10 regenerated cellulose membrane k(PCACTM membrane, Millipore),
then, diafiltered against 5mM ammonium acetate buffer,
pH°5.0, using a PelliconTM system (Millipore) with a
Masterflex peristaltic pump at a recirculation rate of
200 ml/min. The process was performed at room temperature.
15 Ion exchange chromatography (IEX). Pediocin PA-1
purification was achieved using a modification of the
procedure described by Gaussier et al. (2002). Pediocin PA-1
from the concentrated supernatant fluid (1200 ml) was
captured using a SP-Sepharose Fast FlowTM can on exchange
20 column (Amersham). The C26/40 column was packed with 80 ml of
resin and connected to a FPLC chromatography system coupled
to an UVl-detector (Amersham; reading at OD280 nm). The
column was equilibrated with 10 CV (CV = column volume) of
5mM ammonium acetate buffer, pH 5.0 (Buffer A). The pediocin
25 PA-1 solution was applied to the column at a rate of
226 cm/h, then, the column was washed successively with
Buffer A and Buffer A containing 0.25M NaCl. Pediocin PA-1
was eluted from the column with Buffer A containing 0.5M
NaCl.
30 Hydrophobic interaction chromatography (HIC)
Screening of chromatography media. Several HIC media with
varying hydrophobicity were tested, namely: Butyl-SepharoseTM,
Amersham; Butyl-650M, Tosohaas; Octyl-SepharoseTM CL 4B,
CA 02485178 2004-11-22
31
Amersham; Phenyl-SepharoseTM, Amersham; Phenyl-650M, Tosohaas.
Media (1 ml) were equilibrated with 5mM ammonium acetate
buffer, pH 5.0 (Buffer A) containing 0.5M NaCl, mixed with 5
ml of the SP-SepharoseTM FF eluate (previous step) and the
mixtures were incubated for 1 h at room temperature. A first
wash with Buffer A containing 0.5M NaCl was performed
followed by elution with Buffer A (5mM ammonium acetate
buffer, pH 5.0) and then, by a second elution with Buffer A
containing 500 (v/v) acetonitrile. Biorad Econo-ColumnsTM were
used for the screening work and elution was performed by
gravity. Eluate fractions were analyzed for pediocin PA-1's
biological activity by an agar spot test on a MRS agar medium
(Rodriguez et al, 2002).
Refinement of the HIC method. The active fractions
collected from the SP-SepharoseTM column were pooled and
applied to an Octyl Sepharose~~~M CL-4B hydrophobic interaction
column (HR5/5, 1 ml CV, Amer~;ham) using the FPLC system. The
column was equilibrated with Buffer A containing 0.5M NaCl.
The pediocin solution was applied to the HIC column at the
rate of 306 cm/h, the column was washed with the same
equilibration buffer, followed by a second wash with Buffer
A. Pediocin PA-1 was eluted with Buffer A containing 500
(v/v) acetonitrile. All of the initial developmental work was
performed using 1 ml CV HR5/5 HIC columns (Amersham). Later,
a 40 ml CV XK26 column (Amersham) was used to generate more
pediocin PA-1 material. The active fractions were pooled and
concentrated (13-fold) using a 350 ml Stir Cell system
(Amicon) and a lkDa (cut-off) regenerated cellulose membrane
(YMl, DiafloTM ultrafiltration membrane, Amicon) ending with
diafiltration against water (HPLC grade). The retentate was
lyophilized (Flexi-dryTM, FTS Systems Inc.) and the material
was stored at 4°C under a NZ atmosphere.
CA 02485178 2004-11-22
32
Example 2 - Production of recombinant pediocin PA-I in Pichia
pastoris
Strains, plasmids and culture conditions.
Escherichia coli TOP10 was grown in Luria-Bertani (LB) broth
(lo tryptone, 1o NaCl, 0.50 yeast extract, pH 7.0). E.coli
transformants were grown in L,ow Salt Luria-Bertani (LSLB)
medium, supplemented with zeocin (25~g/ml) overnight at 37°C.
Pediococcus acidilactici PAC1.0 and Pediococcus pentosaceus
FBB63 were grown in Lactobacilli MRS broth (Oxoid) for 18h at
37°C. Yeast strains were cultured in YPD (lo yeast extract,
2% peptone, 2o glucose) medium, whereas yeast transformants
were grown in baffled shake flasks under selective conditions
in buffered glycerol-complex medium (BMGY: to yeast extract,
2o peptone, 100mM potassium phosphate, pH 6.0, 1.340 yeast
nitrogen base without amino acids, 4X10-So biotin, to
glycerol) until cultures reached an OD6oonm between 2 and 6.
The cultures were harvested by centrifugation (5000 g,
15 min, 4°C) and the cells were grown for another 3 days at
30°C in buffered methanol complex medium (BMMY: to yeast
extract, 2% peptone, 100mM potassium phosphate, pH 6.0, 1.340
yeast nitrogen base without amino acids, 4X10-50 biotin, to
methanol) for the induction of pediocin PA-1 gene expression.
Samples were taken at different times (0, 6, 12, 24, 48,
72°h) for analysis. Experiments were performed according to
the guidelines supplied by the EasySelectTM Pichia Expression
Kit (Invitrogen Corporation).
Cloning of pedA and plasmid construction. A DNA
fragment encoding the mature domain of pedi.ocin PA-1 was
obtained by polymerase chain reaction (PCR) amplification of
pSRQl1 DNA isolated from Pediococcus acidilactici PAC1.0
using the QIAprepTM Spin Miniprep Kit protocol (QIAgen) and
with an alkaline lysis procedure (2001 of a cold solution
CA 02485178 2004-11-22
33
containing 50mM glucose, lOmm EDTA, 25mM Tris pH 8.0 and
4 mg/ml of lysozyme, from Sigma, were added after step 3).
The sequence of the 5' primer used for amplification (5'-AAA
AAA CTC GAG AAA AGA GAG GTC GAA GCT AAA TAC TAC GGT AAT GGG-
3' [SEQ ID NO: 3]) begins with 6xA nucleotide clamp, followed
by a XhoI restriction enzyme site (CTCGAC), the Kex2 signal
cleavage sequence (AAAAGAGAGGTCGAAGCT [SEQ ID N0: 4]) and
ends with a 18 nucleotide sequence complementary to the first
nucleotides of the pedA gene encoding for the mature peptide
form. The 3' primer (5'-AAA AAA GTC GAC TTA TCA CTA GCA TTT
ATG ATT ACC TTG ATG TC-3'[SEQ ID NO: 5]) contains 6xA
nucleotide clamp, followed by an AccI restriction enzyme site
(GTCGAC), and the stop codons to end with a 23 nucleotide
sequence complementary to the final eight codons of the pedA
gene. PCR amplification was performed with 40U of rTaq DNA
polymerase (Amersham) per ml, 1.35 pg of pSRQll template per
ml, 1.25mM of each nucleoside triphosphate, and 2 ~g of each
primer per ml. PCR was performed to amplify DNA with a hot
start at 96°C for 1 min and then 30-cycles of amplification
(96°C, 30s for strand denaturation, 55°C, 30s for primer
annealing, and 72°C, 60s for primer extension) followed by a
final extension at 72°C for 2 min. The PCR product was
purified using the QIAquickTM PCR Purification Kit Protocol
(QIAgen) and digested with XhoI and AccI restriction
endonucleases (Amersham and New England Biolabs,
respectively). The resulting fragment was purified by agarose
gel electrophoresis using the QIAexTM II agarose gel
extraction kit (QIAgen) and ligated with T4 DNA Ligase (New
England Biolabs) between the XhoI and AccI restriction sites
within the pPICZaA vector. pPICZaA was pre-digested with the
same enzymes. Ligation mixtures were transformed into
competent E.coli TOP10 cells (TOPO TA Cloning, Version K,
Invitrogen). In vitro DNA manipulation (digestion, ligation)
CA 02485178 2004-11-22
34
for cloning in E. coli were performed as described by
Sambrook et al. (1989). The pPICZaA-pedA expression plasmid,
isolated using the QIAgen Plasmid Maxi Protocol (QIAgen), was
identified by restriction enzyme digestion with XhoI/AccI and
BamHI/SacI (New England Biolabs). The pedA gene region was
confirmed by double-stranded DNA sequencing with AmpliTaq DNA
polymerase (ABI PRISMTM Dye Terminator Cycle Sequencing Ready
Reaction Kit) using an ABI 377XL DNA Sequencer (Applied
Biosystems). For PCR sequencing reactions, 5' and 3' AOXl
primers were used to confirm that the pedA gene was in frame
with the C-terminal of the a-factor (EasySelectTM Pichia
Expression Kit, Invitrogen Corporation). PCR reactions were
purified on a Centri-SepTM column (Applied Biosystems) prior
to performing sequence analysis. pPICZaA (10 fig) and pPICZaA-
pedA (10 fig) vectors were linearized with 40U of SacI
restriction endonuclease (Amersham) following the
recommendations given by the EasySelectTM Pichia Expression
Kit (Invitrogen Corporation). Linearized vectors were
electroporated into P. pastoris. Electro-competent cells
(80 ~l) were mixed with DNA solution (10 fig) in a 0.2 cm gap
cuvette chilled on ice. Electroporation was carried out using
a Gene PulserTM (Bio-Rad) with the following parameters:
1.5 kV, 40052, 25 ~F to a final field strength of 7.5 kV cm-1.
After cells had been pulsed, 1 ml of ice-cold sterile 1M
sorbitol was immediately added to the cuvette, the cell
suspension was transferred into a test tube, and the mixture
incubated at 30°C for 1h30. Transformed clones were selected
on Yeast Extract Peptone Dextrose Sorbitol medium (YPDS: 1%
yeast extract, 2o peptone, 2o dextrose, 1M sorbitol, 2% agar)
containing zeocin (100 ~g/ml). Transformants were also tested
on both Minimal Dextrose Medium, with and without histidine,
(1.340 yeast nitrogen base without amino acids, 4X10-5o
CA 02485178 2004-11-22
biotin, 2o dextrose, with and without 0.0040 histidine, 1.50
agar) and Minimal Methanol Medium, with and without
histidine, (1.340 yeast nitrogen base without amino acids,
4X10-5o biotin, 2o methanol, with and without 0.004%
5 histidine, 1.50 agar) plates to confirm the Mut+ phenotype of
the X-33 and GS115 strains.
Detection of activity of pediocin PA-1 by agar spot
test. All transformants were grown in shake flasks as
mentioned before and tested by agar spot test on MRS medium
10 (Parrot et al., 1990) for detection of biological activity. A
MRS plate (1.50 (w/v) agar, 20 ml) was covered with 5 ml of
soft agar (0.80 (w/v) agar) containing an overnight MRS-grown
culture of the indicator strain P. pentosaceus (adjusted to
an OD6oonm of 0.1, Beckman DU 640 Spectrophotometer).
15 Supernatant fluid samples were filtered using a 0.22 ~m
membrane (Syringe Driven Filter Unit, Millex-HV, 4 mm) and
5 ~l of each sample was added to the surface of the soft
agar. The plates were incubated for 18h at 37°C and examined
for the presence of clear zones indicating growth inhibition.
20 The recombinant Pichia was grown in baffled shake
flasks in BMGY medium until glycerol was exhausted and, then,
in BMMY medium with methanol (0.50 (v/v)) for 3 days. The
cultures were harvested by centrifugation (5000g, 15 min,
4°C) and the supernatant fluids analyzed. Expression levels
25 were monitored by quantitative ELISA testing. A pediocin
concentration of 74 ~g/ml was achieved in Pichia comparison
with 6 ~g/ml for the natural producing strain of P.
acidilactici (Example 1). However, biological activity
against P. pentosaceus was not detected in the supernatant
30 fluids of the P. pastoris cultures tested several times over
a 72 h induction period (0, 6, 12, 24, 48 and 72h).
CA 02485178 2004-11-22
36
Example 3 - Materials and Methods - Anticancer activity of
natural and recombinant pediocin PA-1
Pediocin PA-1. Both highly purified pediocin PA-1
(Example 1), obtained from the naturally producing
Pediococcus acidilactici PAC 1.0 strain (Quest International,
Sarasota, Fla.), and semi-purified recombinant pediocin PA-1
(Example 2), produced by Pichia pastoris (KMH71, MutS
phenotype, Invitrogen) were tested in this study. Production
and purification was done as described earlier for natural
pediocin PA-1 (Example 1) and for the recombinant pediocin
PA-1 (Example 2).
Cell culture. The human lung carcinoma cell line A-
549 and the human colon adenc>carcinoma cell line DLD-1 were
obtained from the American Type Culture Collection (ATCC).
I5 Both cell lines were cultured in minimum essential medium
containing Earle's salts and L-glutamine (Mediatech Cellgro,
VA), to which were added loo fetal bovine serum (Hyclone),
vitamins (1X), penicillin (100 I.U./ml) and streptomycin
(100 ~g/ml), essential amino acids (1X) and sodium pyruvate
(1X) (Mediatech Cellgro, VA). Cells were kept at 37°C in a
humidified environment containing 5o CO2.
Anticancer activity assay. Exponentially growing
cells were plated in 96-well microplates (Costar, Corning
inc.) at a density of 5 x 103 cells per well in 100 ~l of
culture medium and were allowed to adhere for 16 h before
treatment. Increasing concentrations of both natural and
recombinant pediocin PA-1 in culture medium (minimum
essential medium, described previously) were then added
(100 ~l per well). The cells were incubated for 48 h in the
presence or absence of pediocin PA-1. The cell growth was
assessed using the resazurin reduction test (0'Brien et al.,
2000). Fluorescence was measured on an automated 96-well
Fluoroskan Ascent F1TM plate reader (Labsystems) using
CA 02485178 2004-11-22
37
excitation and emission wavelengths of 350 nm and 590 nm,
respectively. Anticancer activity was expressed as the
concentration of natural or recombinant pediocin PA-1
inhibiting cell growth by SOo (ICSO). The experiment was
repeated and produced very similar results.
Example 4 - Results - Anticancer activity of natural and
recombinant pediocin PA-I
Anticancer drug discovery using human tumor cell
lines has attracted increasing interest since the mid-1980s
(Baguley and Marshall, 2004). Some antimicrobial peptides are
known to exhibit antitumor activity in tumor-derived cell
lines (melittin, cecropin, magainin) (Winder et al., 1998).
The potential anticancer activity of pediocion PA-1 was
evaluated herein. Both natural pediocin PA-1 and recombinant
pediocin PA-l, obtained in an inactive form from the yeast
Pichia pastoris, were assayed for their anticancer activity
on two carcinoma cell lines using the resazurin reduction
test (0'Brien et al., 2000). The two cell lines tested were
a human lung carcinoma cell line (A-549) and a human colon
adenocarcinoma cell line (DLD-1). Natural, highly purified
pediocin PA-1 showed a antiproliferative effect on both cell
lines (two separate experiments) while no growth inhibition
was observed for recombinant, semi-purified pediocin PA-1
(Table 1 - below). Natural pediocin PA-1 showed very similar
ICSO (~M) values for both human cell lines, which were 1.66 ~M
for lung tumoral cells and 1.61 ~M for colon tumoral cells.
Such values are indicative of significant clinical potential
and have also been reported for other therapeutic agents of
medicinal importance (DNA-reactive compounds,
antimetabolites, mitotic poisons, topoisomerase
poisons)(Luber and Hardy, 2001).
CA 02485178 2004-11-22
38
TABLE 1: Anticancer activity (ICso) of highly purified
natural pediocin PA-1*
Human cell line Pediocin PA-1 ICso (~g/ml)
A-549a 7.66 ~ 0.69 (1.66 ~ 0.15 uM)
DLD-1° 7.43 ~ 0.45 (1.61 ~ 0.10 uM)
* Recombinant pediocin PA-1 produced by P. pastoris showed no
anticancer activity under the same conditions.
Human lung carcinoma cell line.
Human colon adenocarcinoma cell line.
In the past, some authors have demonstrated that
partially purified bacteriocins (PPBs) (Farkas-Himsley, 1988;
Hill and Farkas-Himsley, 1991.) obtained from certain non-
lactic acid bacteria (colicins from E. coli, vibriocins from
V. cholera and pyocins from P. aeruginosa) (Farkas-Himsley et
al., 1975; Farkas-Himsley and Cheung, 1976; Farkas-Himsley
and Musclow, 1980) under specific growth conditions (Farkas-
Himsley and Yu, 1985), contained materials which were active
on various malignant cells, both in vitro (Farkas-Himsley,
1980; Farkas-Himsley et al., 1975; Farkas-Himsley and Cheung,
1976) and in vivo (Hill and Farkas-Himsley, 1991). The PPBs
demonstrated selective killing characteristic,
differentiating malignant from normal cells (Farkas-Himsley,
1980; Musclow et al., 1987). These authors also noted that
the malignant cells which interacted with PPBs contained less
DNA than the control cells not treated with PPBs (Farkas-
Himsley and Yu, 1985). It was also shown that PPBs interacted
with the cell surface, possibly via transferrin receptors
CA 02485178 2004-11-22
39
(Farkas-Himsley and Musclow, 1986). And finally, they
reported that PPBs most likely belonged to the group of
proteins that interact with external cell surface receptors
and are able to signal the activation of endonucleolytic
processes. This supported the hypothesis that PPBs cause
programmed cell death by apoptosis (Farkas-Himsley et al.,
1992) .
However, the partially purified bacteriocins
studied were rather ~~crude" preparations and it possible that
contaminating materials (e.g. endotoxins) may have been the
cause of variability in the results, primarily for the in
vivo experiments. It was recognized that much better
purification of the bacteriocins would be required before
establishing more definitive conclusions (Fumorola, 1978;
Fumorola et al., 1977). In the case of the highly purified
pediocin PA-1 preparation tested herein, the results obtained
are noticeably much more valuable due to the quality and the
purity of the pediocin PA-1 preparation used.
Further, some bacteriocins can be toxic to
mammalian cells (Murinda et al., 2003). Thus, some
bacteriocins often intended for use as biopreservatives need
to be evaluated for toxicity to mammalian cells.
In contrast, LAB and products derived therefrom are
generally considered safe. Indeed, studies carried out with
nisin A and pediocin PA-1/AcH have indicated that both
bacteriocins are nontoxic to laboratory animals and humans
when used at the recommended levels. As such, the uses of
LAB-derived bacteriocins described herein provide the
advantage of being both safe and having anticancer activity.
In the present study, natural pediocin (e. g.
produced by Pediococcus sp.) showed anticancer activity
whereas the recombinant pediocin produced by Pichia sp.
showed no activity. The latter observation is consistent with
CA 02485178 2004-11-22
previous results showing the absence of biological activity
for recombinant pediocin PA-1 (Example 2).
Example 4 - Production of recombinant pediocin PA-1 in
5 Escherichia coli
Strains, plasmids and culture conditions. The
sources and relevant genotypes of bacterial strains, as well
as the plasmids used in this study are listed in Table I.
Escherichia coli DHSa, used for standard cloning procedures,
10 and the Origami (DE3) cell line, used for gene expression
experiments, were grown in Luria-Bertani (LB) broth (1.0o
tryptone, 1.0% NaCl, 0.5o yeast extract, pH 7.0).
Transformants of E.coli were selected onto LB medium
supplemented with ampicillin (60 ~g/ml). Pediococcus
15 acidilactici PAC 1.0, the producing strain of pediocin PA-1,
and Pediococcus pentosaceus FBB63, used as indicator strain,
were grown in Lactobacilli MRS broth (Oxoid) for 18h at 37°C.
Cloning of pedA and plasmid construction. In vitro
DNA manipulations (digestion, ligation, transformation) for
20 cloning in E. coli were performed as described by Sambrook et
al. (1989). A DNA fragment encoding the mature domain of
pediocin PA-1 was obtained by polymerase chain reaction (PCR)
amplification of pSRQll DNA isolated fram Pediococcus
acidilactici PAC 1.0 using the QIAprepTM Spin Miniprep Kit
25 protocol (QIAgen) with an alkaline lysis procedure (2001 of
a cold solution containing 50mM glucose, lOmM EDTA, 25mM Tris
pH 8.0 and 4mg/ml of lysozyme (Sigma) were added after step
3). The sequence of the 5' primer used for amplification (5'-
AAC CCC AGA TCT CGA CGA CGA CAA GAA ATA CTA CGG TAA TGG G-3'
30 [SEQ ID NO: 6]) begins with AACCCC nucleotides clamp followed
by a BglII restriction enzyme site (AGATCT), the enterokinase
cleavage sequence (CGACGACGACAAGAA [SEQ ID N0: 7]) and ends
with a 18 nucleotide sequence complementary to the first
CA 02485178 2004-11-22
41
nucleotides of the pedA gene encoding for the mature peptide
form. The 3' primer (5'-CCC GGG CTC GAG CTA TTA TCA GCA TTT
ATG ATT ACC TTG ATG TCC A-3' [SEQ ID NO: 8]) contains CCCGGG
nucleotides clamp, followed by an XhoI restriction enzyme
site (CTCGAG), the stop colons and ends with a 25 nucleotide
sequence complementary to the final eight colons of the pedA
gene. PCR amplification was performed using 40U of Deep VentTM
(exo-) DNA polymerase (New Englands Biolabs) per ml, 1.35 pg
of pSRQll template per ml, 1.25mM of each nucleoside
triphosphate, and 2 ~g of each primer per ml. PCR was
performed to amplify DNA with a hot start at 96°C for 1 min
and, then, 30 cycles of amplification (96°C, 30s for strand
denaturation; 60°C, 30s, for primer annealing and 72°C, 60s,
for primer extension) followed by a final extension at 72°C
for 1 min. The PCR product was purified using the QIAquickTM
PCR Purification Kit Protocol (QIAgen) and digested with
BglII and XhoI restriction endonucleases (New England
Biolabs). The resulting fragment was purified by agarose gel
electrophoresis using the QIAex IITM agarose gel extraction
kit (QIAgen) and ligated with T4 DNA Ligase (New England
Biolabs) between the BglII and XhoI restriction sites within
the pET32b vector. pET32b was pre-digested with the same
enzymes. Ligation mixtures were transformed into competent
E.coli DHSa cells employing the CaCl2 approach. The pET32b-
pedA expression plasmid, isolated using the QIAprepTM Spin
Miniprep Kit protocol (QIAgen), was identified by restriction
enzyme digestion with XbaI/XhvI (New England Biolabs).
Vectors (pET32b and pET32b-pedA) were transferred into E.coli
Origami (DE3) cells. Transformants were selected on Luria
Bertani (LB) medium containing ampicillin (60 ~g/ml). The
pET32b vector and the pET32b-pedA expression plasmid were
isolated using the QIAprepTM Spin Miniprep Kit protocol
CA 02485178 2004-11-22
42
(QIAgen). The pedA gene region was confirmed by double-
stranded DNA sequencing with AmpliTaqTM DNA polymerase (ABI
PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit)
using an ABI 3777XL DNA Sequencer (Applied Biosystems). For
PCR sequencing, the sequence of the 5' primer used for
amplification was (5'-TTC CTT TCG GGC TTT GTT AGC AGC-3' [SEQ
ID N0: 9]) and the sequence of the 3' primer was (5'-TAA ATT
CGA ACG CCA GCA CAT GGA-3' [SEQ ID N0: 10]). These primers
corresponded to nucleotide sequences complementary to the
upstream and downstream regions of the pedA gene to confirm
that its position was in frame with the thioredoxin gene. PCR
reaction products were purified using Centri-SepTM columns
(Applied Biosystems) prior to sequencing.
Expression of pediocin PA-1 in E.coli.
Transformants (clone Bl) of E.coli Origami (DE3), stored in
15% glycerol kept at -80°C (0.7 ml), were grown in 2 separate
shake flasks of 2L, each with 500 ml of LB medium
supplemented with ampicillin ;100 ~g/ml) until the culture
reached an OD600 of 0.6-0.8 (approximately 7h). Protein
expression was induced by addition of isopropyl ~-D-
thiogalactoside (IPTG, Sigma) to a final concentration of
20 ~M. The culture was harvested four hours after induction
by centrifugation (30008, 25 min, 4°C). The cell pellets were
stored frozen at -20°C prior t.o analysis.
Preparation of cleared cell lysates. Cell lysates
were prepared using frozen cells from one liter of culture.
Cells were resuspended in 100 ml of 50 mM sodium phosphate
buffer, pH 8.0, containing 300 mM NaCl. The mixture was
homogenized using a high-pressure homogenizer (Microfluidics
International Corporation, Newton, MA) to disrupt the cells.
Cell breakage, done on ice, was initially performed using a
resting pressure of 60psi (0.4MPa) corresponding to an
internal pressure of 17000psi (117MPa) and it was repeated 3
CA 02485178 2004-11-22
43
times. The resulting cell lysate was centrifuged at 140008
for 30 min at 4°C to remove the insoluble fraction. Cleared
cell lysate was filtered using a 0.45~m, low protein binding
membrane (Cellulose acetate, Corning) and stored at -20°C
until needed.
Affinity purification of fusion protein under
native conditions. Fusion protein (Trx-pedA) present in the
cleared cell lysate was captured using a Ni-NTA agarose resin
(QIAgen). The VL11 X 250 Amicon column was packed with lOml
of resin and connected to a GradiFracTM Chromatography system
integrated with an UV1-detector (UV lamp, OD280 nm,
Amersham). The column was equilibrated with 10 CV (column
volume) of 50mM sodium phosphate buffer, 300 mM NaCl, pH 8.0
(Buffer A). Cleared cell lysate solution (100 ml) was applied
to the column at a rate of 30b cm/h, then, the column washed
with Buffer A. The fusion protein was eluted with Buffer A
containing 250mM imidazole (ICN Biomedicals, Inc.).
Concentration and diafiltration. Fractions
containing Trx-pedA were pooled and concentrated in a 350m1
Stir Cell system (Amicon) using a 3kDa (cut-off) regenerated
cellulose membrane (YM3, Diaflo ultrafiltration membrane,
Amicon) and diafiltered against 20mM Tris-HCl, 50mM NaCl, 2mM
CaCl2 buffer, pH 7.4 (cleavage buffer).
Site-specific cleavage of Trx-pedA using
recombinant enterokinase. The retentate (26 ml) obtained
after the concentration step was treated with 80U of
recombinant enterokinase (rek, Novagen) overnight at room
temperature under gentle rotational agitation (Adams
Nutator). The reaction mixture was centrifuged (5 min, 18118
at 20°C) and supplemented with NaCl to reach a concentration
of 0.5M. The pH was adjusted with HC1 (0.1N) to 5.0 prior to
hydrophobic interaction chromatography (HIC).
CA 02485178 2004-11-22
44
Hydrophobic interaction chromatography. The
retentate after recombinant enterokinase treatment, which
contained biologically active pediocin PA-l, was applied to
an Octyl Sepharose CL-4B hydrophobic interaction column
(HR5/5, 1 ml CV, Amersham) using a GradiFrac system as
mentioned above (Example 1). The column was equilibrated with
5 mM ammonium acetate buffer, pH 5.0, (Buffer B) containing
0.5 M NaCl. The retentate was applied to the column at
306 cm/h, washed with the same buffer (Buffer B + 0.5 M NaCl)
followed by another wash with Buffer B. Pediocin PA-1 was
eluted with Buffer B containing 50% (v/v) acetonitrile. After
removal of the acetonitrile by evaporation (Rotavapor,
Buchi), the preparation was loaded onto a semi-preparative
reversed-phase HPLC column.
Semi-preparative HPLC purification. The active
recombinant pediocin PA-1 fraction eluated from the Octyl-
Sepharose column was loaded onto a C18 reversed-phase column
(Semi-prep column CSC-Inertsil 150A/ODS2, 5 um, 25 x l.Ocm)
and purified by high-pressure liquid chromatography (Waters
Millennium32, Waters Scientific, Mississauga, Ontario,
Canada). The system was equipped with a Waters 996 Photo
Diode Array detector, a Waters 600E solvent delivery pump, a
Waters 717 autosampler, and a Waters temperature control
module (TCM). A method incorporating a 10 minute non-linear
gradient (curve profile number 5) going from 24% (v/v)
acetonitrile in 5 mM HCl to 600 (v/v) acetonitrile in 5 mM
HC1 was utilized. The sample was eluted at a flow rate of
3 ml/min with a column temperature of 39°C. The natural (used
as standard) and recombinant pediocin PA-1 peak appeared at a
retention time of 6 min, corresponding to an acetonitrile
concentration of 510 (v/v). Active fractions were pooled and
concentrated with a 50 ml Stir Cell system (Amicon) using a
1 kDa (cut-off) regenerated cellulose membrane (YM1, Diaflo
CA 02485178 2004-11-22
ultrafiltration membrane, Amicon) and diafiltered against
water (HPLC grade). The retentate was freeze-dried (Flexi-
dry, FTS Systems Inc.) and kept at 4°C under a NZ atmosphere.
Detection of pedioci_n PA-1. Purification was
5 followed using a non-competitive indirect ELISA method
(Martinez et al. 2000). Microtiter plate wells (Maxisorp,
Nunc) were coated with 100 ~l samples diluted in CB (CB =
Coating Buffer: 0.1 M sodium carbonate-bicarbonate buffer, pH
9.6). The plates were incubated overnight at 4°C, blocked for
10 1h at 37°C with 300 ~1 of to (w/v) ovalbumin (Sigma, grade
III) in PBS (phosphate-buffered saline O.OlM, pH 7.4) and
washed with 0.050 Tween 20 in PBS. Then, 50 ~l of polyclonal
antibodies against pediocin PA-1 (antiserum provided by
Professor P. E. Hernandez; Martinez et al. 1998) diluted 1000
15 times in PBS were added and the plates incubated for 1h at
37°C. Goat anti-rabbit IgG-HRP (Horseradish Peroxidase)
(Biorad) diluted 500 times in ovalbumin solution (lo (w/v) in
PBS; blocking solution) was added and the complex was
revealed by adding 100 ~l of ABTS (2,2'-azino-bis(3-
20 ethylbenzthiazoline-6-sulphonic acid) substrate (Sigma).
Absorbance was read at 405 nm using a SPECTRAFluor Plus
Spectrophotometer (TECAN Austria Gmbh, Grodig, Austria).
Activity test of pediocin PA-1. Samples were
analyzed by agar spot test on MRS agar medium (Parrot et al.
25 1990). A MRS 1.5o agar plate (20 ml) was covered with 5m1 of
soft agar (0.8% (w/v) agar) containing a MRS-grown overnight
culture of the indicator strain Pediococcus pentosaceus
(adjusted to an optical density of 0.1 at 600 nm, Beckman DU
640 Spectrophotometer). Samples were filtered using a 0.22 ~m
30 membrane (Syringe Driven Filter Unit, Millex-HV, 4mm) prior
to addition of 5 ~l onto the surface of the soft agar. The
CA 02485178 2004-11-22
46
plates were incubated for 18h at 37°C and examined for growth
inhibition zones.
Biological activity of the recombinant pediocin PA-
1 was also analyzed after Trx-PedA cleavage using the agar
spot test method (Parrot et a1.1990). Table 2 shows the dish
overlay results. An inhibition zone of growth was observed
for section 1 corresponding to the natural pediocin PA-1
standard (Example 1). Biological activity was not detected
for the uncleaved Trx-pedA. A very marked inhibition zone was
noted for the recombinant ped:iocin PA-1 after cleavage by the
enterokinase, which was comparable to that observed with
natural pediocin PA-1.
Table 2 - Measurements of natural and recombinant pediocin
activity using the dish overlay assay
Type of pediocin used Inhibition of growth of
Pediococcus pentosaceus
Natural ~+++
Recombinant uncleaved
Recombinant cleaved I+++
Throughout this application, various references are
referred to describe more fully the state of the art to which
this invention pertains. The disclosures of these references
are hereby incorporated by reference into the present
disclosure.
CA 02485178 2004-11-22
47
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