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 for example be more effective, more selective towards cancerous cells, may not cause drug
resistance and/or may be used to halt the progression of multi-drug resistant cancerous cells.
SUMMARY 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 a further aspect, the present invention provides a composition for preventing or treating cancer in an animal, said composition comprising a bacteriocin derived from a lactic acid bacteria and a carrier.
In a further aspect, the present invention provides a composition for inhibiting proliferation of a cancerous cell in an animal, said composition comprising a bacteriocin derived from a lactic acid bacteria and a carrier.
In a further aspect, the present invention provides a composition for inhibiting proliferation of a cancerous cell, said composition comprising a bacteriocin derived from a lactic acid bacteria and a carrier.
In a further aspect, the present invention provides a 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 a 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 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) instructions for use of said agent in the treatment or prevention of cancer in an animal.
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 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 above-mentioned agent is cytotoxic to the cancerous cell of the cancer. In another
embodiment, the above-mentioned agent and/or composition is administered by or adapted for administration by (e.g., the above-mentioned carrier is adapted for administration by) 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 above-mentioned agent is bacteriocin. In an 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 bacteriocin 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 and, in a further embodiment, the bacteriocin is pediocin PA-I. In yet another embodiment, the pediocin PA-I 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 1.0.
In an embodiment, the above-mentioned agent is the above-mentioned composition.
In an embodiment, the above-mentioned 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 administration through an oral route. In an embodiment, the above-mentioned 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 above-mentioned animal is a mammal and, in yet a further embodiment, the mammal is a human. In an embodiment, the above-mentioned 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, 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 an embodiment, the above-mentioned cancerous cell is located in or is derived from 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, fallopian tube, ovaries, vagina, vulva, larynges, oropharynges, immune cell, immune system, lung, lymph node and plasma cell. In another embodiment, the cancerous cell is a lung cancerous cell and/or a colon cancerous cell.
BRIEF DESCRIPTION OP THE DRAWINGS
Figure 1. Pediococcus acidilactici bacteriocin complete coding sequence (SEQ ID NO: 1, GenBank accession number M83924)
Figure 2. Pediocin PA-I polypeptide sequence (SEQ ID NO: 2, GenBank accession number AAA25559)
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, thyroid cancer) , immune system (e.g acute lymphocytic leukemia (ALL), acute myelogenous 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 comprising a bacteriocin derived from a lactic acid bacteria and a carrier.
Antimicrobial peptides, such as bacteriocins, possess cationic and amphipathic properties which allow interactions with the membrane of living cells, and have been studied for their potential in various applications (Marshall and Arenas, 2003; Reisch, 2002; Hancock and Lehrer, 1998; Crescenzi et al., 2000; Chen et al., 2000; Wachsman et al., 1999; Wachsman et al., 2003; 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 HoIo, 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 have been described, belonging to the class Ha (pediocin, lactacine, etc.) .
Pediocin PA-I, one of the known class Ha bacteriocins, is produced by Pediococcus acidilactϊci PAC 1.0 and is strongly active against Listeria monocytogenes (Bhunia et al. , 1988) . The primary structure of pediocin PA-I consists of 44 amino acids and its theoretical molecular weight is 4624 Da (Figure 2, SEQ ID NO: 2), in the presence of its two disulfide bonds, which have been studied for their role in its biological activity (Gaussier, 2003; Henderson et al., 1992; Rodriguez et al., 2002) .
The activity of partially purified bacteriocins (PPBs) obtained from certain non-lactic acid bacteria (colicins from E. coli, vibriocins from V. cholera and pyocins from P. aeruginosa) , under specific growth conditions, have been studied, both in vitro and in vivo (Farkas-Himsley, 1988; Hill and Farkas-Himsley, 1991; Farkas- Himsley et al. , 1975; Farkas-Himsley and Cheung, 1976; Farkas-Himsley and Musclow, 1980; Farkas-Himsley and Yu,
1985; Farkas-Himsley, 1980; Farkas-Himsley, 1980; Musclow et al., 1987; Farkas-Himsley and Musclow, 1986; Farkas-Himsley et al., 1992) . However, the partially purified bacteriocins studied were rather "crude" preparations and contaminating materials (e.g. endotoxins) may have been the cause of variability in the results. It was recognized that much better purification of the bacteriocins would be required for such studies (Fumorola, 1978; Fumorola et al., 1977) . 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-l/AcH have indicated that both bacteriocins are nontoxic to laboratory animals and humans when used at the recommended levels. As such, an advantage of the uses of LAB-derived bacteriocins described herein is safety/lack of toxicity.
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 immunofluorescent compounds or an enzyme that enables colorimetric 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-I. Pediocin AcH and pediocin PA-I are used herein interchangeably. In yet another embodiment, pediocin PA-I 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 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 substitution" refers to the substitution of an 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 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); GIu (+3.0); Ser (+0.3); Asn (+0.2); GIn (+0.2); GIy (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); VaI (-1.5); Leu (-1.8); He (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4) . In alternative embodiments, conserved amino acid 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: He (+4.5); VaI (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); GIy (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); GIu (-3.5); GIn (-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, VaI, Leu, He, Phe, Trp, Pro, Met; acidic: Asp, GIu; basic: Lys, Arg, His; neutral: GIy, Ser, Thr, Cys, Asn, GIn, Tyr.
Conservative amino acid substitutions 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 substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3- diamino propionic acid, alpha-aminoisobutyric acid, 4-axnino- 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 hydrophobicity, 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. MoI. Bio. 179:125-142, 1984) . Genetically encoded hydrophobic amino acids include GIy, Ala, Phe, VaI, Leu, lie, Pro, Met and Trp, and genetically encoded hydrophilic amino
acids include Thr, His, GIu, Gin, 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 hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic 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, - NO, -NH2, -NHR, -NRR, -C(O)R, -C(O)OH, -C(O)OR, -C(O)NH2, - C(O)NHR, -C(O)NRR, etc., where R is independently (C1-C6) alkyl, substituted (Cl-Cβ) alkyl, (Cl-Cβ) alkenyl, substituted (C1-C6) alkenyl, (C1-C6) alkynyl, substituted (Cl-Cβ) 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- fIuorophenylalanine3-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 GIy, Leu, VaI, lie, 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, VaI, and lie, 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 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 GIn, 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 GIu. 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 classified 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 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 O-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 (Cl- Cβ) alkyl, (Cl-Cβ) alkenyl, (C1-C6) alkynyl, substituted (Cl- Cβ) alkyl, substituted (C1-C6) alkenyl, or substituted (Cl- Cβ) alkynyl) or isostere of an amide linkage (for example, - CH2NH-, -CH2S, -CH2CH2-, -CH=CH- (cis and trans), -C(O)CH2-, -CH(OH)CH2-, or -CH2SO-) .
In embodiments, the invention further relates to nucleic acid and polypeptide variants or fragments thereof, which are homologous or substantially identical to a nucleic acid or polypeptide of the invention or fragment thereof (e.g., any of SEQ ID Nos: 1-10), and uses thereof for
preventing or treating cancer or for inhibiting proliferation of a cancerous cell. Such variants may differ from a nucleic acid or polypeptide of the invention by substitution, deletion and/or addition of one or more residues (nucleotide or amino acid, as appropriate) . Such variants may be homologous or substantially identical to a nucleic acid or polypeptide of the invention or fragment thereof.
"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 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 identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. As such, in embodiments, a sequence which is substantially identical to any of SEQ ID NOs. 1 to 10 may have at least 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with any of SEQ ID NOs 1 to 10. 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 preferably less than about 25 % identity
(e.g., with any of SEQ ID NOs. 1 to 10) .
Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, of any one of SEQ ID NOs. 1 to 10. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. MoI.
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 GAP, 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. MoI. Biol. 215:403-10 (using the published default settings) . Software for performing BLAST analysis may be available through the National Center for Biotechnology 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 (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 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 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 NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% 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 NaHPO4, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 680C (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 50C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
In another aspect, the 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 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 1.0.
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 60%, more generally 75% or over 90%, 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. 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 (e.g., see 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 D-amino acids by L-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 fermented food product or a fermented milk. The incorporation of bacteriocins into a food product or nutraceutical may prevent the degradation of the bacteriocins.
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
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 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 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 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 prophylactically 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 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 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 a further aspect of the invention, a bacteriocin 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 247:1465-1468) . A delivery apparatus (e.g., a "gene 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 DNA to a cation, 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 el al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.
5,166,320) . Binding of the DNA-ligand complex to the 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 Curiel el 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. (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 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) 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. MoI. 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) MoI. 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) MoI. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) MoI. 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. 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.
EXAMPLES
In the studies described herein, the potential of growth inhibition of pediocin PA-I on the A-549 human lung carcinoma cell line and the DLD-I human colon adenocarcinoma cell line was evaluated.
Example 1 - Production of native/natural pediocin PA-I
Pediococcus acidilactici strain PAC 1.0 (Quest International, Sarasota, FIa.) was cultivated in a 20 L fermentor (Chemap, Switzerland) equipped with pH and pθ2 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 additional aeration was provided and no chemical antifoam was added during cultivation.
The cells were separated from the broth by centrifugation (500Og, 15 min, 20°C) . The supernatant fluid was concentrated 13-fold using a 0.5m2 1 kDa (cut-off) regenerated cellulose membrane (PCAC™ membrane, Millipore) , then, diafiltered against 5mM ammonium acetate buffer, pH°5.0, using a Pellicon™ system (Millipore) with a Masterflex peristaltic pump at a recirculation rate of 200 ml/min. The process was performed at room temperature. Ion exchange chromatography (IEX) . Pediocin PA-I purification was achieved using a modification of the procedure described by Gaussier et al. (2002) . Pediocin PA-I from the concentrated supernatant fluid (1200 ml) was captured using a SP-Sepharose Fast Flow™ cation exchange 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 PA-I 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-I was eluted from the column with Buffer A containing 0.5M NaCl.
Hydrophobic interaction chromatography (HIC)
Screening of chromatography media. Several HIC media with varying hydrophobicity were tested, namely: Butyl-Sepharose™, Amersham; Butyl-650M, Tosohaas; Octyl-Sepharose™ CL 4B, Amersham; Phenyl-Sepharose™, 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-Sepharose™ 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 50% (v/v) acetonitrile. Biorad Econo-Columns™ were used for the screening work and elution was performed by gravity. Eluate fractions were analyzed for pediocin PA-I' 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-Sepharose™ column were pooled and applied to an Octyl Sepharose™ CL-4B hydrophobic interaction column (HR5/5, 1 ml CV, Amersham) 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-I was eluted with Buffer A containing 50% (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-I material. The active fractions were pooled and concentrated (13-fold) using a 350 ml Stir Cell system (Amicon) and a IkDa (cut-off) regenerated cellulose membrane
(YMl, Diaflo™ ultrafiltration membrane, Amicon) ending with diafiltration against water (HPLC grade) . The retentate was lyophilized (Flexi-dry™, FTS Systems Inc.) and the material was stored at 4°C under a N2 atmosphere.
Example 2 - Production of recombinant pediocin PA-I in Pichia pastoris
Strains, plasmids and culture conditions. Escherichia coli TOPlO was grown in Luria-Bertani (LB) broth (1% tryptone, 1% NaCl, 0.5% yeast extract, pH 7.0) . E. coli transformants were grown in Low Salt Luria-Bertani (LSLB) medium, supplemented with zeocin (25μg/ml) overnight at 370C. Pediococcus acidilactici PACl.0 and Pediococcus pentosaceus FBB63 were grown in Lactobacilli MRS broth (Oxoid) for 18h at 37°C. Yeast strains were cultured in YPD (1% yeast extract, 2% peptone, 2% glucose) medium, whereas yeast transformants were grown in baffled shake flasks under selective conditions in buffered glycerol-complex medium (BMGY: 1% yeast extract, 2% peptone, 10OmM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base without amino acids, 4X10~5% biotin, 1% glycerol) until cultures reached an ODeoonm 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 300C in buffered methanol complex medium (BMMY: 1% yeast extract, 2% peptone, 10OmM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base without amino acids, 4X10~5% biotin, 1% methanol) for the induction of pediocin PA-I 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 EasySelect™ Pichia Expression Kit (Invitrogen Corporation) .
Cloning of pedA and plasmid construction. A DNA fragment encoding the mature domain of pediocin PA-I was
obtained by polymerase chain reaction (PCR) amplification of pSRQll DNA isolated from Pediococcus acidilactici PACl.0 using the QIAprep™ Spin Miniprep Kit protocol (QIAgen) and with an alkaline lysis procedure (200μl of a cold solution containing 5OmM glucose, 10mm 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 Gee¬ s'' [SEQ ID NO: 3]) begins with 6xA nucleotide clamp, followed by a Xhol restriction enzyme site (CTCGAC) , the Kex2 signal cleavage sequence (AAAAGAGAGGTCGAAGCT [SEQ ID NO: 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 Accl 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 4OU of rTaq DNA polymerase (Amersham) per ml, 1.35 pg of pSRQll template per ml, 1.25mM of each nucleoside triphosphate, and 2 μg of each primer per ml. PCR was performed to amplify DNA with a hot start at 960C for 1 min and then 30-cycles of amplification (9β°C, 30s for strand denaturation, 55°C, 30s for primer annealing, and 720C, 60s for primer extension) followed by a final extension at 720C for 2 min. The PCR product was purified using the QIAquick™ PCR Purification Kit Protocol (QIAgen) and digested with Xhol and Accl restriction endonucleases (Amersham and New England Biolabs, respectively) . The resulting fragment was purified by agarose gel electrophoresis using the QIAex™ II agarose gel extraction kit (QIAgen) and ligated with T4 DNA Ligase (New England Biolabs) between the Xhol and Accl restriction sites
within the pPICZαA vector. pPICZαA was pre-digested with the same enzymes. Ligation mixtures were transformed into competent E.coli TOPlO cells (TOPO TA Cloning, Version K, Invitrogen) . In vitro DNA manipulation (digestion, ligation) for cloning in E. coli were performed as described by
Sambrook et al. (1989) . The pPICZαA-pedA expression plasmid, isolated using the QIAgen Plasmid Maxi Protocol (QIAgen) , was identified by restriction enzyme digestion with Xhol/Accl and BamRI/SacI (New England Biolabs) . The pedA gene region was confirmed by double-stranded DNA sequencing with AmpliTaq DNA polymerase (ABI PRISM™ 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 α-factor (EasySelect™ Pichia
Expression Kit, Invitrogen Corporation) . PCR reactions were purified on a Centri-Sep™ column (Applied Biosystems) prior to performing sequence analysis. pPICZαA (10 μg) and pPICZαA- pedh (10 μg) vectors were linearized with 4OU of Sacl restriction endonuclease (Amersham) following the recommendations given by the EasySelect™ Pichia Expression Kit (Invitrogen Corporation) . Linearized vectors were electroporated into P. pastoris. Electro-competent cells
(80 μl) were mixed with DNA solution (10 μg) in a 0.2 cm gap cuvette chilled on ice. Electroporation was carried out using a Gene Pulser™ (Bio-Rad) with the following parameters: 1.5 kV, 400Ω, 25 μF to a final field strength of 7.5 kV cm"1. After cells had been pulsed, 1 ml of ice-cold sterile IM sorbitol was immediately added to the cuvette, the cell suspension was transferred into a test tube, and the mixture incubated at 300C for lh30. Transformed clones were selected on Yeast Extract Peptone Dextrose Sorbitol medium (YPDS: 1%
yeast extract, 2% peptone, 2% dextrose, IM sorbitol, 2% agar) containing zeocin (100 μg/ml) . Transformants were also tested on both Minimal Dextrose Medium, with and without histidine, (1.34% yeast nitrogen base without amino acids, 4X10"5% biotin, 2% dextrose, with and without 0.004% histidine, 1.5% agar) and Minimal Methanol Medium, with and without histidine, (1.34% yeast nitrogen base without amino acids, 4X10"5% biotin, 2% methanol, with and without 0.004% histidine, 1.5% agar) plates to confirm the Mut+ phenotype of the X-33 and GS115 strains.
Detection of activity of pediocin PA-I by agar spot test. All transformants were grown in shake flasks as mentioned before and tested by agar spot test on MRS medium (Parrot et al., 1990) for detection of biological activity. A MRS plate (1.5% (w/v) agar, 20 ml) was covered with 5 ml of soft agar (0.8% (w/v) agar) containing an overnight MRS-grown culture of the indicator strain P. pentosaceus (adjusted to an ODβoonm of 0.1, Beckman DU 640 Spectrophotometer) . Supernatant fluid samples were filtered using a 0.22 μm membrane (Syringe Driven Filter Unit, Millex-HV, 4 mm) and 5 μl 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. The recombinant Pichia was grown in baffled shake flasks in BMGY medium until glycerol was exhausted and, then, in BMMY medium with methanol (0.5% (v/v) ) for 3 days. The cultures were harvested by centrifugation (5000gr, 15 min, 40C) and the supernatant fluids analyzed. Expression levels were monitored by quantitative ELISA testing. A pediocin concentration of 74 μg/ml was achieved in Pichia comparison with 6 μg/ml for the natural producing strain of P. acidilactici (Example 1) . However, biological activity against P. pentosaceus was not detected in the supernatant
fluids of the P. pastoris cultures tested several times over a 72 h induction period (0, 6, 12, 24, 48 and 72h) .
Example 3 - Materials and Methods - Anticancer activity of natural and recombinant pediocin PA-I
Pediocin PA-I. Both highly purified pediocin PA-I (Example 1), obtained from the naturally producing Pediococcus acidilactici PAC 1.0 strain (Quest International, Sarasota, FIa.) , and semi-purified recombinant pediocin PA-I (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-I (Example 1) and for the recombinant pediocin PA-I (Example 2) . Cell culture. The human lung carcinoma cell line A-
549 and the human colon adenocarcinoma cell line DLD-I were obtained from the American Type Culture Collection (ATCC) . Both cell lines were cultured in minimum essential medium containing Earle's salts and L-glutamine (Mediatech Cellgro, VA) , to which were added 10% fetal bovine serum (Hyclone) , vitamins (IX), penicillin (100 I.U. /ml) and streptomycin (100 μg/ml) , essential amino acids (IX) and sodium pyruvate (IX) (Mediatech Cellgro, VA) . Cells were kept at 37°C in a humidified environment containing 5% 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 μl of culture medium and were allowed to adhere for 16 h before treatment. Increasing concentrations of both natural and recombinant pediocin PA-I in culture medium (minimum essential medium, described previously) were then added (100 μl per well) . The cells were incubated for 48 h in the presence or absence of pediocin PA-I. The cell growth was
assessed using the resazurin reduction test (O'Brien et al. ,
2000) . Fluorescence was measured on an automated 96-well Fluoroskan Ascent Fl™ plate reader (Labsystems) using excitation and emission wavelengths of 350 nm and 590 nm, respectively. Anticancer activity was expressed as the concentration of natural or recombinant pediocin PA-I inhibiting cell growth by 50% (IC50) . 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) . The potential anticancer activity of pediocion PA-I was evaluated herein. Both natural pediocin PA-I and recombinant pediocin PA-I, 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 (O'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-I showed a antiproliferative effect on both cell lines (two separate experiments) while no growth inhibition was observed for recombinant, semi-purified pediocin PA-I (Table 1 - below) .
Natural pediocin PA-I showed very similar IC50 (μM) values for both human cell lines, which were l.ββ μM for lung tumoral cells and 1.61 μM 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) .
TABLE 1: Anticancer activity (IC50) of highly purified natural pediocin PA-I*
Human cell line Pediocin PA-I IC50 (μg/ml)
A-549a 7 . 66 ± 0 . 69 ( 1 . 66 ± 0 . 15 μM)
DLD-lb 7 . 43 ± 0 . 45 ( 1 . 61 + 0 . 10 μM)
* Recombinant pediocin PA-I produced by P. pastoris showed no anticancer activity under the same conditions. a Human lung carcinoma cell line. b Human colon adenocarcinoma cell line.
A high quality/purified preparation of PA-I was used in the studies described herein. It was found that 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 results showing the absence of biological activity for recombinant pediocin PA-I (Example 2) .
Example 4 - Production of recombinant pediocin PA-I in 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 1.
Escherichia coli DH5α, used for standard cloning procedures, and the Origami (DE3) cell line, used for gene expression experiments, were grown in Luria-Bertani (LB) broth (1.0% tryptone, 1.0% NaCl, 0.5% yeast extract, pH 7.0) .
Transformants of E. coli were selected onto LB medium
supplemented with ampicillin (60 μg/ml) . Pediococcus acidilactici PAC 1.0, the producing strain of pediocin PA-I, and Pediococcus pentosaceus FBB63, used as indicator strain, were grown in Lactobacilli MRS broth (Oxoid) for 18h at 370C. Cloning of pedA and plasmid construction. In vitro
DNA manipulations (digestion, ligation, transformation) for cloning in E. coli were performed as described by Sambrook et al. (1989) . A DNA fragment encoding the mature domain of pediocin PA-I was obtained by polymerase chain reaction (PCR) amplification of pSRQll DNA isolated from Pediococcus acidilactici PAC 1.0 using the QIAprep™ Spin Miniprep Kit protocol (QIAgen) with an alkaline lysis procedure (200μl of a cold solution containing 5OmM glucose, 1OmM 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' [SEQ ID NO: 6]) begins with AACCCC nucleotides clamp followed by a BgIII restriction enzyme site (AGATCT) , the enterokinase cleavage sequence (CGACGACGACAAGAA [SEQ ID NO: 7]) 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'-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 Xhol restriction enzyme site (CTCGAG) , the stop codons and ends with a 25 nucleotide sequence complementary to the final eight codons of the pedA gene. PCR amplification was performed using 4OU of Deep Vent™ (exo-) DNA polymerase (New England Biolabs) per ml, 1.35 pg of pSRQll template per ml, 1.25mM of each nucleoside triphosphate, and 2 μg of each primer per ml. PCR was performed to amplify DNA with a hot start at 960C for 1 min and, then, 30 cycles of amplification (960C, 30s for strand denaturation; 6O0C, 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 QIAquick™ PCR Purification Kit Protocol (QIAgen) and digested with BgIII and XhoT restriction endonucleases (New England Biolabs) . The resulting fragment was purified by agarose gel electrophoresis using the QIAex II™ agarose gel extraction kit (QIAgen) and ligated with T4 DNA Ligase (New England Biolabs) between the BgIII and Xhol restriction sites within the pET32b vector. pET32b was pre-digested with the same enzymes. Ligation mixtures were transformed into competent E.coli DH5α cells employing the CaCl2 approach. The pET32b- pedA expression plasmid, isolated using the QIAprep™ Spin Miniprep Kit protocol (QIAgen) , was identified by restriction enzyme digestion with Xbal/Xhol (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 μg/ml) . The pET32b vector and the pET32b-pedA expression plasmid were isolated using the QIAprep™ Spin Miniprep Kit protocol (QIAgen) . The pedA gene region was confirmed by double- stranded DNA sequencing with AmpliTaq™ 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 NO: 9]) and the sequence of the 3' primer was (5'-TAA ATT CGA ACG CCA GCA CAT GGA-3' [SEQ ID NO: 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-Sep™ columns (Applied Biosystems) prior to sequencing.
Expression of pecilocin PA-I 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 μg/ml) until the culture reached an OD600 of 0.6-0.8 (approximately 7h) . Protein expression was induced by addition of isopropyl β-D- thiogalactoside (IPTG, Sigma) to a final concentration of 20 μM. The culture was harvested four hours after induction by centrifugation (300Og, 25 min, 4°C) . The cell pellets were stored frozen at -200C prior to 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 times. The resulting cell lysate was centrifuged at 1400Og for 30 min at 40C to remove the insoluble fraction. Cleared cell lysate was filtered using a 0.45μm, low protein binding membrane (Cellulose acetate, Corning) and stored at -2O0C 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 VLIl X 250 Amicon column was packed with 10ml of resin and connected to a GradiFrac™ Chromatography system integrated with an UVl-detector (UV lamp, OD280 nm, Amersham) . The column was equilibrated with 10 CV (column volume) of 5OmM 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 306 cm/h, then, the column washed with Buffer A. The fusion protein was eluted with Buffer A containing 25OmM imidazole (ICN Biomedicals, Inc.) . Concentration and diafiltration. Fractions containing Trx-pedA were pooled and concentrated in a 350ml Stir Cell system (Amicon) using a 3kDa (cut-off) regenerated cellulose membrane (YM3, Diaflo ultrafiltration membrane, Amicon) and diafiltered against 2OmM Tris-HCl, 5OmM NaCl, 2mM CaC12 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 8OU of recombinant enterokinase (rek, Novagen) overnight at room temperature under gentle rotational agitation (Adams
Nutator) . The reaction mixture was centrifuged (5 min, Iδllg at 200C) and supplemented with NaCl to reach a concentration of 0.5M. The pH was adjusted with HCl (0.1N) to 5.0 prior to hydrophobic interaction chromatography (HIC) . Hydrophobic interaction chromatography. The retentate after recombinant enterokinase treatment, which contained biologically active pediocin PA-I, 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-I was eluted with Buffer B containing 50% (v/v) acetonitrile. After removal of the acetonitrile by evaporation (Rotavapor, Bϋchi) , the preparation was loaded onto a semi-preparative reversed-phase HPLC column.
Semi-preparative HPLC purification. The active recombinant pediocin PA-I fraction eluated from the Octyl- Sepharose column was loaded onto a C18 reversed-phase column (Semi-prep column CSC-Inertsil 150A/ODS2, 5 μm, 25 x 1.0cm) 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 60% (v/v) acetonitrile in 5 mM HCl 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-I peak appeared at a retention time of 6 min, corresponding to an acetonitrile concentration of 51% (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 (YMl, Diaflo 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 N2 atmosphere.
Detection of pediocin PA-I. Purification was followed using a non-competitive indirect ELISA method (Martinez et al. 2000) . Microtiter plate wells (Maxisorp, Nunc) were coated with 100 μl 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 Ih at 370C with 300 μl of 1% (w/v) ovalbumin (Sigma, grade III) in PBS (phosphate-buffered saline 0.01M, pH 7.4) and washed with 0.05% Tween 20 in PBS. Then, 50 μl of polyclonal antibodies against pediocin PA-I (antiserum provided by Professor P. E. Hernandez; Martinez et al. 1998) diluted 1000
times in PBS were added and the plates incubated for Ih at
370C. Goat anti-rabbit IgG-HRP (Horseradish Peroxidase) (Biorad) diluted 500 times in ovalbumin solution (1% (w/v) in PBS; blocking solution) was added and the complex was revealed by adding 100 μl of ABTS (2, 2' -azino-bis (3- ethylbenzthiazoline-6-sulphonic acid) substrate (Sigma) . Absorbance was read at 405 run using a SPECTRAFluor Plus Spectrophotometer (TECAN Austria Gmbh, Grodϊg, Austria) . Activity test of pediocin PA-I. Samples were analyzed by agar spot test on MRS agar medium (Parrot et al. 1990) . A MRS 1.5% agar plate (20 ml) was covered with 5ml 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 ran, Beckman DU 640 Spectrophotometer) . Samples were filtered using a 0.22 μm membrane (Syringe Driven Filter Unit, Millex-HV, 4mm) prior to addition of 5 μl onto the surface of the soft agar. The plates were incubated for 18h at 370C 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 al.1990) . Table 2 shows the dish overlay results. An inhibition zone of growth was observed for section 1 corresponding to the natural pediocin PA-I standard (Example 1) . Biological activity was not detected for the uncleaved Trx-pedA. A very marked inhibition zone was noted for the recombinant pediocin PA-I after cleavage by the enterokinase, which was comparable to that observed with natural pediocin PA-I.
Table 2 - Measurements of natural and recombinant pediocin activity using the dish overlay assay
Type of pediocin used Inhibition of growth of Pediococcus pen.tosa.ceus
Natural +++
Recombinant uncleaved —
Recombinant cleaved +++
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.
REFERENCES
Baguley, B. C. and Marshall, E. S. (2004) Eur J Cancer. 40:
794-801. Bhunia, A. K., Johnson, M. C. et Ray, B. (1988) J Appl
Bacterid. 65(4) : 261-268. Breukink, E. and de Kruijff, B. (1999) Biochim Biophys Acta.
1462: 223-234.
Chen, J., et al. (2000) Biopolymers (Peptide Science) . 55:88- 98.
Crescenzi, O. , et al. (2000) Biopolymers. 53:257-264. Farkas-Himsley, H. (1988) Microbios Lett. 39: 57-66. Farkas-Himsley, H. (1980) Microbios Lett. 15:89-96. Farkas-Himsley, H., Cheung, R. (1976) Cancer Res. 36(10) : 3561-3567.
Farkas-Himsley, H., Cheung, R. and Tompkins, W. A. F. (1975)
IRCS Med Sci. 3: 148 Farkas-Himsley, H. and Musσlow, C. E. (1980) IRCS Med Sci. 8:
497-498. Farkas-Himsley, H. and Musclow, C. E. (1986) Cell MoI Biol.
35 (5) : 607-617.
Farkas-Himsley, H. and Yu, H. (1985) Cytobios. 42: 193-207. Farkas-Himsley, H., et al. (1992) Cell MoI Biol. 38(6) : 643-
651. Fumarola, D. (1978) Boll ist sieroter Milan, pp. 101-102. Fumarola, D., et al. (1977) Giornale di Batteriologica,
Virologica ed Immunologia. 70 (1-6) : 87-93. Gaussier, H., Lavoie, M. and Subirade, M. (2003) Int J Biol
Macromol. 32: 1-9. Guder, A., Wiedemann, I. and Sahl, H-G. (2000) Biopolymers
(Peptide Science) . 55: 62-73.
Hancock, R. E. W. (1997) Lancet 349 : 418-422. Hancock, R. E. W. and Lehrer, R. (1998) Tibtech. 16: 82-88.
Henderson, J. T., Chopko, A. L. and van Wassenaar, P. D.
(1992) Arch Biochem Biophys. 295(1) : 5-12. Hill, R. P. and Farkas-Himsley, H. (1991) Cancer Research.
51: 1359-1365. Johnsen, L., Fimlan, G., Eijsink, V. and Nissen-Meyer, J.
(1998) Appl. Env. Microbiol. 66(11) : 4798-4802. Luber, A. D. and Hardy, W. D. (2001) HIV Newsline. 7(1) : http: //www.thebody.com/hivnews/newsline/feb2001/optimal2
.html. Marshall, S. H. and Arenas, G. (2003) Elect J Biotechnol.
6(2) : 271-284. Mi-Kyung, S., et al. (2003) Kor J Microbiol Biotechnol.
31(4) : 355-361.
Miller, K. W. , Schamber, R. , Osmanagaoglu, O. and Bibek, R. (2000) Appl. Env. Res. 64(6) : 1997-2005.
Murinda, S. E., Rashid, K. A. and Roberts, R. F. (2003) J
Food Protect. 66(5) : 847-853. Musclow, C. E., Farkas-Himsley, H., Wetzman, S. S. and
Herridge (1987) Eur J Cancer Clin Oncol. 23(4) : 411- 418.
Nes, I. F. and HoIo, H. (2000) . Biopolymers (Peptide
Science) . 55: 50-61. O'Brien, J., Wilson, I., Orton, T. and Pognan, F. (2000) Eur
J Biochem. 267: 5421-5426. Reisch, M. S (2002) C & EN Northeast news bureau.
Rodriguez, J. M., Martinez, M. I. and Kok, J. (2002) Crit Rev
Food Sci. 42(2) : 91-121. Ross, R. P., et al. (1999) Antonie van Leeuwenhoek. 76: 337-
346. ϋteng, M., et al. (2002) Appl Environ Microb. 68(2) : 952-
956. Wachsman, M. B., et al. (1999) Int J Antimicrob Ag. 12: 293-
299.
Wachsman, M. B.; efc al. (2003) Antiviral Research. 58: 17-24.
Winder, D., Giinzburg, W. H., Erfle, V. and Salmons, B. (1998) Biochemical and Biophysical Research communications. 242: 608-612. Gaussier, H., H. Morency, M. C. Lavoie, and M. Subirade.
(2002) Appl. Environ. Microb. 68: 4803-4808. Parrot, M., Caufield, P. W. and Lavoie, M. C. (1990) Can J
Microbiol. 36: 123-130.
Rodriguez, J. M., M. I. Martinez, and J. Kok. (2002) Grit. Rev. Food Sci. 42: 91-121.