RU2526511C2 - Cell-penetrating peptides and polypeptides for microorganism cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used for methane-producing cell permeability control. What is prepared is a polypeptide able to permeate into a methane-producing cell and to increase its permeability, characterised by an amino acid sequence SEQ ID NO:117, 118 or 119 or being at least 90% identical to the above sequence, or at least 15 sequential amino acids of the above sequence. What is also prepared is a polynucleotide coding the above polypeptide cloning and expressing vectors used for producing host cells producing the polypeptide or used for the vector replication. The polypeptide can contain a fluorescent tag on an N-terminal amino acid residue.

EFFECT: invention enables providing higher methane-producing cell permeability.

18 cl, 35 dwg, 3 ex

Description

RELATED APPLICATIONS

This application claims priority of US application No. 60/975104, filed September 25, 2007, US application No. 60/989840, filed November 22, 2007, and US application No. 60/989841, filed November 22, 2007, the contents of each of which are incorporated into this description by as a link in full.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the delivery of inhibitory molecules to microbial cells, in particular methane-producing cells. In particular, the present invention relates to signal peptides and polypeptides containing these peptides, as well as polynucleotides that encode these peptides or polypeptides. The present invention also relates to expression vectors and host cells for the production of these peptides or polypeptides. The present invention further relates to methods for determining the targeted delivery, penetration and inhibition of microbial cells, in particular methane-producing cells, using the described peptides or polypeptides, polynucleotides, expression vectors and host cells.

BACKGROUND OF THE INVENTION

In New Zealand, agricultural activity accounts for the majority of greenhouse gas emissions. Therefore, reducing agricultural greenhouse gas emissions is important for New Zealand's compliance with the Kyoto Protocol. Under this protocol, greenhouse gases are required to be reduced to 1990 levels by the end of the first commitment period (2008-2012). Towards the end of this period, the Agricultural Sector Groups and the Government of New Zealand approved the Pastoral Greenhouse Gas Research Consortium (PGGRC) to identify ways to reduce New Zealand's agricultural greenhouse gas emissions.

An important part of PGGRC's activity was research on methane emissions from New Zealand pastures. Reducing methane emissions from ruminants is of commercial interest for two reasons. First, non-compliance with Kyoto commitments will force the government to acquire carbon credits. Currently, the cost of this is estimated at $ 350 million. Secondly, the formation of methane leads to a loss of 8-12% of the total energy produced in the rumen. This energy could be used, instead, to improve the productivity of the ruminant.

Methane is produced in the rumen by microorganisms called methane producers, which are part of the taxonomic type Euryarchaeota in the kingdom of Archaea . Most methanogens grow in CO ₂ and H ₂ as the only sources of energy, but some can use acetate or methyl compounds for growth. There are several different genera of archaea methane producers in the rumen, but species of the genus Methanobrevibacter , in particular M. ruminantium and M. smithii, are considered to be the predominant methane producers in ruminants of New Zealand. M. ruminantium is currently the subject of a genome sequencing project funded by PGGRC. This project represents the first sequencing of the genome of rumen methane producers, and its goal is to improve understanding of the biology of Methanobrevibacter to detect targets for inhibiting the formation of methane.

Inhibition of methane producers or inactivation of their methane production pathway is required to reduce the production of methane in the rumen. A method of inhibiting the formation of methane is the delivery of specific inhibitory molecules into the cells of methane producers. This can be achieved, for example, by combining inhibitory molecules with peptides entering the cell. In microbial cells, signal peptides mediate the translocation of extracellular proteins from the cell’s internal environment to the external one, and are suitable for the transfer of inhibitory molecules. Therefore, it would be useful to identify signal peptides that have the ability to penetrate the cells of methane producers and deliver inhibitors.

Signal peptides, or signal sequences, are typically included in precursor proteins secreted from prokaryotic and eukaryotic cells. Signal peptides are part of the cell-penetrating extension at the N-terminus of the precursor. The primary amino acid sequence of signal peptides is not conserved, except for the cleavage site for signal peptidase (von Heijne, 1985). In addition, signal peptides have structural similarities. Signal peptides typically include one to five positively charged N-terminal amino acid residues (n-region) followed by 10 to 15 hydrophobic amino acid residues (h-region). The glycine or proline residue is usually located within the hydrophobic domain, and the threonine and / or serine residue (s) form the polar domain (c region) near the cleavage site (Inouye and Halegoua, 1980; Vlasuk et al., 1983, von Heijne, 1985).

A loop model of signal peptide translocation has been proposed (Inouye et al., 1977; Inouye and Halagoua, 1980) by which the positively charged N-terminus of the signal peptide interacts with the negatively charged inner surface of the cell membrane. The hydrophobic domain is then immersed in a hydrophobic lipid bilayer membrane by looping. This loop ultimately includes a cleavage site that is exposed to signal peptidase to remove the signal peptide. One of the obstacles to inhibiting or limiting the formation of methane is the ability to deliver inhibitory components to the cells of methane producers. Thus, there is a need for the identification of signal peptides that are able to attach to cell membranes and transfer molecules through the lipid bilayer, in suitable carriers for cell inhibitors.

Summary of the invention

The invention relates to an isolated signal peptide or polypeptide containing this peptide, which contains at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172. In a particular aspect, the peptide or polypeptide comprises at least one amino acid sequence KKLIIILLLLILLLSI of the sequence SEQ ID NO: 117, or at least one amino acid sequence KKIIIILLLLILLLISI of the sequence SEQ ID NO: 119. In another aspect, the peptide or polypeptide comprises a fragment, for example, containing at least one amino acid sequence comprising amino acids 3-14, 3-16, or 2-16 of the sequence SEQ ID NO: 117, or at least one amino acid sequence, containing amino acids 3-15, 3-17, or 2-17 of the sequence of SEQ ID NO: 119. In an additional aspect, this peptide or polypeptide contains a fragment containing at least one conserved core sequence of the sequence SEQ ID NO: 1-172 described in this application. In yet a further aspect, the peptide or polypeptide is encoded by at least a polynucleotide fragment selected from the group consisting of SEQ ID NO: 173-341 or SEQ ID NO: 342-533.

The present invention also relates to an isolated polynucleotide containing the coding sequence of at least one signal peptide or polypeptide containing the peptide. In one aspect, the polynucleotide comprises a coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172. In a particular aspect, the polynucleotide comprises a coding sequence of at least one amino acid sequence KKLIIILLLLILLLSI of the sequence SEQ ID NO: 117, or a coding sequence of at least one amino acid sequence KKIIIILLLLILLLISI of the sequence SEQ ID NO: 119. In another aspect, the polynucleotide comprises a fragment of a coding sequence, for example, a coding sequence of at least one amino acid sequence comprising amino acids 3-14, 3-16, or 2-16 of SEQ ID NO: 117, or a coding sequence of at least one amino acid a sequence containing amino acids 3-15, 3-17, or 2-17 of the sequence of SEQ ID NO: 119. In an additional aspect, the polynucleotide comprises a fragment of a coding sequence, for example, a nucleotide sequence encoding at least one conserved core sequence described in the sequence SEQ ID NO: 1-172.

In a further aspect, the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 173-341 or SEQ ID NO: 342-533. In a particular aspect, the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 531, 532 or 533. In another aspect, the polynucleotide is a fragment or oligonucleotide, for example, comprising a nucleic acid sequence extending from nucleotides 7-42, 7-48 or 4 -48 sequences of SEQ ID NO: 531, 532 or 533. In addition, the invention encompasses an isolated polynucleotide or fragment thereof that hybridizes to any of the nucleic acid sequences of SEQ ID NO: 173-341 or SEQ ID NO: 342-533. The present invention further encompasses an isolated polynucleotide comprising a complementary, backward complementary, reverse sequence or fragments thereof, any of the nucleic acid sequences encoding a signal peptide or polypeptide containing the peptide.

The present invention relates to an expression vector containing a polynucleotide that contains the coding sequence of at least one signal peptide or polypeptide containing this peptide. In one aspect, the expression vector comprises a coding sequence of at least one amino acid sequence selected from the group consisting of sequences of SEQ ID NO: 1-172. In a particular aspect, the expression vector comprises a coding sequence of at least one amino acid sequence KKLIIILLLLILLLSI of the sequence SEQ ID NO: 117, or a coding sequence of at least one amino acid sequence KKIIIILLLLILLLISI of the sequence SEQ ID NO: 119. In another aspect, the expression vector comprises a coding sequence of at least one amino acid sequence extending from amino acids 3-14, 3-16 or 2-16 of SEQ ID NO: 117, or a coding sequence of at least one amino acid sequence containing amino acids 3 -15, 3-17, or 2-17 of the sequence of SEQ ID NO: 119. In yet another aspect, the present invention relates to a host cell, for example, a microbial host cell containing at least one expression vector.

The present invention in particular relates to an antibody directed to the peptide, polypeptide, or polynucleotide described in this application. In certain aspects, the antibody is directed to at least one signal peptide sequence selected from the group consisting of SEQ ID NO: 1-172, or a modified sequence thereof. In alternative aspects, the antibody is directed to at least a fragment of a signal peptide sequence, for example, a conserved core sequence of a sequence selected from the group consisting of SEQ ID NO: 1-172. In an additional aspect, this antibody binds to a polypeptide containing the signal peptide sequence of any of the sequences of SEQ ID NO: 1-172. In alternative aspects, the antibody is directed to at least a polynucleotide fragment selected from the group consisting of SEQ ID NO: 173-341 or SEQ ID NO: 342-533, or a complementary or modified sequence thereof. In another aspect, the antibody comprises one or more fusions or conjugates with at least one cellular inhibitor, for example, anti-methane compounds (e.g. bromoethanesulfonic acid), antibodies and antibody fragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides and other antibiotics described in detail in this application.

The present invention also relates to modified signal peptides and polypeptides containing these peptides and antibodies directed to these peptides or polypeptides, including biologically active changes, fragments, variants and derivatives described in this application. Also described are polynucleotides encoding these modified peptides or polypeptides, as well as changes, fragments, variants and derivatives of the described polynucleotides, expression vectors containing these nucleic acid sequences, and host cells containing these vectors. In particular aspects, the modified polynucleotides, polypeptides or antibodies, or the corresponding expression vectors or host cells, are used in the compositions and methods of the invention. In specific aspects, the peptides or polypeptides are obtained in the form of fusions or conjugates with at least one cellular inhibitor, for example, anti-methane compounds (e.g. bromoethanesulfonic acid), antibodies and antibody fragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides and other antibiotics described in detail in this application.

The invention further relates to a composition containing an isolated signal peptide (for example, at least one of SEQ ID NO: 1-172, or a modified sequence thereof) or a polypeptide containing this peptide, or an antibody directed to this peptide or polypeptide. Also described is a composition containing an isolated polynucleotide (for example, at least one of SEQ ID NO: 173-341 or SEQ ID NO: 342-533, or a complementary or modified sequence thereof). Additionally described is a composition that contains an expression vector, or a host cell containing an expression vector, in accordance with the present invention. The composition may contain any biologically active changes, fragments, variants and derivatives described in this application. The compositions may additionally contain at least one cellular inhibitor, and can be obtained, for example, in the form of pharmaceutical compositions or in the form of food additives, in particular, components of feed for ruminants.

In a particular aspect, the invention relates to a composition of the invention as part of a kit for determining and / or measuring, or targeted delivery, penetration and / or inhibition of microbial cells, in particular methane producer cells, in accordance with the described methods. These kits contain: a) at least one composition described herein; and b) optionally, instructions for use, for example, for targeted delivery or penetration into cells or inhibition of cell growth or replication for methane producers or other microorganisms. In particular aspects, the peptide or polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or a modified sequence thereof.

The present invention relates to a method for producing a signal peptide or polypeptide containing this peptide, the method comprises: a) culturing an expression vector or host cell containing an expression vector that contains the coding sequence of at least one signal peptide or polypeptide containing this peptide in conditions suitable for expression of this peptide or polypeptide; and b) recovering the peptide or polypeptide from the culture. Methods for preparing the described compositions are also described. In particular aspects, the peptide or polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or a modified sequence thereof.

The present invention also relates to a method for producing a signal peptide or polypeptide containing this peptide, which contains a fusion or conjugate with at least one cellular inhibitor, for example, compounds against the formation of methane (e.g. bromoethanesulfonic acid), antibodies and antibody fragments, lytic enzymes, peptide-nucleic acids, antimicrobial peptides and other antibiotics described in detail in this application. Such a method comprises: a) culturing an expression vector or host cell containing an expression vector that contains the coding sequence of at least one peptide or polypeptide under conditions suitable for expression of the peptide or polypeptide; b) the formation of a fusion or conjugate (for example, by expression of a fused sequence or chemical conjugate with a cell inhibitor); and c) recovering a fusion or conjugate. In particular aspects, the signal peptide or polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or a modified sequence thereof.

The present invention relates to a method for penetrating into a microbial cell, in particular, a methanogen producer cell, the method comprising: a) optionally, obtaining or isolating at least one signal peptide or polypeptide containing this peptide; and b) contacting the cell with a signal peptide or polypeptide. In a particular aspect, the peptide or polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or a modified sequence thereof. In further aspects, the peptide or polypeptide comprises a fusion or conjugate with at least one cellular inhibitor, for example, one or more anti-methane compounds (e.g. bromoethanesulfonic acid), antibodies and antibody fragments, lytic fragments, peptide nucleic acids, antimicrobial peptides or other antibiotics described in detail in this application.

The present invention also relates to a method for inhibiting a microbial cell (e.g., inhibiting growth or replication), in particular methane producer cells, the method comprising: a) optionally, obtaining or isolating at least a signal peptide or polypeptide containing this peptide, which further comprises at least at least one cell inhibitor; and b) contacting the cell with a signal peptide or polypeptide. In a particular aspect, the peptide or polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or a modified sequence thereof. In an additional aspect, the cellular inhibitor is selected from compounds against the formation of methane (e.g. bromoethanesulfonic acid), antibodies and antibody fragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides and other antibiotics described in detail in this application.

The present invention also relates to a method for inhibiting a microbial cell (e.g., inhibiting growth or replication), in particular a methanogen cell, comprising: a) optionally, obtaining or isolating at least one signal peptide or polypeptide containing this peptide, which further comprises at least one cellular inhibitor; and b) contacting the cell with a signal peptide or polypeptide. In a particular aspect, the peptide or polypeptide contains at least one amino acid sequence from the group consisting of SEQ ID NO: 1-172, or modified sequences thereof. In an additional aspect, the cell inhibitor is selected from compounds against the formation of methane (e.g. bromoethanesulfonic acid), antibodies and antibody fragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides and other antibiotics described in detail in this application.

The present invention also relates to a method for determining and / or measuring levels of a signal peptide or corresponding polypeptide or polynucleotide, comprising: 1) contacting a sample obtained from an individual with an antibody directed to a signal peptide (e.g., at least one of SEQ ID NO : 1-172, or their modified sequences) or the corresponding polypeptide or polynucleotide; and 2) determining the presence or levels of an antibody complex formed with a signal peptide or corresponding polypeptide or polynucleotide in a sample. Such methods can also be used to determine and / or measure the levels of microbial cells, in particular methane producer cells.

The present invention also relates to a method for determining and / or measuring levels of a polynucleotide signal sequence (e.g., a coding sequence of a signal peptide, or a corresponding coding sequence of a polypeptide), comprising: 1) contacting a sample obtained from an individual with a complementary polynucleotide (e.g., a sequence complementary any of the sequences of SEQ ID NO: 173-341, or their modified sequences); and 2) determining the presence or levels of a hybridization complex formed with the polynucleotide signal sequence in this sample. Such methods can also be used to determine and / or measure the levels of microbial cells, in particular methane producer cells.

In particular aspects, the methods of the invention utilize in vivo or in vitro expression components. In other aspects, the methods utilize peptides or polypeptides produced by recombinant, synthetic or semi-synthetic methods, or peptides or polypeptides produced by endogenous methods.

Other aspects and embodiments of the present invention are described below.

Brief Description of the Drawings

The present invention is described with reference to specific embodiments thereof and with reference to the drawings.

FIG. 1A-1C: Comparison of the genomes of methanobacteria (FIG. 1A); genome statistics of M. ruminantium (FIG. 1B); genes allegedly involved in the formation of methane in species of methanobacteria (FIG. 1C).

FIG. 2: Alignment of the signal peptide of Methanobrevibacter ruminantium . The conserved cortical region of each peptide is shown in bold.

FIG. 3A: A 102-sequence logo protein sequence created using the LogoBar. FIG. 3B: A 102 protein logo logo sequence created using the LogoBar showing the most conserved amino acid residues. FIG. 3C: Crustal consensus signal protein sequence for M. ruminantium . FIG. 3D: Amino acid sequence of M. ruminantium cell penetrating peptide with the addition of an N-terminal lysine-fluorescein.

FIG. 4: Permeability in cells of M. ruminantium fluorescein-labeled peptide.

FIG. 5: Venn diagram showing preliminary definitions of the SignalP 3.0-HMM signal peptide using three different models for the M. ruminantium signal sequence of the M1093 ORFome.

FIG. 6: M. ruminantium genes and corresponding indicators of signal peptides.

FIG. 7: M. ruminantium genes and corresponding signal peptides.

FIG. 8: Coding sequences of signal peptides of FIG. 7.

FIG. 9: Coding sequences of signal peptides of FIG. 7, with codons optimized for expression in E. coli.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Altered" nucleic acid sequences encoding signal peptides, in the context of the present invention, include sequences with deletions, insertions or substitutions of various nucleotides, resulting in a polynucleotide that encodes the same or functionally equivalent peptides. The encoded peptide may also be “modified” and contain deletions, insertions or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent peptide. Intentional amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, provided that biological activity (e.g., association with the cell or penetration into the cell) is preserved, or the immunogenic activity of this peptide. For example, negatively charged amino acids may include aspartic and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar terminal groups having similar hydrophobicity values may include leucine, isoleucine and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.

"Amino acid sequence" in the context of the present invention, refers to an oligopeptide, peptide, polypeptide or protein sequence, or fragments thereof, and to natural, recombinant, synthetic or semi-synthetic molecules. The sequence of the invention (for example, SEQ ID NO: 1-172) contain at least 5, 6, 7, 8, 9, 10, 11, 12, 15, 17, 19, or 22 amino acids, preferably at least 5 -10, 5-15, 10-15, 12-15, 15-17, 17-19 or 17-22 amino acids, and preferably retain biological activity (e.g., association with a cell or penetration into a cell) or immunological activity (e.g. at least one antibody binding site) of the original sequence. In cases where the “amino acid sequence” cited in the present description refers to the amino acid sequence of a molecule of a natural peptide or polypeptide, it is believed that the amino acid sequence and similar terms do not limit the amino acid sequence to a complete, natural amino acid sequence linked to a full-sized molecule.

"Amplification" in the context of the present invention refers to the production of additional copies of a nucleic acid sequence and is mainly carried out using polymerase chain reaction (PCR) techniques well known in the art (Dieffenbach, CW and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY).

The term “antibody” should be understood in its broadest sense and the inclusion of intact monoclonal antibodies and polyclonal antibodies is contemplated. The term also encompasses antibody fragments and derivatives, provided that they exhibit the desired biological activity. Antibodies encompass immunoglobulin molecules and immunologically active parts of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunologically interacts) with the antigen. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fc, Fab, Fab ', and Fab ₂ fragments and the Fab expression library.

Antibody molecules belong to any of the classes of IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chain located in the molecule. These molecules also include subclasses such as IgG1, IgG2, and others. The light chain may be a kappa chain or a lambda chain. The reference in the present description to antibodies includes a reference to all classes, subclasses and types. Also included are chimeric antibodies, for example, monoclonal antibodies or fragments thereof that are specific for more than one source, for example, one or more, mouse, human, or ruminant sequences. Additionally included camelid antibodies or nanobodies. It will be understood that each reference to “antibodies” or any similar term, as used herein, includes intact antibodies, as well as any fragments, measurements, derivatives or variants thereof.

The terms “biologically active” or “functional”, as used herein, refers to a peptide or polypeptide that retains one or more structural, immunogenic or biochemical functions (eg, association with a cell or penetration into a cell) of a natural sequence. As one example, a functional sequence includes at least one of the conserved conserved regions described herein.

The terms “cell inhibitor” or “inhibitor,” as used herein, refers to agents that reduce or block the growth or replication of microbial cells, especially methanogen producing cells. A cell inhibitor can act to reduce or block, for example, cell division. An inhibitor can reduce or block, for example, DNA synthesis, RNA synthesis, protein synthesis, or post-translational modifications. An inhibitor can also reduce or block the activity of enzymes involved in the methane formation cascade. An inhibitor can target a cell for recognition by components of the immune system. Cell inhibition also includes cell killing and cell death, for example, by lysis, apoptosis, necrosis, etc. Suitable inhibitors include, but are not limited to, anti-methane compounds (e.g., bromoethanesulfonic acid), antibodies and antibody fragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides and other antibiotics described in detail herein.

The terms "complementary" or "complementarity", in the context of the present invention, refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing. For the A-G-T sequence, the complementary sequence is T-C-A, the backward complementary sequence is A-C-T, and the reverse sequence is T-G-A. The complementarity between two single-stranded molecules can be partial, in which only some of the nucleic acids bind, or can be complete when there is absolute complementarity between single-stranded molecules. The degree of complementarity between nucleic acid chains has significant effects on the efficiency and strength of hybridization between nucleic acid chains. This is of particular importance in amplification reactions, which depend on the binding between nucleic acid chains and in the creation and use of RNA molecules.

The term "derivative" in the context of the present invention refers to the chemical modification of a nucleic acid encoding a signal peptide, or a nucleic acid complementary to it. Such modifications include, for example, replacing hydrogen with an alkyl, acyl, or amino group. In preferred aspects, the nucleic acid derivative encodes a peptide that retains the biological or immunological function of a naturally occurring molecule. A derived peptide is a peptide that is modified by glycosylation, pegylation, or any similar method that retains one or more biological functions (e.g., association with a cell or penetration into a cell) or an immunogenic function of a sequence that it is derived from.

The term "homology" in the context of the present invention refers to the degree of complementarity. Homology may be partial (i.e., identity less than 100%) or complete (i.e., 100% identity). A partially complementary sequence that at least partially inhibits the hybridization of an identical sequence with a target nucleic acid is called using the functional term “substantially homologous”. Inhibition of hybridization of a fully complementary sequence to a target sequence can be investigated by hybridization analysis (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or hybridization probe will compete for or inhibit the binding of a fully homologous sequence to the target sequence under conditions of low stringency. This is not to say that low stringency conditions are such that non-specific binding is allowed; low stringency conditions require that the binding of two sequences to each other be a specific (i.e., selective) interaction.

The term "hybridization" in the context of the present invention refers to any process by which a nucleic acid strand binds to a complementary strand via base pairing.

“Insertion” or “addition” in the context of the present invention refers to a change in the amino acid or nucleotide sequence, resulting in the addition of one or more amino acid residues or nucleotides, respectively, compared with a natural molecule.

"Methanoproducer" in the context of the present invention refers to microorganisms that produce methane gas, which include Methanobrevibacter , Methanothermobacter , Methanomicrobium , Methanobacterium , and Methanosarcina . Typical metanoprodutsenty include, but not limited to, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter acididurans, Methanobrevibacter thaueri, Methanobacterium bryantii, Methanobacterium formicicum, Methanothermobacter marburgensis, Methanothermobacter wolfeii, Methanosphaera stadtmanae, Methanomicrobium mobile, Methanosarcina barkeri, Methanosarcina mazei, Methanococcoides burtonii, and Methanolobus taylorii . All genders and species of methane producers are covered by this term.

"Microbial" cells in the context of the present invention relate to natural or genetically modified microbial cells, including archebacteria, such as methanoproducers, halophiles and thermoacidophils, and eubacteria, such as cyanobacteria, spirochetes, proteobacteria, as well as gram-positive and gram-negative bacteria.

The term “modified” refers to altered sequences and to fragments, variants and derivatives of the sequences described in the present invention.

A "nucleic acid sequence" or "nucleotide sequence" in the context of the present invention refers to a sequence of a polynucleotide, oligonucleotide or fragments thereof, and to DNA or RNA of natural, recombinant, synthetic or semi-synthetic origin, which may be single-stranded or double-stranded, and may be semantic or antisense strand, and coding or non-coding regions. The sequences of the present invention most preferably include sequences encoding a polypeptide (for example, SEQ ID NO: 173-341 or 342-533, or their complementary or modified sequences) that contain at least 15, 18, 21, 24, 27, 30 , 33, 36, 39, 45, 51, 57 or 66 nucleotides, preferably at least 15 to 30, 15 to 45, 30 to 45, 36 to 45, 45 to 51, 51 to 57 , or from 51 to 66 nucleotides, or at least 100 nucleotides, or at least 1000 nucleotides. It will be understood that each reference to a “nucleic acid sequence” or “nucleotide sequence” in the present invention will include a native full-length sequence (for example, SEQ ID NO: 173-341 or 342-533), as well as any of its complementary sequences, fragments, changes, derivatives or variations.

The term "oligonucleotide" refers to a nucleic acid sequence containing at least 6, 8, 10, 12, 15, 18, 21, 25, 27, 30 or 36 nucleotides, or at least 12 to 36 nucleotides, or at least at least 15 to 30 nucleotides (for example, at least a fragment of the sequences of SEQ ID NO: 173-341 or 342-533, or a complementary sequence thereof) that can be used in PCR amplification, sequencing or hybridization assays. In the context of the present invention, the oligonucleotide is essentially equivalent to the terms “amplifiers”, “primers”, “oligomers”, “oligomers” and “probes”, as commonly defined in this field.

The term "polynucleotide", used in the singular or plural, generally refers to any nucleic acid sequence, for example, any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or a modified RNA or DNA. This term includes, without limitation, single-stranded and double-stranded DNA, DNA containing single-stranded and double-stranded regions, single-stranded and double-stranded RNA and RNA, containing single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, which can be single-stranded or, more often, double-stranded , or contain single and double helical regions. Also included are RNA or DNA containing three-helix regions, or both RNA and DNA. In particular, mRNA, cDNA and genomic DNA, and any fragments thereof are included. This term includes DNA and RNA that contain one or more modified bases, such as tritium labeled bases, or unusual bases, such as inosine. Polynucleotides of the present invention can encompass coding or non-coding sequences, or sense or antisense sequences, or iRNAs, such as siRNAs. It will be understood that each reference to a “polynucleotide” or similar term in the context of the present invention will include full-length sequences, as well as any complementary sequences, fragments, measurements, derivatives or variants thereof.

"Peptide-nucleic acid" or "PNA" in the context of the present invention refers to an antisense molecule, or an antigenic agent that contains bases linked by a peptide skeleton.

The term "ruminant" in the context of the present invention refers to animals that have a scar as a special type of digestive organ. Ruminants include, but are not limited to, cattle, sheep, goats, bulls, moose, caribou, and deer.

“Signal peptides” in the context of the present invention refers to the isolated peptides of the invention obtained from any species, preferably microorganisms, from any source, whether natural, synthetic, semisynthetic or recombinant. In particular, the signal peptide can be obtained from methanogen producing cells, such as Methanobrevibacter cells, in particular M. ruminantium , or M. smithii cells. For recombinant production, the signal peptide of the invention can be obtained from microbial or eukaryotic cells, for example, Escherichia , Streptomyces , Bacillus , Salmonella , yeast, insect cells such as Drosophila , animal cells such as COS and CHO cells, or plant cells. It will be understood that each reference to a “peptide” in the context of the present invention will include a full-length sequence (for example, SEQ ID NO: 1-172), as well as any changes, fragments, derivatives or variants thereof.

The terms “stringency conditions” or “stringency” in the context of the present invention refer to hybridization conditions defined by nucleic acid, salt and temperature. These conditions are well known in the art and can be modified in order to identify or identify identical or related polynucleotide sequences. See, for example, Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. Numerous equivalent conditions involving either low or high stringency depend on factors such as length and nature of the sequence (DNA, RNA, nucleotide composition), nature of the target (DNA, RNA, nucleotide composition), environmental conditions (in solution or immobilized on solid substrate), the concentration of salts or other components (e.g. formamide, dextran sulfate and / or polyethylene glycol), and the reaction temperature (within a range of about 5 ° C below the melting point of the probe to about 20 ° C-25 ° C below temperature ur melting). One or more factors can be modified to create conditions of either low or high rigidity, different from the conditions listed above, but equivalent to them.

The term "individual" includes humans and animals that are not related to humans. Non-human animals include, but are not limited to, birds and mammals such as ruminants, and in particular mice, rabbits, cats, dogs, pigs, sheep, goats, cows and horses.

The terms "substantially purified" or "isolated" in the context of the present invention refers to nucleic or amino acid sequences that are isolated from their cellular, recombinant or synthetic medium, and are at least 60% free, preferably 75% free, and most preferably at least 90% free or at least 99% free of other components with which they are associated in a cellular, recombinant or synthetic medium.

"Transformation" in the context of the present invention describes a process by which exogenous DNA enters and modifies a recipient cell. This can occur under natural or artificial conditions using various methods well known in the art. The transformation may be based on any known method of inserting sequences of foreign nucleic acids into a prokaryotic or eukaryotic host cell. The method is selected based on the type of transformable host cell, and it may include, but not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of a host chromosome. They also include cells that transiently express inserted DNA or RNA for limited periods of time.

A “variant” of a peptide or polypeptide in the context of the present invention refers to an amino acid sequence that is altered by one or more amino acids. A polynucleotide variant is altered by one or more nucleotides. A variant may result in “conservative” changes in which the substituted amino acid has similar structural or chemical properties, for example, replacing leucine with isoleucine. Less commonly, an option may result in “non-conservative” changes, such as replacing glycine with tryptophan. Similar minor changes may also include amino acid deletions or insertions, or both. Guidance on determining which amino acid residues can be replaced, inserted or removed without loss of biological or immunogenic activity can be found using computer programs well known in the art, for example, LASERGENE (DNASTAR) software.

The present invention also encompasses variants that retain at least one biological activity (eg, association with a cell or penetration into a cell) or functional activity of a peptide or polypeptide. A preferred embodiment is one whose sequence is at least 80%, and more preferably at least 90%, identical to the described sequence. The most preferred option is one whose sequence is at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99 , 8%, or at least 99.9% identical to the sequence described in the present invention. The percentage of identity is determined by aligning the two compared sequences, as described below, determining the number of identical residues in the aligned part, dividing this number by the total number of residues in the sequence of the invention (desired), and multiplying this result by 100. A suitable alignment program is AlignX (Vector NTI).

Description of the invention

Methane is formed in the front of the digestive tract of ruminants by methane producers, which act as the final carbon reducers in the rumen system. The multi-stage pathway for methane production is well illuminated, mainly from the study of methane producers of non-ruminant animals, but the adaptations that allow methane producers to grow and persist in the rumen are not entirely understood. Methanobrevibacter ruminantium is a well-known methanogen producer in New Zealand ruminants. As described in this application, the size of the preliminary genomic sequence of M. ruminantium is approximately 3.0 Mb, and the content of GC is 33.68%. As important information, it was found that the M. ruminantium genome contains signal peptide sequences for use in targeted delivery and penetration into cells. The present invention therefore relates to signal peptides, including those that contain SEQ ID NO: 1-172, as well as polypeptides containing these peptides, and their changes, fragments, variants and derivatives.

The present invention relates to the use of these peptides or polypeptides for targeted delivery and penetration into microbial cells, in particular methane producer cells. The present invention further relates to the use of peptides or polypeptides for inhibiting the growth or replication of such cells. The peptides and polypeptides of the present invention can be expressed and used in various assays to determine their biological activity. These peptides and polypeptides can be used for large-scale synthesis and isolation protocols, for example, for commercial production. Such peptides and polypeptides can be used to induce antibodies, to isolate appropriate antibody sequences, and to quantify levels of amino acid sequences.

The polypeptides of the present invention can also be used as compositions, for example, pharmaceutical compositions, and as feed additives, for example, feed components for ruminants. The peptides and polypeptides of the present invention also benefit health. For example, in health-related aspects, methane-producing inhibitors can be used to restore energy to an individual, which is usually lost as methane. In specific aspects, rumen slow-release devices can be used in conjunction with the peptides, polypeptides and compositions (e.g., pharmaceutical compositions and feed additives) of the present invention.

The peptides and polypeptides of the present invention contain at least one sequence selected from the group consisting of: (a) peptides or polypeptides containing at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or its changes, fragments, variants or derivatives; (b) peptides or polypeptides containing a functional domain (for example, a conservative core region described herein) of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or changes, fragments, variants thereof or derivatives; and (c) peptides or polypeptides containing at least a specific amount of contiguous residues of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1-172, or changes, fragments, variants or derivatives thereof. All of these sequences together in the present description are called peptides and polypeptides of the present invention. In one embodiment, the invention relates to an isolated peptide or polypeptide containing the amino acid sequence of at least one of SEQ ID NO: 1-172.

The present invention also relates to polynucleotides that encode at least one signal peptide, including SEQ ID NO: 1-172, as well as to polypeptides containing such peptides, and their changes, fragments, variants or derivatives.

The present invention encompasses the use of these polynucleotides for the production of expression vectors and host cells for targeted delivery and penetration into microbial cells, especially methane producing cells. The present invention further encompasses the use of these polynucleotides to inhibit the growth or replication of such cells. The isolated polynucleotides of the present invention are also useful in genome mapping, physical mapping, and in cloning genes of more or less related bacteria. The probes obtained using the polynucleotides of the present invention can be used to detect the presence and study of gene expression profiles in any organism that has sufficiently homologous DNA and RNA sequences in their cells using techniques that are well known in the art, such as slot microchip blotting or analysis. Primers prepared using the polynucleotides of the present invention can be used for sequencing and PCR amplifications.

The polynucleotides of the present invention can also be used as compositions, for example, pharmaceutical compositions, and as feed additives, for example, feed components for ruminants. Polynucleotides of the present invention also benefit health. For such use, polynucleotides can be presented as expression vectors or host cells containing expression vectors. In specific aspects, rumen slow release devices may be used in conjunction with polynucleotides, vectors, host cells, and compositions (e.g., pharmaceutical compositions and feed additives) of the present invention.

The polynucleotides of the present invention contain at least one sequence selected from the group consisting of: (a) sequences containing the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or changes thereof , fragments, variants or derivatives; (b) complementary sequences, reverse sequences, and backward complementary to coding sequences of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or changes, fragments, variants or derivatives thereof; (c) open reading frames contained in the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or changes, fragments, variants or derivatives thereof; (d) functional domains (e.g., conserved conserved regions described herein) of a coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1-172, or changes, fragments, variants or derivatives thereof; and (e) sequences containing at least a specific amount of contiguous residues of a coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1-172, or changes, fragments, variants or derivatives thereof. Oligonucleotide probes and primers are also provided. All of these polynucleotide and oligonucleotide probes and primers together in the present description are called polynucleotides of the present invention. In one embodiment, the present invention encompasses an isolated polynucleotide comprising a coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172.

It will be understood by those skilled in the art that as a result of the degeneracy of the genetic code, a plurality of nucleotide sequences encoding the peptides of the present invention can be obtained, to some extent carrying minimal homology to the nucleotide sequences of any known and natural gene. Therefore, the present invention considers all, without exception, possible variants of the nucleotide sequence that could be obtained by selecting combinations based on possible choices of codons. These combinations are obtained in accordance with the standard triplet genetic code, in relation to natural amino acid sequences, and all such variants are considered specifically described.

The nucleotide sequences that encode signal peptides or polypeptides, or their modified sequences, are preferably able to hybridize to the nucleotide sequence of a natural sequence under suitably selected stringency conditions. However, it may be preferable to obtain nucleotide sequences encoding a peptide or its derivatives having a substantially different codon usage frequency. Codons can be selected to increase the rate at which expression of this peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which specific codons are used by that host. For example, codons can be optimized for expression in E. coli , for example, in accordance with SEQ ID NO: 342-533. Other reasons for substantially altering the nucleotide sequence encoding the peptides and its derivatives without altering the encoded amino acid sequences include obtaining RNA transcripts having more desirable properties, such as a longer half-life, than transcripts derived from a natural sequence.

The present invention also encompasses the production of DNA sequences, or fragments thereof, that encode peptides or polypeptides, or their modified sequences, exclusively by synthetic chemistry. Once obtained, the synthetic sequence can be integrated into any of the many available expression vectors and cell systems using reagents that are well known in the art. Moreover, synthetic chemistry can be used to introduce mutations into a sequence encoding a peptide or polypeptide, or any changes, variants, derivatives or fragments thereof. The invention also encompasses polynucleotide sequences that are capable of hybridizing with the claimed nucleotide sequences, and, in particular, shown in SEQ ID NO: 173-341 or 342-533, or their complements, under different stringency conditions, as described by Wahl, GM and SL Berger (1987; Methods Enzymol. 152: 399-407) and Kimmel, AR (1987; Methods Enzymol. 152: 507-511).

DNA sequencing methods that are well known and generally available in the art, and can be used to practice any of the embodiments of the present invention. Enzymes such as Klenow DNA polymerase I fragment, SEQUENASE (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase Amersham Pharmacia Biotech (Piscataway, NJ), or a combination of polymerases and correcting exonucleases can be used in the methods. such as those on the ELONGASE Amplification System sold by Life Technologies (Gaithersburg, MD). Preferably, this process is automated by devices such as Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer), or Genome Sequencer 20 ^TM (Roche Diagnostics).

Nucleic acid sequences encoding these peptides can be extended using an incomplete nucleotide sequence and using various methods known in the art to identify upstream sequences, such as promoters and regulatory elements. For example, in one method that can be used, restriction site PCR, universal primers are used to search for an unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2: 318-322). In particular, genomic DNA was first amplified in the presence of a primer to a linker sequence and a primer specific for a known site. The amplified sequences were then subjected to a second round of PCR with the same linker primer and another specific primer located inside the first. The products of each round of PCR are transcribed with a suitable RNA polymerase followed by reverse transcriptase.

Capillary electrophoresis systems that are commercially available can be used to analyze the size or confirmation of the nucleotide sequence of sequencing or PCR products. In particular, in capillary sequencing, flowing polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are activated by a laser, and the determination of the emitted wavelengths using a charge-coupled camera can be used. The output / light intensity can be converted into an electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer) and the whole process from loading samples to computer analysis and electronic data display can be controlled by a computer. Capillary electrophoresis is particularly preferred for sequencing small parts of DNA, which may be in limited quantities in a particular sample.

In another embodiment of the present invention, polynucleotides or fragments thereof that encode peptides or polypeptides can be used in recombinant DNA molecules to direct the expression of peptides, polypeptides or their modified sequences in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences can be obtained that encode essentially the same or functionally equivalent amino acid sequence, and these sequences can be used for cloning and expression of signal peptides or polypeptides. The nucleotide sequences of the present invention can be constructed using methods generally known in the art for changing amino acid coding sequences for a variety of reasons, including but not limited to modifications that modify the cloning, processing and / or expression of gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to construct nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, change glycosylation profiles, change codon preferences, introduce mutations, and so on.

In another embodiment of the present invention, natural, modified, or recombinant nucleic acid sequences encoding peptides or polypeptides may be ligated to a heterologous sequence to encode a fusion protein. For example, it may be appropriate to encode a chimeric sequence that can be recognized by a commercially available antibody. A fusion protein can also be designed to contain a cleavage site between the peptide or polypeptide of the invention and the heterologous protein sequence, so that the peptide or polypeptide can be cleaved and purified from the heterologous portion.

In another embodiment, the sequences encoding the peptides or polypeptides can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, MH et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the peptide or polypeptide itself can be prepared using chemical methods to synthesize an amino acid sequence or fragment thereof. For example, peptide synthesis can be carried out using various solid phase methods (Roberge, JY et al. (1995) Science 269: 202-204; Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154) and automated synthesis can be achieved, for example, using an ABI 431A Peptide Synthesizer (Perkin Elmer). Various fragments of peptides or polypeptides can be chemically synthesized separately and combined using chemical methods to obtain a full-sized molecule.

The newly synthesized peptide or polypeptide can be isolated using preparative high performance liquid chromatography (e.g. Creighton, T. (1983) Proteins Structures and Molecular Principles, WH Freeman and Co., New York, NY). The composition of synthetic peptides or polypeptides can be confirmed by amino acid analysis or sequencing (for example, using the Edman cleavage technique; Creighton, supra). Additionally, the amino acid sequence of the peptide or polypeptide, or any part thereof, can be changed during direct synthesis and / or combined using chemical methods with sequences from other proteins, or any parts thereof, to obtain a modified molecule.

For the expression of biologically active peptides, nucleotide sequences encoding a peptide or functional equivalents can be inserted into the corresponding expression vector, i.e. a vector that contains the necessary elements for transcription and translation of the built-in coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding this peptide and the corresponding transcriptional and translational control elements. These methods include in vitro recombinant DNA technologies, synthetic methods, and in vivo genetic recombination. Such technologies are described by Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, and Ausubel, FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY.

A variety of expression vectors / host systems can be used to contain and express sequences encoding the peptides of the invention. They include, but are not limited to microorganisms, such as bacteria transformed with a recombinant bacteriophage, a plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; cellular systems of insects infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g. Ti or pBR322 plasmids); or animal cell systems. For bacteria, suitable plasmids include pET, pRSET, pTrcHis2, and pBAD plasmids from Invitrogen, pET and pCDF plasmids from Novagen, and Director ^TM plasmids from Sigma-Aldrich. For methane producers, suitable plasmids include, but are not limited to, pME2001, pMV15, and pMP1. In particular, Escherichia coli can be used with the pET expression vector. The present invention is not limited to the expression vector used or the host cell.

“Control elements” or “regulatory sequences” are untranslated regions of enhancer vectors, 5 ′ and 3 ′ promoters of untranslated regions that interact with host cell proteins to transcribe and translate. Such elements may vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcriptional and translational elements, including constitutive and inducible promoters, can be used. For example, when cloning into bacterial systems, inducible promoters, such as the hybrid lacZ promoter BLUESCRIPT phagemids (Stratagene, LaJolla, CA) or pSPORT1 plasmids (Life Technologies) and the like, can be used. The polyhedrin promoter of baculovirus can be used in insect cells. Promoters or enhancers derived from plant cell genomes (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into this vector.

In bacterial systems, the number of expression vectors can be selected depending on the use of this peptide. For example, in cases where large amounts of peptide are needed, vectors that direct a high level of expression of fusion proteins that are easy to clean can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which the peptide coding sequence can be ligated into a vector in frame with sequences for the amino terminal Met and subsequent 7 β-galactosidase residues so that a fusion protein is formed; pIN vectors (Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509); and the like.

pGEX vectors (Promega, Madison, WI) can also be used to express foreign peptides as fusion proteins with glutathione S-transferase (GST). Basically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption on glutathione-agarose beads, followed by elution in the presence of free glutathione. Proteins obtained in such systems can be created containing heparin, thrombin, or factor Xa protease cleavage sites so that the cloned peptide of interest can be freed from the GST portion as desired. In yeast, Saccharomyces cerevisiae , a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For a review, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153: 516-544.

Specific initiation signals can also be used to achieve more efficient translation of sequences encoding the peptides of the invention. Such signals include the ATG start codon and adjacent sequences. In cases where the sequences encoding the peptide, their start codon and overlying sequences are inserted into the corresponding expression vector, additional transcriptional or translational control signals may not be necessary. However, in cases where only the coding sequence, or a fragment thereof, is inserted, exogenous translational control signals, including the ATG start codon, must be provided. Moreover, the start codon must be in the correct reading frame to ensure translation of the entire insert. Exogenous translation elements and starting codons can be of various origins, both natural and synthetic. Expression efficiency can be increased by including enhancers that are appropriate for the particular cell system used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).

In addition, the strain of the host cell can be selected by its ability to modulate the expression of the inserted sequences or process the expressed peptide or polypeptide in the desired manner. Such sequence modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves the “prepro” form of the peptide or polypeptide, can also be used to facilitate proper incorporation, formation of a folded structure and / or function. Various host cells that have a specific cellular mechanism and characteristic mechanisms of post-translational activities are available from the American Type Culture Collection (ATCC; Bethesda, MD) and can be selected to ensure the correct modification and processing of the sequence. Specific host cells include, but are not limited to, methanogen producing cells, such as Methanobrevibacter cells, in particular M. ruminantium , or M. smithii cells. Host cells of interest include, for example, Rhodotorula , Aureobasidium , Saccharomyces , Sporobolomyces , Pseudomonas , Erwinia and Flavobacterium ; or other organisms such as Escherichia , Lactobacillus , Bacillus , Streptomyces , and the like. Particular host cells include Escherichia coli , which is particularly suitable for use in the present invention, Saccharomyces cerevisiae , Bacillus thuringiensis , Bacillus subtilis , Streptomyces lividans , and the like.

There are several techniques for introducing nucleic acids into eukaryotic cells cultured in vitro . These techniques include chemical methods (Felgner et al., Proc. Natl. Acad. Sci., USA, 84: 7413 7417 (1987); Bothwell et al., Methods for Cloning and Analysis of Eukaryotic Genes, Eds., Jones and Bartlett Publishers Inc., Boston, Mass. (1990), Ausubel et al., Short Protocols in Molecular Biology, John Wiley and Sons, New York, NY (1992); and Farhood, Annal. NY Acad. Sci., 716: 23 34 (1994)), the use of protoplasts (Bothwell, supra) or electrical impulses (Vatteroni et al., Mutn. Res., 291: 163 169 (1993); Sabelnikov, Prog. Biophys. Mol. Biol., 62: 119 152 (1994); Bothwell et al., Supra; and Ausubel et al., Supra), the use of attenuated viruses (Davis et al., J. Virol. 1996, 70 (6), 3781 3787; Brinster et al. J. Gen Virol. 2002, 83 (Pt 2), 369 381; Moss, Dev. Biol. Stan., 82:55 63 (1994); and Bothwell et al., Supra), as well as physical methods (F ynan et al., supra; Johnston et al., Meth. Cell Biol., 43 (Pt A): 353 365 (1994); Bothwell et al., supra; and Ausubel et al., supra).

Successful delivery of nucleic acids to animal tissue can be achieved using cationic liposomes (Watanabe et al., Mol. Reprod. Dev., 38: 268 274 (1994)), by direct injection of naked DNA or RNA into the muscle tissue of the animal (Robinson et al., Vacc., 11: 957 960 (1993); Hoffman et al., Vacc. 12: 1529 1533; (1994); Xiang et al., Virol., 199: 132 140 (1994); Webster et al. , Vacc., 12: 1495 1498 (1994); Davis et al., Vacc., 12: 1503 1509 (1994); Davis et al., Hum. Molec. Gen., 2: 1847 1851 (1993); Dalemans et al. Ann NY Acad. Sci. 1995, 772, 255 256. Conry, et al. Cancer Res. 1995, 55 (7), 1397-1400), and embryos (Naito et al., Mol. Reprod. Dev., 39: 153 161 (1994); and Burdon et al., Mol. Reprod. Dev., 33: 436 442 (1992)), by intramuscular injection of self-replicating RNA vaccines (Davis et al., J Virol 199 6, 70 (6), 3781 3787; Balasuriya et al. Vaccine 2002, 20 (11 12), 1609 1617) or by intracutaneous injection of DNA using the gene gun technology (Johnston et al., Supra).

A number of protocols for determining and measuring the expression of the peptides or polypeptides of the invention using either polyclonal or monoclonal antibodies specific for this protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). A two-site monoclonal antibody immunoassay can be used with monoclonal antibodies reactive with two non-interfering epitopes of a peptide or polypeptide, but a competitive binding assay can also be used. These and other assays are described, inter alia, by Hampton, R. et al. (1990; Serological Methods, a laboratory Manual, APS Press, St Paul, MN) and Maddox, D. E. et al. (1983; J. Exp. Med. 158: 1211-1216).

A number of labels and conjugation methods are known to those skilled in the art and can be used in various nucleic acid and amino acid analyzes. Methods for producing labeled hybridization or PCR probes for detecting sequences associated with polynucleotides include oligolabeling, nick translation, end tagging, or PCR amplification using labeled nucleotide. Alternatively, sequences encoding this peptide or any polypeptides containing this peptide, or any modified sequences thereof, can be cloned into a vector to obtain a probe mRNA. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by adding the appropriate RNA polymerase, such as T7, T3 or SP6 and labeled nucleotides. These techniques can be carried out using a variety of commercially available kits from Amersham Pharmacia Biotech, Promega, and US Biochemical. Suitable reporter molecules or labels that can be used to facilitate detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents such as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression vectors or host cells transformed with expression vectors can be cultured under conditions suitable for expression and extraction of the peptide or polypeptide from the culture. This culture may contain components for in vitro or in vivo expression. Components of in vitro expression include components for rabbit reticulocyte lysates, E. coli lysates, and wheat germ extracts, for example, Invitrogen Expressway ™ or RiPs, Genelator ^™ iNtRON Biotechnology, EcoPro ™ or Novagen STP3 ™, TNT® Quick Coupled by Promega, and EasyXpress by QIAGEN. Peptides or polypeptides derived from culture can be secreted or contained intracellularly depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors that encode these peptides or polypeptides are preferably designed to contain signal sequences that directly secrete this peptide through a prokaryotic or eukaryotic cell membrane.

Other constructs may include an amino acid domain that will facilitate the purification of the peptide or polypeptide. Such domains include, but are not limited to, metal chelating peptides, such as histidine-triprofan modules (e.g. 6X-HIS (SEQ ID NO: 514)), which allow purification on immobilized metals, protein A domains, which allow purification on immobilized immunoglobulin; and the domain used in the FLAG® extension / affinity purification system (Immunex Corp., Seattle, WA). Suitable epitope labels include 3XFLAG®, HA, VSV-G, V5, HSV, GST, GFP, MBP, GAL4, and β-galactosidase. Suitable plasmids include plasmids containing a biotin tag (e.g., PinPoint ™ plasmids from Promega), calmodulin binding protein (e.g. pCAL plasmids from Stratagene), streptavidin binding protein (e.g. InterPlay ™ plasmids from Stratagene), c-myc or FLAG® tag (e.g., Sigma-Aldrich immunoprecipitation plasmids), or a histidine tag (e.g., QIAExpress plasmid from QIAGEN).

To facilitate purification, a cleavable linker sequence, for example, specific for factor Xa or enterokinase (Invitrogen, San Diego, CA), can be used. For example, this vector may include one or more linkers between the purification domain and the peptide or polypeptide. In one aspect, the expression vector can provide expression of a fusion protein containing a peptide or polypeptide of the present invention and a nucleic acid encoding 6 histidine residues (SEQ ID NO: 514) upstream of a thioredoxin or enterokinase cleavage site. Histidine residues facilitate IMAC purification (affinity chromatography with immobilized metal ion described by Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281), while the enterokinase cleavage site provides a means for purifying the peptide or polypeptide from fusion protein. A review of vectors that contain fusion proteins is presented by Kroll, D. J. et al. (1993; DNA Cell Biol. 12: 441-453).

Antibodies of the present invention can be obtained using methods generally known in the art, for example, for use in purification or diagnostic methods. In particular, purified peptides, polypeptides or polynucleotides can be used to produce antibodies in accordance with well-known protocols. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric and single chain antibodies, Fab fragments and fragments obtained using the Fab expression library. Neutralizing antibodies (i.e., antibodies that inhibit function) are particularly preferred for use in the present invention.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans, and others, can be immunized by the administration of a peptide, polypeptide, polynucleotide, or any fragment thereof that has immunogenic properties. Depending on the type of host, various adjuvants may be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronyl polyols, polyanions, peptides, oil emulsions, cochlear lymphocytes hemocyanin and dinitrophenol. Among the adjuvants used in humans, BCG (Calmett-Guerin bacilli) and Corynebacterium parvum are particularly preferred.

Preferably, the peptides, polypeptides or fragments used for the induction of antibodies have an amino acid sequence containing at least five amino acids, and more preferably at least 10 amino acids. It is also preferred that they are identical to part of the amino acid sequence of a natural protein, and that they can contain the entire amino acid sequence of a small natural molecule. Short stretches of amino acids can be fused with those of another protein, for example, snail lymph hemocyanin, and an antibody obtained against this chimeric molecule.

Monoclonal antibodies can be obtained. using any technique that produces antibody molecules using a stable cell line in culture. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV hybridoma technology (Kohler, G. et al. (1975) Nature 256: 495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81: 31-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120). Antibodies can also be obtained by inducing in vivo production in a lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents, as described in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833 -3837; Winter, G. et al. (1991) Nature 349: 293-299).

In addition, techniques can be used to purify “chimeric antibodies,” for example, combining antibody genes to produce molecules with appropriate antigenic specificity and biological activity (Morrison, SL et al. (1984) Proc. Natl. Acad. Sci. 81: 6851 -6855; Neuberger, MS et al. (1984) Nature 312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454). Alternatively, the procedures described for preparing single chain antibodies can be adapted using methods known in the art to produce specific single chain antibodies. Antibodies with related specificity, but of a different idiotype composition, can be obtained by chain shuffling from random combinatorial libraries of immunoglobulins (Burton D. R. (1991) Proc. Natl. Acad. Sci. 88: 11120-3).

Specialists in this field to which the present invention relates, the terms "diatel" and "triatel" will be understood. They are molecules that contain a heavy chain variable domain (VH) coupled to a light chain variable domain (VL) by a short peptide linker that is too short to allow pairing between two domains on the same chain. This promotes pairing with the complementary domains of one or more other chains, and stimulates the formation of dimeric or trimeric molecules with two or more functional antigen-binding sites. The resulting antibody molecules can be monospecific or multispecific (for example, bispecific in the case of diabodies). Such antibody molecules can be obtained from two or more antibodies, using a methodology standard in the field to which the present invention relates; for example, as described by Todorovska et al. (Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods. 2001 Feb 1; 248 (1-2): 47-66).

Antibody fragments that contain specific binding sites can also be obtained. For example, such fragments include, but are not limited to, F (ab ') ₂ fragments that can be obtained by pepsin cleavage of the antibody molecule, and Fab fragments that can be obtained by restoring the disulfide bridges of F (ab') ₂ fragments. Alternatively, Fab expression libraries can be designed to be able to quickly and easily identify monoclonal Fab fragments with the desired specificity (Huse, WD et al. (1989) Science 254: 1275-1281).

Various immunoassays can be used for screening to identify antibodies with binding specificity. Numerous protocols for competitive binding or quantitative radioimmunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically include measuring the formation of a complex between a peptide, polypeptide or polynucleotide and an antibody specific for it. Immunological analysis based on monoclonal antibodies with two sites using monoclonal antibodies reactive against two non-interfering epitopes is preferred, but a competitive binding assay (Maddox, supra) can also be used.

The signal peptides described in the present invention have the ability to enter cells and, therefore, are suitable as carrier molecules for the delivery of inhibitory molecules to microbial cells. The chemical reactions for combining amino acids and compounds are well developed and a number of different types of molecules can be coupled to a signal peptide. The most common connection methods are based on the presence of free amino (alpha-amino or Lys), sulfhydryl (Cys), or carboxyl groups (Asp, Glu, or alpha-carboxyl). Connection methods can be used to connect the peptide to a cellular inhibitor via a carboxy or amino terminal residue. In some cases, the sequence includes numerous residues that may interact with the selected chemistry. This can be used to obtain multimers containing more than one cellular inhibitor. Alternatively, the peptide or polypeptide may be shortened or selected so that the reactive residues are located either at the amino or carboxyl end of the sequence.

For example, a reporter molecule, such as fluorescein, can be specifically included in the lysine residue (Ono et al., 1997) using N - α -Fmoc- Nε -1- (4,4-dimethyl-2,6 dioxocyclohex-1- ylidene-3-methylbutyl) -L-lysine during peptide synthesis. After synthesis, 5- and 6-carboxyfluoresceinsuccinimidyl esters can be combined, after which 4,4-dimethyl-2,6 dioxocyclohex-1-ylidene is removed by treatment with hydrazine. Therefore, the coupling of an inhibitory molecule with a signal peptide or polypeptide can be accomplished by incorporating a lysine residue in the penetrating sequence, then by reaction with a suitable derivative cell inhibitor.

An EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) or carbodiimide compound method can also be used. Carbodiimides can activate the carboxyl groups of the side chain of aspartic and glutamic acid, as well as the carboxy-terminal group, to make them reactive sites for connection with primary amines. The activated peptides are mixed with a cellular inhibitor to obtain the final conjugate. If the cell inhibitor is activated first, by EDC, the cell inhibitor will be linked via the N-terminal alpha amine and, possibly, through the amine in the Lys side chain, if present in the sequence.

M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is a heterobifunctional reagent that can be used to couple peptides to cellular inhibitors via cysteines. The connection occurs with the thiol group of cysteine residues. If the selected sequence does not contain Cys, it is common to place the Cys residue at the N- or C-terminus to obtain a highly controlled compound of the peptide with a cellular inhibitor. For synthesis purposes, for cysteine it may be appropriate to position the peptide at the N-terminus. MBS is particularly suitable for use in the present invention.

Glutaraldehyde can be used as a bifunctional binding reagent that connects two compounds through their amino groups. Glutaraldehyde provides a highly flexible spacer between the peptide and the cell inhibitor for a suitable presentation. Glutaraldehyde is a very reactive compound and will interact with Cys, Tyr, and His to a limited extent. The glutaraldehyde crosslinking method is particularly suitable when the peptide contains only one free amino group at its amino terminus. In cases where the peptide contains more than one free amino group, large multimeric complexes can be formed.

In one aspect, the peptides or polypeptides of the present invention can be fused (e.g., by cloning within the frame) or crosslinked (e.g., by chemical coupling) with cellular inhibitors, such as antimicrobial agents. Among these agents are antimicrobial peptides, for example, a bactericidal / penetration enhancing protein, cationic antimicrobial proteins, lysozymes, lactoferrins and cathelicidins (for example, from neutrophils; see, for example, Hancock and Chapple, 1999, Antimicrob. Agents Chemother. 43: 1317-- 1323; Ganz and Lehrer, 1997, Curr. Opin. Hematol. 4: 53-58; Hancock et al., 1995, Adv. Microb. Physiol. 37: 135-175). Antimicrobial peptides further include defensins (for example, from epithelial cells or neutrophils) and platelet microbicidal proteins (see, for example, Hancock and Chapple, 1999, Antimicrob. Agents Chemother. 43: 1317-1323). Additional antimicrobial peptides include, but are not limited to, gramicidin S, bacitracin, polymyxin B, tachyplezin, bactenecin (e.g., cattle bactenecin), ranalexin, cecropin A, indolicidin (e.g., cattle indolicidin), and nisin (e.g., bacterial lowlands).

Ionophores are also included as antimicrobial agents that facilitate the transfer of an ion (e.g., sodium) through a lipid barrier such as a cell membrane. Two ionophore compounds particularly suitable for the present invention are RUMENSIN ^™ (Eli Lilly) and Lasalocid (Hoffman LaRoche). Other ionophores include, but are not limited to, salinomycin, avoparcin, aridcin, and actaplanin. Other antimicrobials include Monensin ™ and azithromycin, metronidazole, streptomycin, kanamycin and penicillin, as well as, in general, ß-lactams, aminoglycosides, macrolides, chloramphenicol, novobiocin, rifampin and fluoroquinolones (see, for example, 2003 Horn et al. , Applied Environ. Microbiol. 69: 74-83; Eckburg et al., 2003, Infection Immunity 71: 591-596; Gijzen et al., 1991, Applied Environ. Microbiol. 57: 1630-1634; Bonelo et al., 1984, FEMS Microbiol. Lett. 21: 341-345; Huser et al., 1982, Arch. Microbiol. 132: 1-9; Hilpert et al., 1981, Zentbl. Bakteriol. Mikrobiol. Hyg. 1 Abt Orig . C 2: 21-31).

Particularly effective inhibitors are compounds that block or inhibit the formation of methane, including bromoethanesulfonic acid, for example 2-bromoethanesulfonic acid (BES), or their salts, for example, sodium salt. Sodium molybdate (Mo) is an inhibitor of sulfate reduction, and can be used with bromoethanesulfonic acid. Other anti-methane compounds include, but are not limited to, nitrate, formate, methyl fluoride, chloroform, chloral hydrate, sodium sulfate, ethylene and unsaturated hydrocarbons, acetylene, fatty acids such as linoleic and cis-oleic acid, saturated fatty acids such as behenic and stearic acid, as well as lumazine (for example, 2,4-pteridinedione). Additional compounds include 3-bromopropanesulfonate (BPS), propionic acid, and ethyl 2-butinoate.

Additionally included as antimicrobial agents are lytic enzymes, including lysozyme, endolysin, lysozyme, lysine, phage lysine, muralizin, muramidase, and virolisin. Suitable enzymes demonstrate the ability to hydrolyze specific bonds in the bacterial cell wall. Specific lytic enzymes include, but are not limited to, glucosaminidases, which hydrolyze glycosidic bonds between amino sugars (e.g., N-acetylmuramic acid and N-acetylglucosamine) of peptidoglycan, amidases that cleave the N-acetylmuramoyl-L-alaninamide cross-linking glycane chain a peptide, and endopeptidases that hydrolyze an interpeptide bond (e.g., cysteine endopeptidases) and endo isopeptidases that act on the pseudo-murein of methanogen producers of the Methanobacteriacaea family.

Additionally, PNA are included as antimicrobial agents. PNAs are hybrid peptide nucleic acids in which the phosphate backbone has been replaced by an achiral and neutral backbone formed from N- (2-aminoethyl) glycine units (see, for example, Eurekah Bioscience Collection. PNA and Oligonucleotide Inhibitors of Human Telomerase. G Gavory and S. Balasubramanian, Landes Bioscience, 2003). Bases A, G, T, C are attached to the amino nitrogen of the skeleton via methylene carbonyl bonds (P.E. Nielsen et al., Science 1991. 254: 1497-1500; M. Egholm et al., Nature 1993. 365: 566-568). PNAs bind complementary sequences with high specificity and higher affinity for similar DNA or RNA (M. Egholm et al., Supra). PNA / DNA or PNA / RNA hybrids also exhibit higher thermal stability than the corresponding DNA / DNA or DNA / RNA duplexes (M. Egholm et al., Supra). PNA also have high chemical and biological stability due to the unnatural amide skeleton, which is not recognized by nucleases or proteases (V. Demidov et al., Biochem Pharmacol 1994. 48: 1310-1313). Typically, PNAs are at least 5 bases in length, and include terminal lysine. PNAs can be pegylated to further increase their lifespan (Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8: 53-63).

In one specific aspect, the peptides or polypeptides of the invention can be fused (e.g., by cloning within the frame) or crosslinked (e.g., by chemical coupling) with cellular inhibitors, such as antibodies or fragments thereof. Antibodies or antibody fragments can be directed to microbial cells, or specifically methanogen producing cells, or one or more cell components. For example, cell surface proteins, such as receptors, can be targeted. Included are immunoglobulin molecules and immunologically active parts of immunoglobulin (Ig) molecules, i.e. molecules that contain an antigen-binding site that specifically binds (immunologically interacts) with the antigen.

The peptides or polypeptides of the invention find practical use in targeted delivery to a microbial cell, in particular a methane producer cell. In some aspects, peptides and polypeptides can be used to bind to a cell wall or membrane and / or permeability into a cell. As such, peptides or polypeptides can be used for transient or prolonged attachment to a cell, or for penetration of a cell wall or membrane and / or accumulation in an extracellular environment. It is understood that peptides, polypeptides, as well as corresponding polynucleotides, expression vectors, host cells, and antibodies of the invention can be used to target various microorganisms, for example, Methanobrevibacter ruminantium , which is a common methane producer in ruminants, and Methanobrevibacter smithii , which is a widespread methane producer in humans. For targeted action, the microbial cell can be brought into contact with a signal peptide or polypeptide containing this peptide, isolated from one or more natural sources, or obtained using expression vectors and / or host cells, or synthetic or semi-synthetic chemistry, as described in detail described in this application. In specific aspects, the peptide or polypeptide is delivered to individuals in the form of the composition described in detail in this application, for example, by using a device for slow release in ruminants.

In some embodiments, the polypeptide is fused or crosslinked with a cellular inhibitor, for example, an anti-methane compound (e.g., bromoethanesulfonic acid), an antibody or antibody fragment, a lytic enzyme, a peptide nucleic acid, an antimicrobial peptide, or other antibiotic. This inhibitor peptide or inhibitor polypeptide is delivered to the individual in the form of a composition for inhibiting the growth or replication of microbial cells, in particular methane producing cells. This composition contains, for example: a) an isolated signal peptide or polypeptide containing the peptide, or a change, fragment, variant or derivative thereof; b) an isolated polynucleotide, or a change, fragment, variant or derivative thereof; c) an expression vector containing this polynucleotide; or d) a host cell containing this expression vector. The compositions of the invention may be specially packaged as part of kits for the targeted delivery, penetration and / or inhibition of microbial cells, in particular methane producer cells, in accordance with the described methods. These kits contain at least one composition provided herein and instructions for use for cell permeability or inhibition of cell growth or replication of methane producers or other microorganisms.

As an additional embodiment, the invention relates to a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, for use with any of the methods described above. Such pharmaceutical compositions may contain a signal peptide or a polypeptide containing this peptide in combination with a cellular inhibitor. Alternatively, the pharmaceutical compositions may contain an expression vector or a host cell, described in detail in this application. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, physiological saline, buffered saline, dextrose, or water. Compositions can be administered to an individual alone or in combination with other agents, drugs (eg, antimicrobials), or hormones.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers containing excipients and excipients that facilitate the processing of the active compounds into preparations that can be used pharmaceutically. Further details on the production and administration technology can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). The pharmaceutical compositions used in the present invention can be administered by any means, including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, enteric, local, sublingual or .

Pharmaceutical compositions for oral administration can be prepared using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers provide pharmaceutical compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, and the like for oral administration. Pharmaceutical preparations for oral administration can be obtained by combining the active compounds with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, followed by the addition of suitable excipients, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein excipients, such as sugars, including lactose, sucrose, mannitol or sorbitol; starch from corn, wheat, rice, potatoes, or other plants; cellulose, for example methyl cellulose, hydroxypropyl methyl cellulose or sodium carboxymethyl cellulose; gums, including Arabian and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrants or solubilizers, such as crosslinked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate can be added.

Pharmaceutical formulations that can be used orally include easy-to-swallow capsules made from gelatin, as well as soft, sealed capsules made from gelatin, and a coating such as glycerol or sorbitol. Easy-to-swallow capsules may contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid or liquid polyethylene glycol with or without stabilizers. Dragee cores may be used in combination with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, glaze solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings to identify the product or to characterize the amount of active substance, i.e. doses.

Pharmaceutical compositions suitable for parenteral administration can be prepared in aqueous solutions, preferably in physiologically compatible buffers, such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers can also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to enable the preparation of high concentration solutions. For topical or nasal administration, penetrants corresponding to the particular barrier to be penetrated are used in the composition. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be obtained by a method known in the art, for example, by conventionally mixing, dissolving, granulating, making dragees, eluting, emulsifying, encapsulating, incorporating, or lyophilizing processes. The pharmaceutical composition can be presented in the form of salt and can be formed with many acids, including, but not limited to, hydrochloric, sulfuric, acetic, dairy, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base forms. In other cases, the preferred preparation may be lyophilized powder, which may contain any or all of the following: 1-50 mM histidine, 0.1% -2% sucrose and 2-7% mannitol, at a pH in the range from 4.5 to 5.5, combined with buffer before use. After receiving the pharmaceutical compositions, they can be placed in an appropriate container and labeled for the treatment of this condition. For use of the composition of the invention, such a label would contain an amount, frequency and route of administration.

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an effective amount to achieve the intended purpose. For any compound, a therapeutically effective dose can be determined first either in cell studies, for example, in microbial cells, or in particular in methane producer cells, or in animal models, usually mice, rabbits, dogs or pigs, or in ruminant species of animals, such as sheep, cattle, deer and goats. An animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine appropriate doses and routes of administration to the individual. Normal dose amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending on the route of administration. Guidance on specific dosages and delivery methods is provided in the literature and is generally available to practitioners in the art. Specialists in this field will use other compositions for polynucleotides than for peptides or polypeptides. Similarly, the delivery of polynucleotides or polypeptides will be specific to specific cells, conditions, locations, etc.

Peptide and polypeptide based drugs are widely known, and methods for preparing such compositions are generally accepted in the art. Exemplary peptide and polypeptide drugs and their preparation are described, for example, for denileukin diphitox, octeotide, vapreotide, lanreotide, peptides of the RC-3940 series, decapeptil, lupron, zoladex, cetrorelix (see, for example, Lu et al., 2006 , AAPS J 8: E466-472), hemocidins, staphopenes (see, e.g., Dubin et al., 2005, Acta Biochemica Polonica, 52: 633-638), as well as indolicidin, defensins, lantibiotics, microcidin B17, histatin and maganine (see, e.g., Yeaman and Yount, 2003, Pharmacol Rev 55: 27-55). General guidance on peptide and polypeptide drugs can also be found in Degim et al., 2007, Curr Pharm Des 13: 99-117 and Shai et al., 2006, Curr Prot Pept Sci, 7: 479-486. Recently approved peptide-based drugs include Hematide ™ (a peptide-based synthetic drug, stimulating erythropoiesis, Affymax, Inc.), exenatide (synthetic exendin-4, Amylin / Eli Lilly), natrekor (neziritide, natriuretic peptide, Scios) , plenaxis (Abarelix, Praecis Pharmaceuticals), and SecreFlo (secretin, Repligen).

The exact dose will be determined by the practitioner, taking into account factors associated with the individual for whom treatment is required. Dose and administration are established to provide sufficient levels of the active agent or to maintain the desired effect. Factors that can be taken into account include the severity of the pathological condition, the general state of the individual’s health, age, body weight and gender of the individual, diet, time and frequency of administration, drug combination (s), sensitivity and tolerance / response to treatment . Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week or once every two weeks, depending on the half-life and the rate of excretion of a particular composition.

Especially suitable for the compositions of the invention (e.g., pharmaceutical compositions) are slow release formulations or mechanisms. For example, intra-scar devices include, but are not limited to, Time Capsule ™ Bolus from Agri-Feeds Ltd., New Zealand, originally developed by AgResearch Ltd., New Zealand, as described in WO 95/19763 and NZ 278977, and CAPTEC by Nufarm Health & Sciences, a division of Nufarm Ltd., Auckland, New Zealand, as described in AU 35908178, PCT / AU81 / 100082, and Laby et al., 1984, Can. J. Anim. Sci. 64 (Suppl.), 337-8, each of which is incorporated herein by reference. As a specific example, the device may include a spring and a piston that pushes the composition toward the hole at the end of the vessel.

As a further embodiment, the present invention relates to a composition for an aqueous additive, for example, an irrigation composition or a feed additive, for example, a feed for ruminants, for use with any of the methods discussed above. In specific aspects, the feed additive comprises at least one edible plant substance and the peptide or polypeptide of the present invention. Alternatively, the feed additive contains at least one edible plant substance and a polypeptide or peptide or polynucleotide encoding the peptide or polypeptide described herein, for example, as an expression vector or a host cell containing an expression vector. In particular, the composition further includes a cell inhibitor fused or linked to the resulting sequence. Preferred plant material includes any of hay, grass, grain or forage cereals, for example, legumes, hay, grass hay, corn silage, cereal silo, bean silo, corn grain, oats, barley, grains, beer pellets, soy flour and cottonseed flour. In particular, cereal silage is useful as a feed composition for ruminants. The plant material may be genetically modified so that it contains one or more components of the invention, for example, one or more polypeptides or peptides, polynucleotides or vectors.

In another embodiment, antibodies that specifically bind to the peptides, polypeptides or polynucleotides of the present invention can be used to determine the presence of microorganisms, in particular methane producers, or in assays to monitor the levels of such microorganisms. Antibodies suitable for diagnostic purposes can be obtained in the same manner as described above. Diagnostic tests include methods that use an antibody and label to detect a peptide or polypeptide in human body fluids or extracts of cells or tissues. Antibodies can be used with or without modification and can be labeled by combining them, either covalently or non-covalently, with a reporter molecule. A variety of reporter molecules that are known in the art can be used, some of which are described above.

A number of protocols for measuring the levels of a peptide, polypeptide or polynucleotide are known in the art (e.g., ELISA, RIA, FACS, and blotting), and provide a basis for determining the presence or levels of a microorganism, especially methane producers. Normal or standard levels established by combining body fluids or cell extracts taken from healthy individuals, such as healthy people or ruminants, with an antibody under conditions suitable for complexation. The standard complexation can be quantified by various methods, but preferably by a photometric method. The amounts of the peptide, polypeptide or polynucleotide expressed in the individual and in the processed samples (for example, samples from the individuals who have undergone processing) are compared with standard values. The deviation between the standard values and the values obtained from the individual sets the parameters for determining the presence or levels of the microorganism.

In a specific embodiment of the present invention, polynucleotides can be used for diagnostic purposes using specific hybridization and / or amplification techniques. Polynucleotides that may be used include oligonucleotides, complementary RNAs and DNA molecules and PNAs. Polynucleotides can be used to identify and quantify gene expression in samples in which expression may correlate with the presence or levels of the microorganism. Diagnostic analysis can be used to distinguish between the absence, presence and change of levels of microorganisms and to monitor levels during therapeutic intervention.

In one aspect, hybridization with PCR probes can be used to identify nucleic acid sequences, especially genomic sequences that encode the peptides or polypeptides of the present invention. The specificity of the probe, whether it is obtained from a highly specific region, for example, 10 unique nucleotides in the 5'-regulatory region, or a less specific region, for example, in the 3'-coding region, and the rigidity of hybridization or amplification (maximum, high, intermediate or low) will determine if the probe identifies only natural sequences, alleles, or related sequences. Probes can also be used to detect related sequences, and preferably should contain at least 50% of the nucleotides from any of the coding sequences. Hybridization probes of the subject invention can be DNA or RNA and come from the nucleotide sequence of SEQ ID NO: 173-341 or 342-533, or complements, or their modified sequences, or from genomic sequences, including the promoter, enhancer elements, and introns of the natural sequence .

Methods for producing specific DNA hybridization probes include cloning nucleic acid sequences into vectors to obtain mRNA probes. Such vectors are known in the art, commercially available, and can be used for in vitro synthesis of RNA probes by adding the corresponding RNA polymerases and the corresponding labeled nucleotides. Hybridization probes can be labeled with a number of reporter groups, for example, radionuclides, such as ³² P or ³⁵ S, or enzyme labels, such as alkaline phosphatase, coupled to the probe via avidin / biotin coupling systems, and the like. Polynucleotides can be used in Southern or Northern analysis, dot blotting or other membrane techniques; in PCR methods; or in test strips, rods, ELISA assays, or microarrays using liquids or tissues from an individual's biopsy specimens to detect the presence or levels of the microorganism. Such qualitative and quantitative methods are known in the art.

In a particular aspect, nucleic acid sequences can be used in various assays, labeled by standard methods and added to a liquid or tissue sample from an individual under conditions suitable for hybridization and / or amplification. After an appropriate incubation period, the sample is washed and the signal is measured and compared with a standard value. If the signal value in the test sample varies significantly compared to the signal value of the control sample, the presence of altered levels of nucleotide sequences in the sample indicates the presence or levels of the microorganism. Such analyzes can also be used to evaluate the effectiveness of a particular treatment regimen in animal studies, in clinical trials, or when monitoring an individual’s treatment.

To provide a basis for diagnosing the presence or levels of a microorganism, a normal or standard expression profile is established. This can be done by combining body fluids or cell extracts taken from healthy individuals with a polynucleotide or fragment thereof, under conditions suitable for hybridization and / or amplification. Standard levels can be quantified by comparing levels obtained from healthy individuals with levels from an experiment in which a known amount of substantially purified polynucleotide is used. The standard values obtained from normal samples can be compared with the values obtained from samples from individuals who underwent treatment against microbial growth. The deviation between the standard values and the values of the individual is used to establish the presence or levels of the microorganism.

After the microorganism has been identified and the treatment protocol has been started, hybridization and / or amplification analyzes can be repeated regularly to determine if the expression level in an individual begins to decrease compared to that observed in a normal individual. The results obtained from subsequent analyzes can be used to demonstrate the effectiveness of treatment over a period of time ranging from several days to months.

Specific diagnostic applications for oligonucleotides derived from nucleic acid sequences may include the use of PCR. Such oligomers can be synthesized chemically, obtained enzymatically or obtained in vitro . Oligomers will preferably consist of two nucleotide sequences, one with a sense orientation (5'.fwdarw.3 ') and the other with an antisense orientation (3'.fwdarw.5') used under optimized conditions to identify a specific gene or condition. The same two oligomers, a set of oligomers for nested PCR, or even a degenerate pool of oligomers can be used in less stringent conditions to identify and / or quantify closely related DNA or RNA sequences.

Methods that can also be used for quantitative analysis of expression include radioactively labeled or biotinylated nucleotides, co-amplification of a control nucleic acid, and standard curves on which experimental results are interpolated (Melby, PC et al. (1993) J. Immunol. Methods, 159: 235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The speed of quantitative analysis of multiple samples can be increased by conducting this study in the ELISA format, where the oligomer of interest is in various dilutions and the spectrophotometric or colorimetric response gives a quick quantitative determination.

In further embodiments, oligonucleotides or longer fragments derived from any polynucleotides described herein can be used as targets in a microchip. The microchip can be used to monitor the expression level of a huge number of genes simultaneously (to obtain a transcript image), and to identify genetic variants, mutations, and polymorphisms. This information can be used to determine gene function, to understand the genetic basis of a disease, to diagnose a disease, and to develop and monitor the activities of therapeutic agents. In one embodiment, a microchip is prepared and used in accordance with methods known in the art, for example, those described in PCT Application WO 95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619).

In one aspect, oligonucleotides can be synthesized on the surface of a microarray using a chemical coupling technique and an inkjet application device, such as described in PCT Application WO95 / 251116 (Baldeschweiler et al.). In another aspect, a “grid” matrix similar to a dot or slot blot (HYBRIDOT apparatus, Life Technologies) can be used to systematize and connect fragments of cDNA or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical methods binding. In another aspect, the matrix can be obtained manually, or by using available devices, materials, and devices (including multichannel microdosers or robotic tools; Brinkmann, Westbury, NY) and may contain 8, 24, 96, 384, 1536, or 6144 oligonucleotides, or any other multiple of from 2 to 1,000,000, which is suitable for the effective use of commercially available technical means.

To analyze a sample using microarrays, polynucleotides are extracted from a biological sample. Biological samples can be obtained from any body fluid (blood, urine, saliva, sputum, gastric juice, etc.), cultured cells, biopsy specimens, or other tissue preparations. To obtain probes, polynucleotides extracted from a sample are used to obtain nucleic acid sequences that are complementary to nucleic acids on a microchip. If the microchip consists of cDNA, antisense RNAs are suitable probes. Therefore, in one aspect, mRNA is used to obtain cDNA, which, in turn, in the presence of fluorescent nucleotides, is used to obtain fragments or antisense RNA probes. These fluorescently labeled probes are incubated with the microchip, so that the probe sequences hybridize with the cDNA of the microchip oligonucleotides. In another aspect, the nucleic acid sequences used as probes may contain polynucleotides, fragments, and complementary or antisense sequences obtained using restriction enzymes, PCR methods, and oligolabeling kits (Amersham Pharmacia Biotech) well known in the field of hybridization technology.

In another embodiment of the present invention, the peptides or polypeptides of the invention, or their functional or immunogenic fragments or oligopeptides, can be used to screen libraries of compounds in any of a variety of drug screening methods. The fragment used in such a screening can be free in solution, fixed on a solid substrate, transferred to the cell surface or located intracellularly. The formation of binding complexes between the peptide or polypeptide and the test substance can be measured.

One drug screening technique that can be used involves high throughput screening for compounds having binding affinity for a peptide or polypeptide of interest, as described in PCT published application WO 84/03564. In this method, a large number of various small test compounds are synthesized on a solid substrate, for example, plastic pins, or some other surfaces. Test compounds are reacted with a peptide or polypeptide, or fragments thereof, and washed. The bound peptide or polypeptide is then detected by methods well known in the art. The purified peptide or polypeptide may also be applied directly to the tablets for use in the aforementioned drug screening methods. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another method, competitive assays can be used to screen drugs in which neutralizing antibodies capable of binding to a peptide or polypeptide specifically compete with a test compound for binding to this peptide or polypeptide. Thus, antibodies can be used to detect the presence of a test compound that shares one or more antigen binding sites with an antibody.

EXAMPLES

The examples described in this application are intended to illustrate embodiments of the present invention. Other options for implementation, methods and types of analysis are within the competence of specialists in the field of molecular diagnostics and do not need a detailed description. Other embodiments within the scope of the prior art are considered part of this invention.

EXAMPLE 1: Materials and Methods

Genome Size Estimation

The Methanobrevibacter ruminantium M1 ^T strain (DSM1093) was grown in BY + medium (minimal medium, Joblin et al., 1990), which consists of [g / L] NaCl (1), KH ₂ PO ₄ (0.5), (NH ₄ ) ₂ SO ₄ (0.25), CaCL ₂ .2H ₂ O (0.13), MgSO ₄ .7H ₂ O (0.2), K ₂ HPO ₄ (1), clarified scar fluid (300 ml), dH ₂ O (360 ml), NaHCO ₃ (5), resazurin (0.2 ml), L-cysteine-HCl (0.5), yeast extract (2), and Balch trace element solution (10 ml) (added trace elements; Balch et al., 1979), which consists of (g / l) nitrile triacetic acid (1.5), MgSO ₄ .7H ₂ O (3), MnSO ₄ .H ₂ O (0.5), NaCl (1), FeSO ₄ .7H ₂ O (0,1), CoCl ₂ .6H ₂ O (0,1), CaCl ₂ (0,1), ZnSO ₄ .7H ₂ O (0,1), CuSO ₄ .5H ₂ O (0.01), AlK (SO ₄ ) ₂ .12H ₂ O (0.01), H ₃ BO ₃ (0.01), Na ₂ MoO ₄ .2H ₂ O (0.01), NiSO ₄ .6H ₂ O (0.03), Na ₂ SeO ₃ (0.02), and Na ₂ Wo ₄ .2H ₂ O (0.02). Genomic DNA was extracted using the method of grinding by freezing. Cells were harvested by centrifugation, and cell debris was placed in a pre-chilled mortar, frozen with liquid nitrogen, and carefully ground into a fine powder using a pre-chilled sterile mortar and pestle. Cell homogenates were immersed in agarose plugs, and subsequent manipulations were performed in these plugs to reduce the physical fragmentation of genomic DNA. Cleavages were performed with restriction endonucleases, and DNA fragments were separated using pulsed field gradient gel electrophoresis (PFGE).

DNA cloning and sequencing

The DNA of the M. ruminantium genome was sequenced by Agencourt Biosciences Corporation (Massachusetts, USA) using the cloning approach of stochastic genomic fragments (Fleischmann et al., 1995) and Macrogen Corporation (Rockville, MD, USA) using pyrosequencing. M. ruminantium DNA libraries were constructed in Escherichia coli by random physical disruption of genomic DNA and separation of fragments by gel electrophoresis. Large fragments in the range of 40 Kb were extracted from the gel and used to obtain a large insert phosmid library. DNA fragments in the range from 2 to 4 Kb were extracted and used to obtain a small insert plasmid library. Clones obtained from both large and small insertion libraries were grown, their phosmid or plasmid DNA was extracted and sequenced using high-throughput sequencing technology. The corresponding clones were sequenced to theoretically obtain 8-fold coverage of the M. ruminantium genome. Pyrosequencing was performed on randomly split genomic DNA fragments with a final theoretical 10-fold coverage.

Assembly and sequence analysis

DNA sequences were aligned to find overlapping sequences and assembled into continuous (contig) sequences using the Paracel Genome Assembler (Paracel Inc, CA, USA) and the Staden package (Staden et al., 1998) in combination with both standard and inverted PCR. Contigs were analyzed using the finder of the open reading frame (ORF) GLIMMER (G ene L ocator I nterpolated M arkov M odel ER, Salzberg et al., 1998) and each ORF were analyzed by BLAST (B asic L ocal A lignment S earch T ool (Altschul et al., 1997) regarding the replenished nucleotide and protein databases of the National Center for Biotechnological Information (NCBI).

Contigs of an 8-fold preliminary sequence were randomly joined by artificially joining sequences to obtain a “pseudomolecule,” and submitted to the Institute for Genomic Research (TIGR, DC, USA) for auto annotation. Contigs collected by 10-fold pyrosequencing were re-analyzed using GLIMMER, and ORFs were auto-annotated using GAMOLA (Global Annotation of Multiplexed On-site Blasted DNA sequences; Altermann and Klaenhammer, 2003). Automated annotations were subsequently verified manually. ORFs were classified by function using an orthologous protein (COG) cluster database (threshold 1e-02) (Tatusov et al., 2001).

Protein motifs were determined using HMMER (hypertext transfer protocol: //hmmer.wustl.edu), using the PFAM HMM and TIGRFAM libraries, and general and local alignment (hypertext transfer protocol: //pfam.wustl.edu) and standard and fragment models TIGRFAM HMM (hypertext transfer protocol: //www.tigr.org/TIGRFAMs), respectively (threshold 1e-02). tRNAs were identified using TRNASCAN-SE (Lowe and Eddy, 1997) and nucleotide repeats were identified using the KODON software package (Applied Maths, Austin, TX, USA) and REPUTER (Kurtz and Schleiermacher, 1999). A genomic atlas of visualizations was compiled using GENEWIZ (Jensen et al., 1999). The reconstruction path from the predictedM. ruminantium ORFeoma was performed in combination with KEGG (Kyoto Encyclopedia of Genes and Genomes, Kanehisa et al., 2004) on-line databases using proprietary software (PathwayVoyager; Altermann and Klaenhammer, 2005).

Signal Peptide Identification

There is currently no signal peptide model for Archaea . There are just a few experimentally verified secretory proteins available for Archaea to form a specific model. For this reason, open reading frame (ORF) sequences were analyzed for the presence of signal peptides using SignalP Version 3.0 (Bendtsen et al., 2004), prepared for gram-positive, gram-negative and eukaryotic models. SignalP-HMM (Hidden Markov Model) was used to distinguish between the ORF of the signal peptide and the non-signal peptide, while SignalP-NN (neural networks) was used to preliminarily determine the cleavage sites, as described by Emanuelsson et al., 2007.

SignalP predicts the presence and localization of signal peptide cleavage sites in amino acid sequences from various organisms. This method includes the preliminary determination of cleavage sites and the preliminary determination of the signal peptide / non-signal peptide based on a combination of several artificial neural networks and hidden Markov models. Signal peptide sequences identified from gram-positive data sets were aligned and consensus sequences were calculated using the AlignX Vector NTI software (version 9.1.0, Invitrogen Corporation). A conservative hydrophobic core was identified by analysis of amino acid hydrophobicity.

A consensus data set was identified from all three SignalP models, and the corresponding signal peptide sequences were aligned using ClustalW (Larkin et al., 2007) and processed using BioEdit (hypertext transfer protocol: // world wide web.mbio.ncsu.edu/ BioEdit / bioedit.html). The logo protein sequence (FIG. 3A) was obtained using the LogoBar (Perez-Bercoff et al., 2006) to represent the information contained in this multiple sequence alignment. In this study, ORFs containing a signal peptide and three or more transmembrane domains were considered membrane proteins and were excluded from further analyzes. The best Y-criterion from each of the three models was taken as the putative cleavage site (FIG. 6). The optimized codon usage frequency (FIG. 9) was calculated using a proprietary perl script: opt_codons.pl (Altermann, E) based on the codon table of Escherichia coli K12.

Peptide synthesis and fluorescence labeling

The core consensus peptide was synthesized commercially using Invitrogen's custom peptide service (Invitrogen NZ Ltd). This peptide was synthesized using Fmoc chemistry on a small scale (10-12 mg) and purified using HPLC to a purity> 95%. This peptide was labeled with fluorescein on an N-terminal lysine (K).

Cell Permeability Test

The entry of the labeled peptide into M. ruminantium cells was accompanied by fluorescence analysis. A culture of M. ruminantium was grown in 10 ml of BY + medium and collected by centrifugation at 10,000 × g for 10 min at 4 ° C. Cells were transferred to 1.5 ml Eppendorf polypropylene tubes and washed in 1 ml TE buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, pH 8) and collected by centrifugation in a microcentrifuge at 13,000 × g for 10 min at 4 ° C. Cells (approximately 1 × 10 ⁸ ) were resuspended in a total volume of 200 μl of TE buffer and 20 μg of fluorescently labeled peptide was added. This mixture was incubated for 30 minutes at 37 ° C, and then centrifuged at 13000 × g for 10 min at 4 ° C. The liquid over the cell debris was left and the supernatant fraction was made up. Cell debris was washed 3 times with 200 μl TE buffer, by resuspending the cells in the buffer, and centrifuged at 13000 × g for 10 min at 4 ° C.

Wash solutions were collected and the cell wash fraction was made up. The cell debris remaining after the third wash was resuspended in 200 μl of TE buffer containing 1% sodium dodecyl sulfate. Cells were centrifuged at 13,000 × g for 10 min at 4 ° C to pellet the cells, and liquid above the cells was collected and constituted the cell-associated fraction. The remaining cell debris was frozen in liquid N ₂ , and the cells were physically destroyed by grinding the frozen debris with a glass rod. The resulting cell homogenate was centrifuged at 20,000 × g for 30 min at 4 ° C. The liquid above the debris was collected and the intracellular fraction was represented, while the remaining precipitated substance was resuspended in TE buffer and the cell wall / membrane fraction was represented. The fluorescence in each of these fractions was measured by hermetically closing a sample of each fraction in a glass capillary, and the emitted fluorescence was measured at 510-533 nm relative to the fluorescently labeled peptide standards using a fluorescent sensor (Channel 1) Lightcycler (Roche).

EXAMPLE 2: experimental results

Estimation of the genome size of M. ruminantium by digestion with restriction enzymes of genomic DNA, and the determination of fragment sizes by PFGE, showed one chromosome of approximately 2.5-2.9 Mb. The initial sequencing of large and small insertion clones (6-fold) and the assembly of the sequence into contigs showed that the 40 Kb genome region was highly represented (> 20 times), especially within a small insertion library. Due to this large distortion of the sequence, additional sequencing (2-fold theoretical genome coverage) was performed only for large insertion clones with the final 8-fold coverage resulting from Senger sequencing. The 8-fold phase sequence was assembled into 756 contigs, which were connected via 105 scaffolds. Further pyrosequencing was carried out until an additional ~ 10-fold coverage and inclusion of these sequences in the assembly, resulting in a number of contigs decreasing to 27. Subsequent closing of the breaks using inverted PCR and PCR technology of long fragments, reduced the number of contigs to 14.

The combined length of the 14-contig sequence indicates that the genome is slightly larger (2937347 nucleotides) than the size estimated by PFGE (FIG. 1A) and significantly larger than that of its closest relative, M. smithii (1.9 Mb). % G + C 32.64% is close to the indicated range from 27.5% to 31.6% described for M. ruminantium strains (Balch et al, 1979). The sequence analysis assumes 2239 ORF and the total number of hits in the protein family (TIGRFam and PFam) and clusters of orthologous groups (Clusters of Orthologous Groups (COG)) are presented in FIG. 1B. All genes presumably involved in the formation of methane from H ₂ + CO ₂ and formate are presented (FIG. 1C). However, the draft sequence of M. ruminantium lacks the methyl coenzyme reductase II system ( mcr II or mrt ). In other metanoprodutsentov, mcrII cluster encodes methyl CoM reductase isozyme I, an enzyme that is activated during the growth at high partial pressures of H ₂ (Reeve et al., 1997). H _{2 is} quickly used in the rumen and does not accumulate to high levels, therefore, M. ruminantium is apparently adapted to use low H ₂ levels only through the mcr I system.

A total of 169 signal peptides containing ORF were identified in the M. ruminantium genome. (FIG. 7). Of these, 102 signal peptides were identified using all three SignalP models, and the amino acid sequences of these signal peptides were aligned (FIG. 2) and the logo protein sequence (FIG. 3A) was obtained. A core sequence of 17 hydrophobic amino acids has been identified (KKIIIILLLLILLLISI; SEQ ID NO: 119). SignalP-HMM calculates the probability of the content of a signal peptide in this sequence. The probability of this signal peptide is from 0 to 1, with a set cutoff value of 0.5 to distinguish between the signal peptide and the non-signal peptide for this analysis. SignalP-NN Y-criterion gives the best estimate of the SP cleavage site (FIG. 6). The Y-criterion is defined as the geometric mean C-criterion (raw cleavage site score) and the aligned slope of the S-criterion (signal peptide criterion) obtained using SignalP-NN. The Y-criterion is a value from 0 to 1 with higher rates indicating the correct prediction of the cleavage site.

The consensus amino acid sequence (FIG. 3C) was synthesized and conjugated with a fluorescent label fluorescein through an additional N-terminal lysine residue (FIG. 3D) to obtain a final peptide length of 17 amino acids. The purified FITC peptide was tested for permeability in M. ruminantium cells (FIG. 4). In the analysis of cell permeability of M. ruminantium, 23.5% of the peptide remained in the supernatant unattached to the cells after 30 minutes at 37 ° C. An additional 3.4% of the peptide could be removed from the cells by 3 washes with buffer. Approximately 62.9% of the peptide was recovered after 1% SDS cell extraction, indicating that most of the peptide was associated with cells. Of the remaining peptide, 5.8% was found in the intracellular fraction and 4.4% was associated with the cell wall / membrane fraction. Therefore, 5.8% of the original peptide (equivalent to 1.16 μg) was able to bind to M. ruminantium and pass through the cell membrane to enter the cell cytoplasm, which represents approximately 2.3 × 10 ⁶ peptide molecules per cell.

EXAMPLE 3: Overview

Methanobrevibacter ruminantium was chosen for genome sequencing, due to dominance in the rumen under various nutritional conditions (based on cultivation data and molecular determination), accessibility of cultures, convenience for routine cultivation in a laboratory, and a relatively large number of preliminary studies and primary sources available for this organism. A significant number of sequences in M. ruminantium was assigned a function, and thus, it was possible to obtain a detailed picture of the lifestyle of this microorganism in the rumen. The dependence of M. ruminantium on simple substrates (H ₂ + CO ₂ , formate) and its interaction with the environment of the rumen through surface proteins and exopolysaccharides are targets for inhibition. Data on these sequences explain the metabolism of this organism and its interaction with other microorganisms, and indicate conservative systems and components among methane producers that can be inactivated to prevent or reduce the formation of methane in the rumen.

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All publications and patents mentioned in the above description are incorporated by reference.

Where reference to the above description is given to integers having known equivalents, these equivalents are included as separately indicated.

Although the present invention has been described in relation to particularly preferred embodiments, it should be understood that the present invention, as claimed, should not be inappropriately limited to such specific embodiments.

It is understood that further modifications may be made with respect to the present invention, as described herein, without departing from the spirit and scope of the invention.

Claims (18)

1. The selected polypeptide or peptide that is able to penetrate into a methane-producing cell characterized by the amino acid sequence of SEQ ID NO: 117, 118 or 119. 2. An isolated polypeptide or peptide that is able to penetrate into a methane-producing cell, characterized by:
a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 117;
b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 118; or
c) an amino acid sequence that is at least 90% identical to SEQ ID NO: 119. 3. An isolated polypeptide or peptide that is able to penetrate into a methane-producing cell characterized by:
a) at least 15 consecutive amino acids of the sequence of SEQ ID NO: 117 or 118; or
b) at least 15 consecutive amino acids of the sequence of SEQ ID NO: 119. 4. The selected polynucleotide that encodes a polypeptide or peptide that is able to penetrate into a methane producing cell, characterized by:
a) a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 117, 118 or 119; or
b) a nucleotide sequence complementary to the sequence (a). 5. The selected polynucleotide that encodes a polypeptide or peptide that is able to penetrate into a methane producing cell, characterized by:
a) a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 117;
b) a nucleotide sequence that encodes an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 118;
c) a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 119; or
d) a nucleotide sequence complementary to any one of (a) to (c). 6. The selected polynucleotide that encodes a polypeptide or peptide that is able to penetrate into a methane producing cell, characterized by:
a) a nucleotide sequence that encodes 15 consecutive amino acids of the sequence SEQ ID NO: 117 or 118;
b) a nucleotide sequence that encodes at least 15 consecutive amino acids of the sequence of SEQ ID NO: 119; or
c) a nucleotide sequence complementary to a sequence according to any one of (a) or (b). 7. The selected polynucleotide that encodes a polypeptide or peptide that is able to penetrate into a methane producing cell characterized by the nucleotide sequence of SEQ ID NO: 511, 512 or 513 or a nucleotide sequence complementary to them. 8. A cloning vector that contains a polynucleotide according to any one of claims 4 to 7, or a polynucleotide that encodes a polypeptide or peptide according to any one of claims 1 to 3. 9. An expression vector that contains a polynucleotide according to any one of claims 4 to 7, or a polynucleotide that encodes a polypeptide or peptide according to any one of claims 1 to 3. 10. A host cell transformed with a vector according to claim 8 or 9, for the production of a polypeptide or peptide according to any one of claims 1 to 3, or a polynucleotide according to any one of claims 4 to 7. 11. A host cell for replicating a vector according to claim 8 or claim 9, transformed with a vector according to claim 8 or 9. 12. The host cell of claim 10 or 11, which is selected from a prokaryotic cell or methanogen cell. 13. The host cell of claim 12, which is selected from Escherichia coli and Methanobrevibacter ruminantium, including Methanobrevibacter ruminantium strain M1 ^T (DSM1093). 14. A conjugated molecule or a fusion molecule that is capable of penetrating into a methane producing cell and which contains the polypeptide or peptide according to any one of claims 1 to 3 and a fluorescent label at the N-terminal amino acid residue. 15. A method of increasing the permeability of a methane producing cell, which includes: bringing this cell into contact with a polypeptide or peptide according to any one of claims 1 to 3. 16. A method of increasing the permeability of a methane producing cell, comprising: bringing this cell into contact with a conjugated molecule or a fusion molecule according to claim 14. 17. The method according to clause 16, in which the cell is a Methanobrevibacter ruminantium. 18. The method according to 17, in which the cell is a Methanobrevibacter ruminantium strain M1 ^T (DSM1093).

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