WO2008036061A2 - Enzymes et préparations de décontamination d'agents de guerre chimiques et biologiques - Google Patents

Enzymes et préparations de décontamination d'agents de guerre chimiques et biologiques Download PDF

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Publication number
WO2008036061A2
WO2008036061A2 PCT/US2006/013031 US2006013031W WO2008036061A2 WO 2008036061 A2 WO2008036061 A2 WO 2008036061A2 US 2006013031 W US2006013031 W US 2006013031W WO 2008036061 A2 WO2008036061 A2 WO 2008036061A2
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Prior art keywords
seq
sequence
activity
nucleic acid
polypeptide
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PCT/US2006/013031
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English (en)
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WO2008036061A3 (fr
Inventor
Dan E. Robertson
Toby Richardson
Karen Kustedjo
Gabriel Amitai
Keith Lejeune
Jason Berberich
Jennifer Ann Chaplin
Jessica Sinclair
Original Assignee
Verenium Corporation
Agentase, Llc
Life Science Research Israel Ltd.
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Application filed by Verenium Corporation, Agentase, Llc, Life Science Research Israel Ltd. filed Critical Verenium Corporation
Priority to CA002602185A priority Critical patent/CA2602185A1/fr
Priority to EP06851338A priority patent/EP1928560A2/fr
Priority to JP2008535508A priority patent/JP2009517002A/ja
Priority to IL186399A priority patent/IL186399A0/en
Publication of WO2008036061A2 publication Critical patent/WO2008036061A2/fr
Publication of WO2008036061A3 publication Critical patent/WO2008036061A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/02—Chemical warfare substances, e.g. cholinesterase inhibitors

Definitions

  • This invention was produced in part using funds from the Federal government under Defense Threat Reduction Agency contract # HDTRA-04-C-0046. Accordingly, the Federal government has certain rights in this invention.
  • the invention provides enzymes and methods for enzyme-based active decontamination, e.g., nerve agent detoxification.
  • the invention provides enzymes having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non- heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, and methods of making and using them,
  • an esterase activity e.g., an organophosphohydrolase activity (such as an organophosphoeste
  • the invention provides polypeptides, nucleic acids, infective vehicles and methods of using them to generate self-protecting, pesticide-resistant cells or plants.
  • the invention provides an enzyme mixture or composition that provides broad-spectrum, agent-specific oxidation and hydrolysis activities as well as hypohalite generation for oxidation or hydrolysis of V-agents, G-agents and H-agents and which generates oxidants such as chlorine dioxide and reactive radicals for killing of biological agents, including anthrax spores.
  • the invention provides novel polypeptides (e.g., enzymes, peptides and antibodies) and methods for use in enzyme-based active decontamination, e.g., of toxins and poisons, including synthetic substances such as nerve gases, e.g. agent VX, mustard gases and the like, and toxic biological agents, e.g., Bacillus and other toxic spores.
  • enzyme-based active decontamination e.g., of toxins and poisons
  • synthetic substances such as nerve gases, e.g. agent VX, mustard gases and the like
  • toxic biological agents e.g., Bacillus and other toxic spores.
  • novel polypeptides for example, enzymes and catalytic antibodies, having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, and mixtures thereof (e.g., as formulations), for decontamination activity, including thermostable and thermotolerant forms of these enzymes (e.g., having oxidoreductase, hydrolase, etc).
  • the invention provides isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550,
  • nucleic acids of the invention include isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ
  • Exemplary nucleic acids of the invention also include isolated, synthetic or recombinant nucleic acids encoding a polypeptide of the invention, including exemplary sequences of the invention, e.g., amino acid sequences as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, etc. all the even numbered SEQ ID NO:s through SEQ ID NO: 194, and subsequences thereof and variants thereof.
  • the polypeptide has a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non- heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, or a decontamination activity or other related activity.
  • the hydrolase, oxidoreductase, or decontamination activity, or other activity is a regioselective and/or chemoselective activity.
  • the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity.
  • the enzymatic activity can comprise hydrolysis of, or decontamination of, a V agent.
  • the enzymatic activity can comprise hydrolysis of, or decontamination of, a V agent, or the enzymatic activity can comprises a haloperoxidase activity, or an activity comprising catalyzing the hydrolysis of a methylphosphonofluoridate or a thiophosphoric ester, or a combination thereof.
  • the haloperoxidase activity can comprise a chloroperoxidase activity.
  • the V agent detoxified, neutralized and/or decontaminated by an polypeptide of the invention can comprises VX (0-Ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate, or methylphosphonothioic acid), VE (O-Ethyl-S-[2- (diethylamino)ethyl] ethylphosphonothioate), VG (O,O-Diethyl-S-[2- (diethylamino)ethyl] phosphorothioate), VM (0-Ethyl-S-[2-(diethylamino)ethyl] methylphosphonothioate), VR (Phosphonothioic acid) Soviet V-gas (Russian VX), Tetriso (0,0-diisopropyl S-(2-diisopropyla
  • the enzyme hydrolyzing or decontaminating a V agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 89; SEQ ID NO:1 17; SEQ ID NO:119; SEQ ID NO: 127; SEQ ID NO: 151; SEQ ID NO: 167; SEQ ID NO: 171; SEQ ID NO: 187 or SEQ ID NO: 191, or having an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:90; SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO: 128; SEQ ID NO: 152; SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 189 or SEQ ID NO: 192.
  • the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity against a G agent, e.g., the enzymatic activity comprises hydrolysis of, or decontamination of, a G agent.
  • the enzymatic activity can comprise hydrolysis of, or decontamination of, a G agent comprises an organophosphoric acid anhydrolase (OPAA) activity.
  • the G agent can comprise tabun (GA), sarin (methylphosphonofluoridic acid) (GB), soman (GD), cyclosarin (GF) or a combination thereof.
  • the enzyme that hydrolyzes or decontaminates a G agent can comprise a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO: 101 ; SEQ ID NO: 103; SEQ ID NO: 105; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127;SEQIDNO:129;SEQIDNO:131;SEQIDNO:133;SEQIDNO:135;SEQID NO:137; SEQIDNO:139; SEQIDNO:141; SEQIDNO:143; SEQIDNO:145; SEQID NO:147; SEQ IDNO:149; SEQ
  • the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity against an H agent, e.g., the enzymatic activity comprises hydrolysis of, or decontamination of, an H agent.
  • the H agent can comprise a chloroperoxidase activity, a dehalogenase activity or a combination thereof.
  • the H agent can comprise mustard gas; 1 I' thiobis [2 chloroethane] bis-(2-chloroethyl) sulphide; beta, beta 1 dichloroethyl sulphide; 2, 2' dichloroethyl sulphide; bis (beta-chloroethyl) sulphide; l-chloro-2 (beta- chlorodiethylthio) ethane; or, a combination thereof.
  • the enzyme hydrolyzing or decontaminating an H agent can comprise a polypeptide encoded by a nucleic acid encoding a chloroperoxidase and having a sequence as set forth in SEQ ID NO:1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively, or a nucleic acid encoding a dehalogenase and having a sequence as set forth in SEQ ID NO: 69 or SEQ ID NO:91 or the enzyme has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:
  • the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity against a biological agent, e.g., the enzymatic activity comprises hydrolysis of, or decontamination of, a biological agent.
  • the enzymatic activity can comprise a chloroperoxidase activity.
  • the biological agent can be a gram negative spore, such as a Bacillus spore, e.g., an anthrax, or Bacillus anthracis, spore.
  • the enzyme hydrolyzing, neutralizing or decontaminating a biological agent can comprise a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO:1, SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID
  • the enzymatic activity can comprise hydrolysis of, or decontamination of, a P-F bond.
  • the polypeptide having P-F bond hydrolyzing, neutralizing or decontamination activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO: 101; SEQ ID NO:
  • SEQ ID NO: 106 SEQ ID NO: 108; SEQ ID NO: 110; SEQ ID NO: 112; SEQ ID NO: 114;
  • SEQ ID NO: 186 SEQ ID NO: 188, SEQ ID NO: 192 or SEQ ID NO: 194.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises hydrolysis of, or decontamination of, a P-S bond.
  • the polypeptide having P-S bond hydrolyzing, neutralizing or decontamination activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:1; SEQ ID NO: 75; SEQ ID NO:
  • SEQ ID NO: 151 SEQ ID NO:167; SEQ ID NO:171; SEQ ID NO:187 or SEQ ID NO:191, or has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:76; SEQ ID NO: 78; SEQ ID NO:90; SEQ ID NO: 118; SEQ ID NO:120; SEQ ID NO: 128; SEQ ID NO: 152;
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises an organophosphoric acid anhydrolase (OPAA) activity.
  • the enzyme having organophosphoric acid anhydrolase (OPAA) activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO: 194.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises an aminopeptidase activity.
  • the enzyme having aminopeptidase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 189, or has an amino acid sequence as set forth in SEQ ID NO: 190.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises a carboxylesterase activity.
  • the enzyme having carboxylesterase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87 or SEQ ID NO:89.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises a heme-based peroxidase activity.
  • the enzyme having heme-based peroxidase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41 ; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises a non-heme-based peroxidase activity.
  • the enzyme having non- heme-based chloroperoxidase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises a dehalogenase activity.
  • the enzyme having a dehalogenase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises a diisopropylfluorophosphatase (DFPase) activity.
  • the enzyme having diisopropylfluorophosphatase (DFPase) activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises an organophosphoesterase and can hydrolyze a P-S or a P-F bond and detoxify an acetylcholinesterase- or butyrylcholinesterase- inhibitor.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises a halogenation reaction to form a hypohalite from hydrogen peroxide and chloride, bromide or iodide.
  • the enzymatic activity comprises catalysis of the transfer of oxygen from hydrogen peroxide to an organic substrate.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises chemical bleaching of lignin.
  • the enzymatic activity can comprise chemical bleaching of lignin in a pulp or paper manufacturing process.
  • the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention comprises detoxifying a pesticide, herbicide and/or insecticide.
  • the pesticide can comprise Demeton-S, Demeton-S-methyl, Demeton-S-methylsulphon, Demeton- methyl, Parathion, Phosmet, Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos- ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate-methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Prothoate, S
  • the invention also provides enzyme-encoding nucleic acids with a common novelty in that they are derived from mixed cultures.
  • the invention provides enzyme-encoding nucleic acids isolated from mixed cultures comprising a nucleic acid of the invention, e.g., a nucleic acid having a sequence at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention over a region of at least about 10, 20, 30,
  • the invention also provides enzyme-encoding nucleic acids with a common novelty in that they are derived from environmental sources, e.g., mixed environmental sources.
  • the invention provides enzyme-encoding nucleic acids isolated from environmental sources, e.g., mixed environmental sources, comprising a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention over a region
  • the invention provides enzyme-encoding nucleic acids isolated from environmental sources, e.g., mixed environmental sources, comprising a nucleic acid of the invention.
  • the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d "nr pataa" -F F, and all other options are set to default.
  • Another aspect of the invention is an isolated, synthetic or recombinant nucleic acid including at least 10 consecutive bases of a nucleic acid sequence of the invention, sequences substantially identical thereto, and the sequences complementary thereto.
  • the isolated, synthetic or recombinant nucleic acid encodes a polypeptide having a enzyme activity, which is thermostable.
  • the polypeptide can retain activity under conditions comprising a temperature range of between about 37 0 C to about 95°C; between about 55 0 C to about 85°C, between about 7O 0 C to about 95°C, or, between about 90 0 C to about 95°C.
  • the isolated or recombinant nucleic acid encodes a polypeptide having an enzyme, structural or binding activity, which is thermotolerant.
  • the polypeptide can retain activity after exposure to a temperature in the range from greater than 37°C to about 95 0 C or anywhere in the range from greater than 55°C to about 85°C.
  • the polypeptide can retain activity after exposure to a temperature in the range between about TC to about 5 0 C, between about 5°C to about 15°C, between about 15°C to about 25°C, between about 25°C to about 37°C, between about 37 0 C to about 95°C, between about 55°C to about 85°C, between about 70 0 C to about 75 0 C, or between about 9O 0 C to about 95 0 C, or more.
  • the polypeptide retains activity after exposure to a temperature in the range from greater than 90 0 C to about 95 0 C at about pH 4.5.
  • the invention provides isolated, synthetic or recombinant nucleic acids comprising a sequence that hybridizes under stringent conditions to a nucleic acid of the invention, e.g., an exemplary nucleic acid of the invention comprising a sequence (or its complement) as set forth in SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, etc. (all of the odd-numbers SEQ ID NO:s set forth in the sequence listing, i.e., SEQ ID NO: 1 through SEQ ID NO: 193), including fragments or subsequences thereof, and complementary sequences thereof.
  • the nucleic acid encodes a polypeptide having an enzyme activity, e.g., a decontamination activity, including having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity.
  • an enzyme activity e.g., a decontamination activity
  • a hydrolase activity e.g., an esterase activity, e.g.
  • the nucleic acid can be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length or the full length of the gene or transcript.
  • the stringent conditions include a wash step comprising a wash in 0.2X SSC at a temperature of about 65 0 C for about 15 minutes.
  • the invention provides a nucleic acid probe, e.g., a probe for identifying a nucleic acid encoding a polypeptide having a enzyme activity, wherein the probe comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more, consecutive bases of a sequence of the invention, or fragments or subsequences thereof, wherein the probe identifies the nucleic acid by binding or hybridization.
  • a nucleic acid probe e.g., a probe for identifying a nucleic acid encoding a polypeptide having a enzyme activity
  • the probe comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,
  • the probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a sequence comprising a sequence of the invention, or fragments or subsequences thereof.
  • the probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a nucleic acid sequence of the invention, or a subsequence thereof.
  • the invention provides an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having an enzyme activity, wherein the primer pair is capable of amplifying a nucleic acid comprising a sequence of the invention, or fragments or subsequences thereof.
  • One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence.
  • the invention provides methods of amplifying a nucleic acid encoding a polypeptide having enzyme activity, comprising amplification of a template nucleic acid with an amplification primer sequence pair capable of amplifying a nucleic acid sequence of the invention, or fragments or subsequences thereof.
  • the invention provides expression cassettes comprising a nucleic acid of the invention or a subsequence thereof.
  • the expression cassette can comprise the nucleic acid that is operably linked to a promoter.
  • the promoter can be a viral, bacterial, mammalian or plant promoter.
  • the plant promoter can be a potato, rice, corn, wheat, tobacco or barley promoter.
  • the promoter can be a constitutive promoter.
  • the constitutive promoter can comprise CaMV35S.
  • the promoter can be an inducible promoter.
  • the promoter can be a tissue- specific promoter or an environmentally regulated or a developmentally regulated promoter.
  • the promoter can be, e.g., a seed-specific, a leaf-specific, a root-specific, a stem-specific or an abscission-induced promoter.
  • the expression cassette can further comprise a plant or plant virus expression vector.
  • the invention provides cloning vehicles comprising an expression cassette (e.g., a vector) of the invention or a nucleic acid of the invention.
  • the cloning vehicle can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome.
  • the viral vector can comprise an adenovirus vector, a retroviral vector or an adeno -associated viral vector.
  • the cloning vehicle can comprise a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage Pl -derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
  • BAC bacterial artificial chromosome
  • PAC bacteriophage Pl -derived vector
  • YAC yeast artificial chromosome
  • MAC mammalian artificial chromosome
  • the invention provides transformed cell comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention, or a cloning vehicle of the invention.
  • the transformed cell can be a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
  • the plant cell can be a potato, wheat, rice, corn, tobacco or barley cell.
  • the invention provides transgenic non-human animals comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention.
  • the animal is a mouse.
  • the invention provides transgenic plants comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention.
  • the transgenic plant can be a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant or a tobacco plant.
  • the invention provides transgenic seeds comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention.
  • the transgenic seed can be rice, a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a peanut or a tobacco plant seed.
  • the invention provides an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid of the invention.
  • the invention provides methods of inhibiting the translation of an enzyme message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid of the invention.
  • the invention provides an isolated, synthetic or recombinant polypeptide comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequence identity to an exemplary polypeptide or peptide of the invention over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600
  • Exemplary polypeptide or peptide sequences of the invention include SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ
  • Exemplary polypeptide or peptide sequences of the invention include sequence encoded by a nucleic acid of the invention.
  • Exemplary polypeptide or peptide sequences of the invention include polypeptides or peptides specifically bound by an antibody of the invention.
  • a polypeptide of the invention has at least one enzyme activity.
  • the activity is a regioselective and/or chemoselective activity.
  • Another aspect of the invention is an isolated, synthetic or recombinant polypeptide or peptide including at least 10 consecutive bases of a polypeptide or peptide sequence of the invention, sequences substantially identical thereto, and the sequences complementary thereto.
  • the enzyme activity can be thermostable.
  • the polypeptide can retain an enzyme activity under conditions comprising a temperature range of between about 37°C to about 95°C, between about 55°C to about 85°C, between about 70 0 C to about 95°C, or between about 90 0 C to about 95°C.
  • the enzyme activity can be thermotolerant.
  • the polypeptide can retain an enzyme activity after exposure to a temperature in the range from greater than 37°C to about 95 0 C, or in the range from greater than 55 0 C to about 85°C.
  • the polypeptide can retain an enzyme activity after exposure to a temperature in the range from greater than 9O 0 C to about 95°C at pH 4.5.
  • the isolated, synthetic or recombinant polypeptide can comprise the polypeptide of the invention that lacks a signal sequence.
  • the isolated or recombinant polypeptide can comprise the polypeptide of the invention comprising a heterologous signal sequence, such as a heterologous enzyme or non-enzyme signal sequence.
  • the invention provides chimeric proteins comprising a first domain comprising a signal sequence of the invention and at least a second domain.
  • the protein can be a fusion protein.
  • the second domain can comprise an enzyme.
  • the enzyme can be an enzyme (e.g., an enzyme of the invention, or, another enzyme).
  • the enzyme activity comprises a specific activity at about 37°C in the range from about 100 to about 1000 units per milligram of protein. In another aspect, the enzyme activity comprises a specific activity from about 500 to about 750 units per milligram of protein. Alternatively, the enzyme activity comprises a specific activity at 37 0 C in the range from about 500 to about 1200 units per milligram of protein. In one aspect, the enzyme activity comprises a specific activity at 37°C in the range from about 750 to about 1000 units per milligram of protein. In another aspect, the thermotolerance comprises retention of at least half of the specific activity of the enzyme at 37 0 C after being heated to the elevated temperature. Alternatively, the thermotolerance can 5 comprise retention of specific activity at 37°C in the range from about 500 to about 1200 units per milligram of protein after being heated to the elevated temperature.
  • the invention provides the isolated, synthetic or recombinant polypeptide of the invention, wherein the polypeptide comprises at least one glycosylation site.
  • glycosylation can be an N-linked glycosylation.
  • the polypeptide o can be glycosylated after being expressed in a P. pas (oris or a S. pombe.
  • the polypeptide can retain an enzyme activity under conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptide can retain an enzyme activity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11. 5
  • the invention provides protein preparations comprising a polypeptide of the invention, wherein the protein preparation comprises a liquid, a solid or a gel.
  • the invention provides heterodimers comprising a polypeptide of the invention and a second domain.
  • the second domain can be a polypeptide and the heterodimer can be a fusion protein.
  • the second domain can be an epitope0 or a tag.
  • the invention provides homodimers comprising a polypeptide of the invention.
  • the invention provides immobilized polypeptides having an enzyme activity, wherein the polypeptide comprises a polypeptide of the invention, a polypeptide encoded by a nucleic acid of the invention, or a polypeptide comprising a polypeptide of the5 invention and a second domain.
  • the polypeptide can be immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
  • the invention provides arrays comprising an immobilized nucleic acid of the invention.
  • the invention provides arrays comprising an antibody of the invention.0
  • the invention provides isolated, synthetic or recombinant antibodies that specifically bind to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
  • the antibody can be a monoclonal or a polyclonal antibody.
  • the invention provides hybridomas comprising an antibody of the invention, e.g., an antibody that specifically binds to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
  • the invention provides food supplements for an animal comprising a polypeptide of the invention, e.g., a polypeptide encoded by the nucleic acid of the invention.
  • the polypeptide in the food supplement can be glycosylated.
  • the invention provides edible enzyme delivery matrices comprising a polypeptide of the invention, e.g., a polypeptide encoded by the nucleic acid of the invention.
  • the delivery matrix comprises a pellet.
  • the polypeptide can be glycosylated.
  • the enzyme activity is thermotolerant. In another aspect, the enzyme activity is thermostable.
  • the invention provides method of isolating or identifying a polypeptide having an enzyme activity comprising the steps of: (a) providing an antibody of the invention; (b) providing a sample comprising polypeptides; and (c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying a polypeptide having an enzyme activity.
  • the invention provides methods of making an anti-enzyme antibody comprising administering to a non-human animal a nucleic acid of the invention or a polypeptide of the invention or subsequences thereof in an amount sufficient to generate a humoral immune response, thereby making an anti-enzyme antibody.
  • the invention provides methods of making an anti-enzyme immune comprising administering to a non-human animal a nucleic acid of the invention or a polypeptide of the invention or subsequences thereof in an amount sufficient to generate an immune response.
  • the invention provides methods of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid of the invention operably linked to a promoter; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide.
  • the method can further comprise transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
  • the invention provides methods for identifying a polypeptide having an enzyme activity comprising the following steps: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing an enzyme substrate; and (c) contacting the polypeptide or a fragment or variant thereof of step (a) with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having an enzyme activity.
  • the invention provides methods for identifying an enzyme substrate comprising the following steps: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as an enzyme substrate.
  • the invention provides methods of determining whether a test compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid comprises a nucleic acid of the invention, or, providing a polypeptide of the invention; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide.
  • the invention provides methods for identifying a modulator of an enzyme activity comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the enzyme, wherein a change in the enzyme activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the enzyme activity.
  • the enzyme activity can be measured by providing an enzyme substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increase in the amount of the substrate or a decrease in the amount of a reaction product.
  • a decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of enzyme activity.
  • An increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of enzyme activity.
  • the invention provides computer systems comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence or a nucleic acid sequence of the invention (e.g., a polypeptide encoded by a nucleic acid of the invention).
  • the computer system can further comprise a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon.
  • the sequence comparison algorithm comprises a computer program that indicates polymorphisms.
  • the computer system can further comprise an identifier that identifies one or more features in said sequence.
  • the invention provides computer readable media having stored thereon a polypeptide sequence or a nucleic acid sequence of the invention.
  • the invention provides methods for identifying a feature in a sequence comprising the steps of: (a) reading the sequence using a computer program which identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence of the invention; and (b) identifying one or more features in the sequence with the computer program.
  • the invention provides methods for comparing a first sequence to a second sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer program which compares sequences, wherein the first sequence comprises a polypeptide sequence or a nucleic acid sequence of the invention; and (b) determining differences between the first sequence and the second sequence with the computer program.
  • the step of determining differences between the first sequence and the second sequence can further comprise the step of identifying polymorphisms.
  • the method can further comprise an identifier that identifies one or more features in a sequence.
  • the method can comprise reading the first sequence using a computer program and identifying one or more features in the sequence.
  • the invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample comprising the steps of: (a) providing an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having an enzyme activity, wherein the primer pair is capable of amplifying a nucleic acid of the invention; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and, (c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the environmental sample, thereby isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample.
  • One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of a sequence of the invention.
  • the invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample comprising the steps of: (a) providing a polynucleotide probe comprising a nucleic acid of the invention or a subsequence thereof; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a); (c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample.
  • the environmental sample can comprise a water sample, a liquid sample, a soil sample, an air sample or a biological sample.
  • the biological sample can be derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.
  • the invention provides methods of generating a variant of a nucleic acid encoding a polypeptide having an enzyme activity comprising the steps of: (a) providing a template nucleic acid comprising a nucleic acid of the invention; and (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid.
  • the method can further comprise expressing the variant nucleic acid to generate a variant enzyme polypeptide.
  • the modifications, additions or deletions can be introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof.
  • GSSM Gene Site Saturation Mutagenesis
  • SLR synthetic ligation reassembly
  • the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair- deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
  • the method can be iteratively repeated until an enzyme having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced.
  • the variant enzyme polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature.
  • the variant enzyme polypeptide has increased glycosylation as compared to the enzyme encoded by a template nucleic acid.
  • the variant enzyme polypeptide has an enzyme activity under a high temperature, wherein the enzyme encoded by the template nucleic acid is not active under the high temperature.
  • the method can be iteratively repeated until an enzyme coding sequence having an altered codon usage from that of the template nucleic acid is produced.
  • the method can be iteratively repeated until an enzyme gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.
  • the invention provides methods for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention encoding a polypeptide having an enzyme activity; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
  • the invention provides methods for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity; the method comprising the following steps: (a) providing a nucleic acid of the invention; and, (b) identifying a codon in the nucleic acid of step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon, thereby modifying codons in a nucleic acid encoding an enzyme.
  • the invention provides methods for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention encoding an enzyme polypeptide; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non- preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
  • the invention provides methods for modifying a codon in a nucleic acid encoding a polypeptide having an enzyme activity to decrease its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention; and (b) identifying at least one preferred codon in the nucleic acid of step (a) and replacing it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in a host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell.
  • the host cell can be a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.
  • the invention provides methods for producing a library of nucleic acids encoding a plurality of modified enzyme active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived from a first nucleic acid comprising a sequence encoding a first active site or a first substrate binding site the method comprising the following steps: (a) providing a first nucleic acid encoding a first active site or first substrate binding site, wherein the first nucleic acid sequence comprises a sequence that hybridizes under stringent conditions to a nucleic acid of the invention, and the nucleic acid encodes an enzyme active site or an enzyme substrate binding site; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the
  • the method comprises mutagenizing the first nucleic acid of step (a) by a method comprising an optimized directed evolution system, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, and a combination thereof.
  • GSSM Gene Site Saturation Mutagenesis
  • SLR synthetic ligation reassembly
  • error-prone PCR shuffling
  • oligonucleotide-directed mutagenesis assembly PCR
  • sexual PCR mutagenesis in vivo mutagenesis
  • cassette mutagenesis cassette mutagenesis
  • recursive ensemble mutagenesis recursive ensemble mutagenesis
  • the method comprises mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modif ⁇ ed DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair- deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
  • the invention provides methods for making a small molecule comprising the following steps: (a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymes comprises an enzyme encoded by a nucleic acid of the invention; (b) providing a substrate for at least one of the enzymes of step (a); and (c) reacting the substrate of step (b) with the enzymes under conditions that facilitate a plurality of biocatalytic reactions to generate a small molecule by a series of biocatalytic reactions.
  • the invention provides methods for modifying a small molecule comprising the following steps: (a) providing an enzyme enzyme, wherein the enzyme comprises a polypeptide of the invention, or, a polypeptide encoded by a nucleic acid of the invention, or a subsequence thereof; (b) providing a small molecule; and (c) reacting the enzyme of step (a) with the small molecule of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the enzyme enzyme, thereby modifying a small molecule by an enzyme enzymatic reaction.
  • the method can comprise a plurality of small molecule substrates for the enzyme of step (a), thereby generating a library of modified small molecules produced by at least one enzymatic reaction catalyzed by the enzyme enzyme.
  • the method can comprise a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurality of enzymatic reactions.
  • the method can further comprise the step of testing the library to determine if a particular modified small molecule which exhibits a desired activity is present within the library.
  • the step of testing the library can further comprise the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by testing the portion of the modified small molecule for the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.
  • the invention provides methods for determining a functional fragment of an enzyme comprising the steps of: (a) providing an enzyme enzyme, wherein the enzyme comprises a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, or a subsequence thereof; and (b) deleting a plurality of amino acid residues from the sequence of step (a) and testing the remaining subsequence for an enzyme activity, thereby determining a functional fragment of an enzyme enzyme.
  • the enzyme activity is measured by providing an enzyme substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product.
  • the invention provides methods for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps: (a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid of the invention; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis.
  • the genetic composition of the cell can be modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene.
  • the method can further comprise selecting a cell comprising a newly engineered phenotype.
  • the method can comprise culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.
  • the invention provides methods of increasing thermo tolerance or thermostability of an enzyme polypeptide, the method comprising glycosylating an enzyme polypeptide, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide of the invention; or a polypeptide encoded by a nucleic acid sequence of the invention, thereby increasing the thermotolerance or thermostability of the polypeptide.
  • the enzyme activity can be thermostable or thermotolerant at a temperature in the range from greater than about 37°C to about 95°C.
  • the invention provides methods for overexpressing a recombinant polypeptide in a cell comprising expressing a vector comprising a nucleic acid comprising a nucleic acid of the invention or a nucleic acid sequence of the invention, wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection, wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.
  • the invention provides detergent compositions comprising a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention, wherein the polypeptide comprises an enzyme activity.
  • the enzyme can be a nonsurface-active enzyme.
  • the enzyme can be a surface-active enzyme.
  • the invention provides methods for washing an object comprising the following steps: (a) providing a composition comprising a polypeptide having an enzyme activity, wherein the polypeptide comprises: a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing an object; and (c) contacting the polypeptide of step (a) and the object of step (b) under conditions wherein the composition can wash the object.
  • the invention provides methods of making a transgenic plant comprising the following steps: (a) introducing a heterologous nucleic acid sequence into the cell, wherein the heterologous nucleic sequence comprises a nucleic acid sequence of the invention, thereby producing a transformed plant cell; and (b) producing a transgenic plant from the transformed cell.
  • the step (a) can further comprise introducing the heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts.
  • the step (a) can further comprise introducing the heterologous nucleic acid sequence directly to plant tissue by DNA particle bombardment.
  • the step (a) can further comprise introducing the heterologous nucleic acid sequence into the plant cell DNA using an Agrobacterium tumefaciens host.
  • the plant cell can be a potato, corn, rice, wheat, tobacco, or barley cell.
  • the invention provides methods of expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps: (a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a nucleic acid of the invention; (b) growing the plant under conditions wherein the heterologous nucleic acids sequence is expressed in the plant cell.
  • the invention provides signal sequences comprising or consisting of a peptide having a subsequence of a polypeptide of the invention, e.g., as described herein.
  • the invention provides a chimeric protein comprising a first domain comprising a signal sequence of the invention and at least a second domain.
  • the protein can be a fusion protein.
  • the second domain can comprise an enzyme.
  • the enzyme can be a hydrolase and/or an oxidoreductase, or enzymes with decontamination activity.
  • the invention provides methods for decreasing the amount of a compound in a composition comprising the following steps: (a) providing a polypeptide having an enzyme activity or encoding a protein of the invention, or a polypeptide encoded by a nucleic acid of the invention; (b) providing a composition comprising the compound; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the enzyme hydrolyzes, dehalogenates, oxidizes, breaks up or otherwise processes the compound in the composition.
  • the invention provides cellulose-comprising compounds comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, and optionally the cellulose-comprising compound comprises a pulp, a paper, a paper product, a wood, a wood product, or a paper or wood waste product.
  • the invention provides methods for chemical bleaching of lignin or a cellulose in a pulp or paper manufacturing process comprising the steps of (a) providing a polypeptide having chloroperoxidase activity of the invention, or a polypeptide having chloroperoxidase activity encoded by a nucleic acid of the invention; (b) providing a compound comprising a lignin or a cellulose; and (c) contacting the compound with the polypeptide under conditions wherein the polypeptide is enzymatically active and the lignin or a cellulose is bleached, wherein optionally the enzymatic activity comprises a heme-based peroxidase activity, and optionally the enzyme having heme-based peroxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 1 1; SEQ ID NO: 13; SEQ ID NO: 15
  • compositions comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the composition is formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a pill, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof, and optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%
  • the invention provides pharmaceutical compositions comprising a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the pharmaceutical composition is formulated as an edible delivery agent, an injectable liquid, a spray, a tablet, a gel, a liposome, a capsule, a geltab, a hydrogel, a lotion, a topical applied liquid, a suppository, an aerosol, a powder, a lyophilized compound, a propellant, a foam, an emulsion, a nanostructure, an implant or a combination thereof; wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
  • the invention provides nanostructures (e.g., nanotubules, nanoimplants) comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the nanostructure comprises a nanotubule or a nanofiber, and optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention.
  • nanostructures e.g., nanotubules, nanoimplants
  • the nanostructure comprises a nanotubule or a nanofiber
  • the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention.
  • the invention provides lyophilized polypeptides having a sequence of the invention, or a polypeptide encoded by a nucleic acid of the invention.
  • the polypeptide is present in the lyophilate at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or more.
  • the invention provides decontaminating, neutralizing or detoxifying compositions comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the decontaminating or detoxifying composition is formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof, wherein optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.
  • the decontaminating, neutralizing or detoxifying compositions further comprises a surfactant, an emulsifier, a foaming agent or a combination thereof.
  • the decontaminating, neutralizing or detoxifying composition can be formulated as a pesticide, herbicide and/or insecticide detoxifying agent, a nerve gas detoxifying agent and the like.
  • the decontaminating, neutralizing or detoxifying composition comprises a haloperoxidase enzyme
  • the composition also comprises halite component, and optionally the halite component comprises a chlorite component, and optionally the chlorite component comprises a sodium chlorite or a sodium iodite.
  • the invention provides products of manufacture comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention.
  • the products of manufacture can comprise a haloperoxidase enzyme, the composition also comprises halite component, and optionally the halite component comprises a chlorite component, and optionally the chlorite component comprises a sodium chlorite or a sodium iodite.
  • the invention provides cloth, clothing, textiles, threads and/or fibers comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the at least one polypeptide is immobilized onto the surface of the cloth, textile, thread or fiber, or the at least one polypeptide is a component of a coating or covering on the surface of the cloth, textile or fiber, or a formulation embedded or washed into or onto the cloth, textile or fiber, wherein optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention.
  • a decontaminating, neutralizing or detoxifying composition comprises a mixture of at least two, three, four, five or six different classes of enzymes, wherein optionally each class of enzyme detoxifies a different toxic agent or biological agent.
  • a decontaminating, neutralizing or detoxifying composition of the invention comprises a mixture of at least three different classes of enzymes comprising at least one dehalogenase, at least one haloperoxidase, and at least one organophosphoric acid anhydrolase (OPAA), and optionally the haloperoxidase is a chloroperoxidase.
  • the dehalogenase is a polypeptide having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively, or the chloroperoxidase is a heme- based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1, or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11;
  • the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO:
  • the decontaminating, neutralizing or detoxifying composition comprises a mixture of at least two different classes of enzymes comprising at least one diisopropylfluorophosphatase (DFPase) or organophosphoric acid anhydrolase (OPAA), and at least one haloperoxidase, and optionally the haloperoxidase is a chloroperoxidase.
  • the composition can comprise a diisopropylfluorophosphatase (DFPase), a organophosphoric acid anhydrolase (OPAA) and a haloperoxidase.
  • the DFPase and/or OPAA can be used to decontaminate, neutralize or detoxify G agents
  • the haloperoxidase or chloroperoxidase (CPO) can be used to decontaminate, neutralize or detoxify V agents, H agents and/or biological agents, wherein optionally the biological agent is a bacterial spore.
  • this decontaminating, neutralizing or detoxifying composition further comprises a dehalogenase, and/or a cholinesterase.
  • the diisopropyl-fluorophosphatase (DFPase) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72.
  • the haloperoxidase is a chloroperoxidase.
  • the chloroperoxidase is a heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 , or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO: 17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31 ; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43;
  • organophosphoric acid anhydrolase (OPAA) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
  • the decontaminating, neutralizing or detoxifying composition comprises a mixture of at least two different classes of enzymes comprising at least one dehalogenase (DH) and at least one haloperoxidase, wherein optionally the haloperoxidase is a chloroperoxidase (CPO).
  • DH dehalogenase
  • CPO chloroperoxidase
  • the dehalogenases and/or haloperoxidases can be used to decontaminate, neutralize or detoxify H agents.
  • the haloperoxidase is a heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1, or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO: 17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ
  • the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO:14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:
  • the haloperoxidase is a non- heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
  • the dehalogenase has a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:
  • this formulation further comprises adding at least one diisopropylfluorophosphatase (DFPase), organophosphoric acid anhydrolase (OPAA) and/or cholinesterase to decontaminate, neutralize or detoxify G agents.
  • DFPase diisopropylfluorophosphatase
  • OPAA organophosphoric acid anhydrolase
  • cholinesterase a diisopropylfluorophosphatase
  • the diisopropylfluorophosphatase (DFPase) can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71 , or has an amino acid sequence as set forth in SEQ ID NO:72.
  • the organophosphoric acid anhydrolase (OPAA) activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
  • the invention provides a decontaminating, neutralizing or detoxifying composition
  • a decontaminating, neutralizing or detoxifying composition comprising a mixture of at least two different classes of enzymes comprising at least one dehalogenase (DH) enzyme and at least one organophosphoric acid anhydrolase (OPAA).
  • the dehalogenases can be used to decontaminate, neutralize or detoxify an H agent
  • the organophosphoric acid anhydrolase can be used to decontaminate, neutralize or detoxify G agents.
  • the composition further comprises at least one haloperoxidase to augment the decontamination, neutralization, detoxification of H agents.
  • the composition further comprises a haloperoxidase, and optionally the composition can also comprise a halite component, wherein optionally the halite component comprises an iodite or a chlorite component; and optionally the iodite or a chlorite component comprises a sodium chlorite or a sodium iodite or equivalent components.
  • the composition further comprises at least one diisopropylfluorophosphatase (DFPase) and/or cholinesterase enzyme to supplement the OPAA decontamination, neutralization or detoxification of a G agent.
  • DFPase diisopropylfluorophosphatase
  • the invention provides foaming agents comprising a decontaminating, neutralizing or detoxifying composition of the invention, wherein optionally an enzyme in the foaming agent is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
  • the invention provides emulsifying agents or surfactants comprising a decontaminating, neutralizing or detoxifying composition of the invention, wherein optionally an enzyme in the foaming agent or surfactant is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
  • the invention provides paints or coatings (including undercoats/ undercoatings, boat hull treatments, and the like) comprising a decontaminating, neutralizing or detoxifying composition of the invention, wherein optionally an enzyme in the paint or coating is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
  • the invention provides methods for decontaminating, neutralizing or detoxifying a toxic agent comprising application or administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention.
  • the composition used in this method can be formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof.
  • the invention provides methods for preventing the toxic effects of a V agent, an H agent, a G agent or a biological agent comprising application or administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention.
  • the invention provides gas masks, air or water filters, and the like, comprising a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention.
  • These filters can be placed, e.g., in ventilation systems and other air flow systems, e.g., on or in buildings, airplanes, boats, cars, trucks, trains, shelters and the like.
  • the invention provides a pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agents comprising a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention, wherein optionally an enzyme in the composition is present at a concentration of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or is present at a w/v of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%.
  • the a pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agents can comprise an organophosphoesterase that hydrolyzes P-S or P-F bonds; and in one aspect, the organophosphoesterase acts as an inhibitor of an acetyl-cholinesterases or a butyrylcholinesterase.
  • the pesticide comprises Demeton-S, Demeton-S- methyl, Demeton-S-methylsulphon, Demeton-methyl, Parathion, Phosmet,
  • the pesticide decontaminating, neutralizing or detoxifying agent can be formulated as or with a coating, a paint, a foam, a liquid, a gel, a lotion, a surfactant, a powder or an emulsifier.
  • the invention provides methods for decontaminating, neutralizing or detoxifying a pesticide-, herbicide- or insecticide-comprising composition or application, or methods for administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agent of the invention.
  • the invention provides methods for preventing the toxic effects of a pesticide, herbicide and/or insecticide comprising application or administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition a of the invention, or the pesticide decontaminating, neutralizing or detoxifying agent of the invention.
  • Figure 1 is a block diagram of a computer system.
  • Figure 2 is a flow diagram illustrating one aspect of a process for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
  • Figure 3 is a flow diagram illustrating one aspect of a process in a computer for determining whether two sequences are homologous.
  • Figure 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence.
  • Figure 5 is an illustration of an anti-CPO antibody western blot of various Pichia and Saccharomyces transformants, as discussed in detail in Example 2, below.
  • Figure 6 illustrates data from a study of tetriso degradation in concentrated sodium chlorite (without any enzyme);
  • Figure 6 A illustrates tetriso levels over time;
  • Figure 6C illustrates chlorite concentration over time;
  • Figure 6D illustrates ClO 2 production over time, and
  • Figure 6B illustrates pH over time, as discussed in detail in Example 2, below.
  • Figure 7 illustrates data from a study of bromide ion formation from dehalogenase-catalyzed hydrolysis of dibromomethane, which was assayed as a function of enzyme concentration in various buffers, as discussed in detail in Example 2, below.
  • Figure 8 illustrates data from a study showing the rate of fluoride produced by DFPase-catalyzed hydrolysis of DFP as a function of DFPase concentration in various buffers, as discussed in detail in Example 2, below.
  • Figure 9 illustrates data from a study showing the capacity of various buffers to resist pH change after titration with acid, as discussed in detail in Example 2, below.
  • Figures 10 and 11 illustrate data from a study showing the rate of VX chemical hydrolysis as measured at different NaOH concentrations, i.e., using either 10 ⁇ M or 0.5 mM VX, respectively, as discussed in detail in Example 2, below.
  • Figure 12 illustrate data from a study describing the concentration dependence of BChE inhibition by VX and GF using either RB or butyrylthiocholine (BTC) as substrates, as discussed in detail in Example 2, below.
  • Figure 13 illustrates studies describing CPO-generated oxidizing radicals concentrations as determined by DPD assay, as discussed in detail in Example 2, below.
  • Figure 14 illustrates studies describing the efficacy of attenuated B. anthracis spores inactivation by CPO-generated oxidizing radicals, as discussed in detail in Example 2, below.
  • Figures 15A and 15B illustrate data displaying the kinetics of VX degradation by environmental library samples, as discussed in detail in Example 3, below.
  • Figures 16A and 16B illustrate data displaying the kinetics of Tetriso degradation by these environmental library samples, as discussed in detail in Example 3, below.
  • Figure 17 illustrates data displaying the kinetics of VX degradation by all nine
  • Figure 18 illustrates an anti-C fumago CPO immunoblot of various samples of a nucleic acid (SEQ ID NO: 1) encoding a C. fumago chloroperoxidase (CPO) (SEQ ID NO:2) subcloned into Saccharomyces, as discussed in detail in Example 3, below.
  • Figures 19A and 19B illustrate Matrix Assisted Laser Desorption Time of Flight
  • Mass Spectrometry (MALDI-TOFMS) analyses performed on samples of C. fumago chloroperoxidase (SEQ ID NO:2), as discussed in detail in Example 3, below.
  • Figure 2OA illustrates electrospray ionization coupled with a quadrupole time of flight mass spectrometry (ESI-QTOFMS) instrument to perform tandem mass spectrometry to determine the sequence of peptides from the C. fumago chloroperoxidase that were changing in abundance between the untreated and treated samples, as illustrated in Figure 2OA; and peaks which disappeared were selected for tandem mass spectrometry in the untreated sample to determine their sequence, as illustrated in Figure 2OB, as discussed in detail in Example 3, below.
  • ESI-QTOFMS quadrupole time of flight mass spectrometry
  • Figure 21 illustrates peptides mapped to the CPO 3D structure using electrospray ionization coupled with a quadrupole time of flight mass spectrometry (ESI-QTOFMS) were are on the outside of the molecule, as discussed in detail in Example 3, below.
  • ESI-QTOFMS quadrupole time of flight mass spectrometry
  • Figure 22 illustrates data showing Tetriso degradation by various concentrations of CPO in 320 mM chlorite and 300 mM sodium phosphate buffer, as discussed in detail in Example 3, below.
  • Figure 23 illustrates bromide ion formation from dehalogenase-catalyzed hydrolysis of dibromoethane as a function of enzyme concentration in various buffers, as discussed in detail in Example 3, below.
  • Figure 24 illustrates the rate of fluoride produced by DFPase catalyzed hydrolysis of DFP as a function of DFPase concentration in various buffers, as discussed in detail in Example 3, below.
  • Figure 25 illustrates corrosion studies comparing chlorine dioxide with exemplary enzymatic decon formulations (DFPase, Chloroperoxidase, 25 mM sodium chlorite), and shows the enzymatic decontaminant to be less corrosive to metals, as discussed in detail in Example 4, below.
  • DFPase Chloroperoxidase
  • 25 mM sodium chlorite exemplary enzymatic decon formulations
  • Figure 26 illustrates studies regarding chloroperoxidase and oxidative decontamination, as discussed in detail in Example 4, below.
  • Figure 27 illustrates studies regarding degradation of tetriso by chlorine dioxide and sodium chlorite, as discussed in detail in Example 4, below.
  • Figure 28 illustrates studies regarding degradation of tetriso by chloroperoxidase, NaClO 2 and ClO 2 , as discussed in detail in Example 4, below.
  • Figure 29 illustrates studies regarding inactivation of B. subtilis var Niger by sodium chlorite and bioxidation, as discussed in detail in Example 4, below.
  • Figure 30 illustrates studies demonstrating inactivation of B. subtilis var Niger spores by sodium chlorite and bioxidation using chloroperoxidase or horseradish peroxidase, as discussed in detail in Example 4, below.
  • Figure 31 illustrates studies demonstrating the stability of chloroperoxidase in buffer, in NaClO 2 , in NaClO 2 with tetriso, and in ClO 2 , as discussed in detail in Example 4, below.
  • Figure 32 illustrates studies demonstrating the stability of horseradish peroxidase in buffer, in NaClO 2 , in NaClO 2 with Tetriso, and in ClO 2 , as discussed in detail in Example 4, below.
  • Figure 33 illustrates studies demonstrating Tetriso degradation by CPO in phosphate buffer with NaClO 2 , as discussed in detail in Example 4, below.
  • Figure 34 illustrates studies demonstrating Tetriso degradation by phosphate buffer with NaClO 2 , as discussed in detail in Example 4, below.
  • Figure 35 illustrates studies demonstrating Tetriso degradation by CPO phosphate buffer) with 160 mM, as discussed in detail in Example 4, below.
  • Figure 36 illustrates studies demonstrating a decrease in ClO 2 absorbance upon the addition of CPO, as discussed in detail in Example 4, below.
  • Figure 37 illustrates studies demonstrating the kinetics of ClO 2 degradation upon addition of CPO or an equivalent weight of BSA, as discussed in detail in Example 4, below.
  • Figure 38 illustrates two photos of blue-tempered spring steel by enzyme-based decontamination solution (left panel) and 340 ppm chlorine dioxide (right panel), as discussed in detail in Example 4, below.
  • Figure 39 illustrates studies demonstrating the tm for sulfur mustard (HD) degradation by the exemplary enzyme of the invention SEQ ID NO:70, as discussed in detail in Example 4, below.
  • HD sulfur mustard
  • Figure 40 illustrates data showing the residual percent sulfur mustard (HD) following enzymatic degradation by the exemplary SEQ ID NO: 70 at various enzyme (DHG) concentrations, as discussed in detail in Example 4, below.
  • Figure 41 illustrates data showing the residual percent sulfur mustard (HD) following enzymatic degradation by the exemplary SEQ ID NO: 70 at various sulfur mustard (HD) concentrations, as discussed in detail in Example 4, below.
  • Figure 42 illustrates data showing the degradation of sulfur mustard (HD by the exemplary SEQ ID NO:70 with polyethyleneglycol-400 (PEG-400), as discussed in detail in Example 4, below.
  • Figure 43 illustrates data displaying that CPOZNaClO 2 causes >99% degradation of sulfur mustard (HD) within 1 minute (min), and illustrates data displaying the time- dependence of HD degradation by the exemplary SEQ ID NO: 70, as discussed in detail in Example 4, below.
  • Figure 44 illustrates data showing the degradation of HD by the exemplary SEQ ID NO:70 at various conditions and times, as discussed in detail in Example 4, below.
  • Figure 45 illustrates data showing the exemplary dehalogenase (DHG) SEQ ID NO:70 was an active hydrolyzing enzyme for degradation of HD-SO, and illustrates the time course of_enzymatic degradation of HD-SO by the exemplary SEQ ID NO: 70, at various conditions and times, as discussed in detail in Example 4, below.
  • DHG dehalogenase
  • Figure 46 illustrates the time-course of VX degradation by C. fitmago chloroperoxidase (CPO) (SEQ ID NO:2) at low activity level
  • Figure 46A shows data generated at VX 10 ⁇ M, NaClO 2 0.03 M
  • Figure 46B shows data generated at VX 1 ⁇ M, NaClO 2 0.02 M, Phosphate 5OmM, pH 7.5, 25 0 C, as discussed in detail in Example 4, below.
  • Figure 47 describes the kinetics of VX degradation measured in 96-well plates containing lysate samples at specified time intervals, as discussed in detail in Example 4, below.
  • Figure 48 illustrates data showing "Tetriso hits” (enzymes that hydrolyze Tetriso) discovered by the robotic screening assay as discussed in detail in Example 4, below.
  • Figure 49 and Figure 50 summarize data of all VX and Tetriso degradation kinetics for all library "hits" discovered by the robotic screening assay as discussed in detail in Example 4, below.
  • Figure 51 summarizes data indicating that VX oxidation by CPO/NaClCh as well as GF (cyclohexyl sarin) and DFP hydrolysis by DFPase in 0.2 M phosphate proceed to completion at a relatively rapid rate, whereas VX degradation in 0.2 M AC is slow and incomplete, as discussed in detail in Example 4, below.
  • Figure 52 illustrates photos of carbon steel exposed to levels of chlorine dioxide "normally” generated when practicing and using these haloperoxidase/ chlorite component decontamination (decon) formulations of the invention, as discussed in detail in Example 4, below.
  • Figure 53 illustrates an exemplary study showing that the exemplary CPO of the invention is effective in decontaminating bacterial spores, as discussed in detail in Example 4, below.
  • Figure 54 illustrates data from a time-course detoxification study using an exemplary OPAA enzyme of the invention, as discussed in detail in Example 7, below.
  • Figure 55 illustrates data from a gas chromatograph analysis of the time- course detoxification study illustrated in Figure 54, as discussed in detail in Example 7, below.
  • Figure 56 illustrates data from a time-course detoxification study of HD using an exemplary dehalogenase of the invention, as discussed in detail in Example 8, below.
  • Figure 57 illustrates data from a time-course detoxification study of VX using an exemplary CPO enzyme of the invention, as discussed in detail in Example 9, below.
  • polypeptides including peptides, enzymes and antibodies, having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides.
  • an organophosphohydrolase activity such as an organophosphoesterase activity
  • the invention provides mixtures/ formulations/ combinations of enzymes of the invention, and in some aspects, these mixtures/ formulations/ combinations also comprise known enzymes, for decontamination or detoxification (including neutralization) activity.
  • the invention is directed to polypeptides, e.g., enzymes, having any of these activities (e.g., hydrolase, dehalogenase, haloperoxidase, oxidoreductase activity, organophosphoric acid anhydrolase, organophosphohydrolase, etc.) including enzymes having thermostable and thermotolerant enzyme activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides.
  • enzymes having any of these activities (e.g., hydrolase, dehalogenase, haloperoxidase, oxidoreductase activity, organophosphoric acid anhydrolase, organophosphohydrolase, etc.) including enzymes having thermostable and thermotolerant enzyme activity,
  • polypeptides of the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts, including active decontamination.
  • the polypeptides of the invention are used to synthesize enantiomerically pure chiral products.
  • the polypeptides of the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts, including active decontamination.
  • the invention provides polypeptides (including enzymes, peptides, antibodies of the invention), and novel mixture/ formulation/ combinations of enzymes (including polypeptides of the invention, known enzymes, or a mixture thereof) for decontamination or detoxification (which includes neutralization), e.g., of toxins such as nerve agents, e.g., V agents (VX agent), G agents (sarin, soman, cyclosarin) or H agents (e.g., mustard gases) and/or biological agents (e.g., anthrax spores), for civilian, military and/or homeland security purposes.
  • Enzymes of the invention can be highly selective catalysts.
  • Enzymes of the invention can be versatile. In various aspects, they can function in organic solvents, operate at extreme pHs (for example, high pHs and low pHs) extreme temperatures (for example, high temperatures and low temperatures), extreme salinity levels (for example, high salinity and low salinity), and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates.
  • extreme pHs for example, high pHs and low pHs
  • extreme temperatures for example, high temperatures and low temperatures
  • extreme salinity levels for example, high salinity and low salinity
  • the invention provides nucleic acids, including expression cassettes such as expression or cloning vectors, encoding the polypeptides (e.g., enzymes, peptides, antibodies) of the invention.
  • the invention also includes methods for discovering new enzyme sequences using the nucleic acids of the invention.
  • methods for modifying the nucleic acids of the invention by, e.g., synthetic ligation reassembly, optimized directed evolution system and/or saturation mutagenesis.
  • nucleic acids capable of inhibiting expression of enzymes including for example antisense or RNAi (double-stranded "interfering" RNA, including miRNA or iRNA) having a sequence of the invention.
  • nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.
  • homologous genes can be modified by manipulating a template nucleic acid, as described herein.
  • the invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.
  • RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly.
  • Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc.
  • nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VoIs. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
  • Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
  • Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet.
  • MACs mammalian artificial chromosomes
  • yeast artificial chromosomes YAC
  • bacterial artificial chromosomes BAC
  • Pl artificial chromosomes see, e.g., Woon (1998) Genomics 50:306-316
  • Pl-derived vectors see, e.g., Kern (1997) Biotechniques 23:120- 124; cosmids, recombinant viruses, phages or plasmids.
  • a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.
  • the invention provides fusion proteins and nucleic acids encoding them.
  • a polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
  • Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-414).
  • histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
  • Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
  • isolated can mean that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
  • Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
  • the invention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.
  • nucleic acid can mean that the nucleic acid is adjacent to a "backbone” nucleic acid to which it is not adjacent in its natural environment.
  • nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules.”
  • Backbone molecules include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
  • the enriched nucleic acids represent 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
  • “Recombinant” polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
  • synthetic polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below.
  • a "coding sequence of or a "sequence encodes" a particular polypeptide or protein is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences.
  • gene can include a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability.
  • Genes can include, inter alia, regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
  • nucleic acid or “nucleic acid sequence” can include an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA, RNAi) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., RNAi (double-stranded "interfering" RNA), ribonucleoproteins (e.g., iRNPs).
  • DNA or RNA e.g., mRNA, rRNA, tRNA, RNAi
  • PNA peptide nucleic acid
  • PNA peptide nucleic acid
  • DNA-like or RNA-like material natural or synthetic in origin, including, e
  • nucleic acids i.e., oligonucleotides, containing known analogues of natural nucleotides.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692- 8698; Straussense Nucleic Acid Drug Dev 6:153-156.
  • Oligonucleotide can include either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • the invention provides nucleic acid (e.g., DNA, iRNA) sequences of the invention operatively linked to expression (e.g., transcriptional or translational) control sequence(s), e.g., promoters or enhancers, to direct or modulate RNA synthesis/ expression.
  • expression control sequence can be in an expression vector.
  • Exemplary bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp.
  • Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I.
  • a promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
  • promoter includes all sequences capable of driving transcription of a coding sequence in a cell, e.g., a plant cell.
  • promoters used in the constructs of the invention include crs-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
  • a promoter can be a c/s-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
  • Constutive promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation.
  • “Inducible” or “regulatable” promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions.
  • a promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
  • tissue-specific promoters are transcriptional control elements that are only active in particular cells or tissues or organs, e.g., in plants or animals. Tissue-specific regulation may be achieved by certain intrinsic factors which ensure that genes encoding proteins specific to a given tissue are expressed. Such factors are known to exist in mammals and plants so as to allow for specific tissues to develop.
  • plant includes whole plants, plant parts (e.g., leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and plant cells and progeny of same.
  • the class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous states.
  • the term "transgenic plant” includes plants or plant cells into which a heterologous nucleic acid sequence has been inserted, e.g., the nucleic acids and various recombinant constructs (e.g., expression cassettes) of the invention.
  • Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lad promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter.
  • Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metallothionein-I promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses may also be used.
  • the invention provides expression cassettes that can be expressed in a tissue- specific manner, e.g., that can express an enzyme of the invention in a tissue-specific manner.
  • the invention also provides plants or seeds that express an enzyme of the invention in a tissue-specific manner.
  • the tissue-specificity can be seed specific, stem specific, leaf specific, root specific, fruit specific and the like.
  • a constitutive promoter such as the CaMV 35S promoter can be used for expression in specific parts of the plant or seed or throughout the plant.
  • a plant promoter fragment can be employed which will direct expression of a nucleic acid in some or all tissues of a plant, e.g., a regenerated plant.
  • constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or T- promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill.
  • Such genes include, e.g., ACTIl from Arabidopsis (Huang (1996) Plant MoI. Biol. 33: 125-139); Ca ⁇ from Arabidopsis (GenBank No. U43147, Zhong (1996) MoI. Gen. Genet. 251:196-203); the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe ( 1994) Plant Physiol. 104: 1 167-1176); GPcI from maize (GenBank No. Xl 5596; Martinez (1989) J. MoI. Biol 208:551-565); the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant MoI. Biol. 33:97-112); plant promoters described in U.S. Patent Nos. 4,962,028;
  • tissue-specific or constitutive promoters derived from viruses which can include, e.g., the tobamo virus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92: 1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer (1996) Plant MoI. Biol. 31: 1129-1139).
  • viruses can include, e.g., the tobamo virus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92: 1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells
  • the plant promoter may direct expression of an enzyme-expressing nucleic acid in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control or under the control of an inducible promoter.
  • tissue-specific promoters examples include anaerobic conditions, elevated temperature, the presence of light, or sprayed with chemicals/hormones.
  • the invention incorporates the drought- inducible promoter of maize (Busk (1997) supra); the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant MoI. Biol. 33:897 909).
  • Tissue-specific promoters can promote transcription only within a certain time frame of developmental stage within that tissue. See, e.g., Blazquez (1998) Plant Cell 10:791-800, characterizing the Arabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77, describing the transcription factor SPL3, which recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter eIF4. Tissue specific promoters which are active throughout the life cycle of a particular tissue can be used.
  • the nucleic acids of the invention are operably linked to a promoter active primarily only in cotton fiber cells. In one aspect, the nucleic acids of the invention are operably linked to a promoter active primarily during the stages of cotton fiber cell elongation, e.g., as described by Rinehart (1996) supra.
  • the nucleic acids can be operably linked to the Fbl2A gene promoter to be preferentially expressed in cotton fiber cells (Ibid). See also, John (1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Patent Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promoters and methods for the construction of transgenic cotton plants.
  • Root-specific promoters may also be used to express the nucleic acids of the invention.
  • Examples of root-specific promoters include the promoter from the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol. 123:39-60).
  • Other promoters that can be used to express the nucleic acids of the invention include, e.g., ovule-specific, embryo-specific, endosperm-specific, integument- specific, seed coat-specific promoters, or some combination thereof; a leaf-specific promoter (see, e.g., Busk (1997) Plant J.
  • a pistil-specific promoter from the potato SK2 gene see, e.g., Ficker (1997) Plant MoI. Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots or fibers; the ovule- specific BELl gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBankNo. U39944); and/or, the promoter in Klee, U.S. Patent No. 5,589,583, describing a plant promoter region is capable of conferring high levels of transcription in meristematic tissue and/or rapidly dividing cells.
  • plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids of the invention.
  • the invention can use the auxin-response elements El promoter fragment (AuxREs) in the soybean ⁇ Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) MoI. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
  • auxin-response elements El promoter fragment AuxREs
  • the nucleic acids of the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem.
  • Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g.
  • transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324).
  • chemically- (e.g., hormone- or pesticide-) induced promoters i.e., promoter responsive to a chemical which can be applied to the transgenic plant in the field
  • expression of a polypeptide of the invention can be induced at a particular stage of development of the plant.
  • the invention also provides for transgenic plants containing an inducible gene encoding for polypeptides of the invention whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, inducible at any stage of development of the crop.
  • Tissue-specific plant promoters may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.
  • the nucleic acids of the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents.
  • These reagents include, e.g., herbicides, synthetic auxins, or antibiotics which can be applied, e.g., sprayed, onto transgenic plants.
  • Inducible expression of the enzyme-producing nucleic acids of the invention will allow the grower to select plants with the optimal starch / sugar ratio. The development of plant parts can thus controlled. In this way the invention provides the means to facilitate the harvesting of plants and plant parts.
  • the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners, is used (De Veylder ( 1997) Plant Cell Physiol.
  • Coding sequences of the invention are also under the control of a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 1 1: 1315-1324).
  • polyadenylation region at the 3 '-end of the coding region should be included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from genes in the Agrobacterial T-DNA.
  • the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the enzymes and antibodies of the invention.
  • Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), Pl -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as
  • Vectors of the invention can include chromosomal, non- chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Exemplary vectors are include: bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda- ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia);
  • Eukaryotic pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).
  • any other plasmid or other vector may be used so long as they are replicable and viable in the host.
  • Low copy number or high copy number vectors may be employed with the present invention.
  • the term "expression cassette" comprises a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as an enzyme of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers. "Operably linked” as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.
  • expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
  • a "vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell.
  • a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Patent No.
  • the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
  • the expression vector may comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • Mammalian expression vectors can comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
  • DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • the expression vectors contain one or more selectable marker genes to permit selection of host cells containing the vector.
  • selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRPl gene.
  • Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers.
  • Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells may also contain enhancers to increase expression levels.
  • Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers.
  • a DNA sequence may be inserted into a vector by a variety of procedures.
  • the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases.
  • blunt ends in both the insert and the vector may be ligated.
  • a variety of cloning techniques are known in the art, e.g., as described in Ausubel and Sambrook.
  • the vector may be in the form of a plasmid, a viral particle, or a phage.
  • Other vectors include chromosomal, non-chromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • a variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by, e.g., Sambrook.
  • Particular bacterial vectors which may be used include the commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEMl (Promega Biotec, Madison, WI, USA) pQE70, pQE60, pQE-9 (Qiagen), pDIO, psiX174 pBluescript II KS, pNH8A, pNHl ⁇ a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233- 3, DR540, pRIT5 (Pharmacia), pKK232-8 and pCM7.
  • Particular eukaryotic vectors include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
  • any other vector may be used as long as it is replicable and viable in the host cell.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses and transiently or stably expressed in plant cells and seeds.
  • One exemplary transient expression system uses episomal expression systems, e.g., cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus by transcription of an episomal mini-chromosome containing supercoiled DNA, see, e.g., Covey (1990) Proc. Natl. Acad. Sci. USA 87:1633-1637.
  • coding sequences, i.e., all or sub-fragments of sequences of the invention can be inserted into a plant host cell genome becoming an integral part of the host chromosomal DNA.
  • Sense or antisense transcripts can be expressed in this manner.
  • a vector comprising the sequences (e.g., promoters or coding regions) from nucleic acids of the invention can comprise a marker gene that confers a selectable phenotype on a plant cell or a seed.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.
  • Expression vectors capable of expressing nucleic acids and proteins in plants are well known in the art, and can include, e.g., vectors from Agrohacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J. 16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene 173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology 169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology 234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993) Microbiol Immunol.
  • cauliflower mosaic virus see, e.g., Cecchini (1997) MoI. Plant Microbe Interact. 10:1094-1101
  • maize Ac/Ds transposable element see, e.g., Rubin (1997) MoI. Cell. Biol. 17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194)
  • Spm maize suppressor-mutator
  • the expression vector can have two replication systems to allow it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector can contain at least one sequence homologous to the host cell genome. It can contain two homologous sequences which flank the expression construct.
  • the integrating vector can be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
  • Expression vectors of the invention may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.
  • selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
  • the invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding an enzyme or an antibody of the invention, or a vector of the invention.
  • the host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Enzymes of the invention can be expressed in any host cell, e.g., any bacterial cell, any yeast cell, e.g., Pichia pastoris, Saccharomyces cerevisiae or Schizosaccharomyces pombe. Exemplary bacterial cells include E.
  • coli Lactococcus lactis, Streptomyces , Bacillus subtilis, Bacillus cereus, Salmonella typhimurium or any species within the genera Bacillus, Streptomyces and Staphylococcus.
  • exemplary insect cells include Drosophila S 2 and Spodoptera Sf? .
  • Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477, U.S. Patent No. 5,750,870.
  • the vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention.
  • the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
  • the nucleic acids or vectors of the invention are introduced into the cells for screening, thus, the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid.
  • the method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO 4 precipitation, liposome fusion, lipofection (e.g., LIPOFECTINTM), electroporation, viral infection, etc.
  • the candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfecting such targets are preferred.
  • Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
  • Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
  • the expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
  • Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated. Polypeptides of the invention may or may not also include an initial methionine amino acid residue.
  • Cell-free translation systems can also be employed to produce a polypeptide of the invention. Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof. In some aspects, the DNA construct may be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
  • an appropriate cell-free translation extract such as a rabbit reticulocyte extract
  • the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • nucleic acids encoding the polypeptides of the invention, or modified nucleic acids can be reproduced by, e.g., amplification.
  • the invention provides amplification primer sequence pairs for amplifying nucleic acids encoding an enzyme, where the primer pairs are capable of amplifying nucleic acids of the invention, including the exemplary SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO.13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO.25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO.33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:
  • Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an array or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample.
  • message isolated from a cell or a cDNA library are amplified.
  • the skilled artisan can select and design suitable oligonucleotide amplification primers.
  • Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241 : 1077; Barringer (1990) Gene 89: 117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl.
  • PCR see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), e
  • the invention also provides amplification primer pairs comprising sequences of the invention, for example, wherein the primer pair comprises a first member having a sequence as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more residues of a nucleic acid of the invention, and a second member having a sequence as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more residues of the complementary strand of the first member. Determining the degree of sequence identity
  • the invention provides nucleic acids having various sequence identities (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to exemplary nucleic acids of the invention, including having complete (100%) sequence identity to a nucleic acid of the invention, e.g., an exemplary nucleic acid of the invention (e.g., having a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO
  • sequence identity can be over a region of at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive residues, or the full length of the nucleic acid or polypeptide.
  • the extent of sequence identity (homology) may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.
  • substantially identical in the context of two nucleic acids or polypeptides, can refer to two or more sequences that have, e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, nucleotide or amino acid residue (sequence) identity, when compared and aligned for maximum correspondence, as measured using one any known sequence comparison algorithm, as discussed in detail below, or by visual inspection.
  • nucleotide or amino acid residue (sequence) identity when
  • the invention provides nucleic acid and polypeptide sequences having substantial identity to a nucleic acid of the invention, e.g., an exemplary sequence of the invention, over a region of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 residues, or a region ranging from between about 50 residues to the full length of the nucleic acid or polypeptide.
  • Nucleic acid sequences of the invention can be substantially identical over the entire length of a polypeptide coding region.
  • substantially identical amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.
  • a conservative amino acid substitution for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).
  • One or more amino acids can be deleted, for example, from an enzyme, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for enzyme activity can be removed.
  • Homologous sequences also include RNA sequences in which uridines replace the thymines in the nucleic acid sequences.
  • the homologous sequences may be obtained using any of the procedures described herein or may result from the correction of a sequencing error. It will be appreciated that the nucleic acid sequences as set forth herein can be represented in the traditional single character format (see, e.g., Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) or in any other format which records the identity of the nucleotides in a sequence.
  • sequence comparison programs identified herein are used in this aspect of the invention. Protein and/or nucleic acid sequence identities (homologies) may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. MoI. Biol. 215(3):403- 410, 1990; Thompson et al, Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. MoI. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Columbia
  • sequence comparison refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence comparison one sequence can act as a reference sequence (e.g., an exemplary nucleic acid or polypeptide sequence of the invention) to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the numbers of contiguous residues.
  • continugous residues ranging anywhere from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention, are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the reference sequence has the requisite sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, e.g., in alternative aspects, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, that sequence is within the scope of the invention.
  • subsequences ranging from about 20 to 600, about 50 to 200, and about 100 to 150 are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequence for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443, 1970, by the search for similarity method of person & Lipman, Proc. Nat'l. Acad. Sci.
  • Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences.
  • a number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (Gibbs, 1995).
  • Several genomes have been sequenced, e.g., M. genitalium (Fraser et al., 1995), M. jannaschii (BuIt et al., 1996), H. influenzae (Fleischmann et al., 1995), E. coli
  • BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389-3402; Altschul (1990) J. MoI. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990) supra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc.
  • BLAST Basic Local Alignment Search Tool
  • five specific BLAST programs can be used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six- frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and, (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
  • the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256: 1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993).
  • the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).
  • the NCBI BLAST 2.2.2 programs is used, default options to blastp. There are about 38 setting options in the BLAST 2.2.2 program. In this exemplary aspect of the invention, all default values are used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a "-F F" setting is used, which disables filtering. Use of default filtering often results in Karlin-Altschul violations due to short length of sequence.
  • the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention.
  • the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
  • the terms "computer,” “computer program” and “processor” are used in their broadest general contexts and incorporate all such devices, as described in detail, below.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
  • a computer system 100 refers to the hardware components, software components, and data storage components used to analyze a nucleotide or polypeptide sequence of the invention.
  • the computer system 100 can include a processor for processing, accessing and manipulating the sequence data.
  • the processor 105 can be any well-known type of central processing unit, such as, for example, the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq, AMD or International Business Machines.
  • the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
  • the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
  • a skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.
  • the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon.
  • the computer system 100 can further include one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110.
  • the data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or a modem capable of connection to a remote data storage system (e.g., via the internet) etc.
  • the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon.
  • the computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.
  • the computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100.
  • Software for accessing and processing the nucleotide or amino acid sequences of the invention can reside in main memory 115 during execution.
  • the computer system 100 may further comprise a sequence comparison algorithm for comparing a nucleic acid sequence of the invention.
  • the algorithm and sequence(s) can be stored on a computer readable medium.
  • a "sequence comparison algorithm" refers to one or more programs which are implemented (locally or remotely) on the computer system 100 to compare a nucleotide sequence with other nucleotide sequences and/or compounds stored within a data storage means.
  • the sequence comparison algorithm may compare the nucleotide sequences of the invention stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies or structural motifs.
  • FIG. 2 is a flow diagram illustrating one aspect of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
  • the database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK that is available through the Internet.
  • the process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100.
  • the memory could be any type of memory, including RAM or an internal storage device.
  • the process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison.
  • the process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer.
  • a comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database.
  • the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered.
  • the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence.
  • one aspect of the invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid sequence of the invention and a sequence comparer for conducting the comparison.
  • the sequence comparer may indicate a homology level between the sequences compared or identify structural motifs, or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes.
  • Figure 3 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.
  • the process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory.
  • the second sequence to be compared is then stored to a memory at a state 256.
  • the process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read.
  • the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U.
  • sequence is a protein sequence, then it can be a single letter amino acid code so that the first and sequence sequences can be easily compared.
  • a determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read.
  • the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user.
  • the level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with an every character in a second sequence, the homology level would be 100%.
  • the computer program can compare a reference sequence to a sequence of the invention to determine whether the sequences differ at one or more positions.
  • the program can record the length and identity of inserted, deleted or substituted nucleotides or amino acid residues with respect to the sequence of either the reference or the invention.
  • the computer program may be a program which determines whether a reference sequence contains a single nucleotide polymorphism (SNP) with respect to a sequence of the invention, or, whether a sequence of the invention comprises a SNP of a known sequence.
  • the computer program is a program which identifies SNPs.
  • the method may be implemented by the computer systems described above and the method illustrated in Figure 3. The method can be performed by reading a sequence of the invention and the reference sequences through the use of the computer program and identifying differences with the computer program.
  • the computer based system comprises an identifier for identifying features within a nucleic acid or polypeptide of the invention.
  • An "identifier" refers to one or more programs which identifies certain features within a nucleic acid sequence.
  • an identifier may comprise a program which identifies an open reading frame (ORF) in a nucleic acid sequence.
  • Figure 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence. The process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100.
  • a database of sequence features is opened.
  • a database would include a list of each feature's attributes along with the name of the feature.
  • a feature name could be "Initiation Codon” and the attribute would be "ATG”.
  • Another example would be the feature name "TAATAA Box” and the feature attribute would be "TAATAA”.
  • An example of such a database is produced by the University of Wisconsin Genetics Computer Group.
  • the features may be structural polypeptide motifs such as alpha helices, beta sheets, or functional polypeptide motifs such as enzymatic active sites, helix-turn- helix motifs or other motifs known to those skilled in the art.
  • the process 300 moves to a state 308 wherein the first feature is read from the database.
  • a comparison of the attribute of the first feature with the first sequence is then made at a state 310.
  • a determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name of the found feature is displayed to the user.
  • the process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324.
  • the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence. If the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.
  • the invention provides a computer program that identifies open reading frames (ORFs).
  • a polypeptide or nucleic acid sequence of the invention may be stored and manipulated in a variety of data processor programs in a variety of formats.
  • a sequence can be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE.
  • many computer programs and databases may be used as sequence comparison algorithms, identifiers, or sources of reference nucleotide sequences or polypeptide sequences to be compared to a nucleic acid sequence of the invention.
  • the programs and databases used to practice the invention include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. MoI. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci.
  • Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
  • the invention provides isolated, synthetic or recombinant nucleic acids that hybridize under various stringent conditions to a nucleic acid of the invention, e.g., an exemplary sequence of the invention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, etc., and subsequences thereof (which include complementary sequences), or a nucleic acid that encodes a polypeptide of the invention.
  • the stringent conditions can be highly stringent conditions, medium stringent conditions, low stringent conditions, including the high and reduced stringency conditions described herein.
  • Hybridization refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below. In alternative aspects, nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein.
  • stringency conditions e.g., high, medium, and low
  • nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of nucleic acid of the invention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues in length. Nucleic acids shorter than full length are also included.
  • nucleic acids of the invention can be useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprises conditions of about 50% formamide at about 37°C to 42°C.
  • nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 3O 0 C to 35°C.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42 0 C in 50% formamide, 5X SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA).
  • nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35 0 C.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50 0 C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide.
  • “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide.
  • a specific example of “low stringency” hybridization conditions is when the above hybridization is conducted at 10% formamide.
  • the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
  • Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions are well known in the art. Hybridization conditions are discussed further, below.
  • the above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used.
  • the hybridization temperature may be decreased in increments of 5 0 C from 68 0 C to 42°C in a hybridization buffer having a Na + concentration of approximately IM.
  • the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization.
  • the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
  • concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50 0 C.
  • 6X SSC 0.5% SDS at 50 0 C.
  • wash conditions used to identify nucleic acids within the scope of the invention include, e.g.: a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50 0 C or about 55°C to about 60 0 C; or, a salt concentration of about 0.15 M NaCl at 72°C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 50 0 C or about 55 0 C to about 60 0 C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1X SSC containing 0.1% SDS at 68oC for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen and Ausu
  • Oligonucleotides probes and methods for using them
  • the invention also provides nucleic acid probes for identifying nucleic acids encoding a polypeptide with an enzyme activity.
  • the probe comprises at least 10 consecutive bases of a nucleic acid of the invention.
  • a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 150, 160, 170, 180, 190, 200 or more, or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention.
  • the probes identify a nucleic acid by binding and/or hybridization.
  • the probes can be used in arrays of the invention, see discussion below, including, e.g., capillary arrays.
  • the probes of the invention can also be used to isolate other nucleic acids or polypeptides.
  • the probes of the invention can be used to determine whether a biological sample, such as a soil sample, contains an organism having a nucleic acid sequence of the invention (e.g., an enzyme-encoding nucleic acid) or an organism from which the nucleic acid was obtained.
  • a biological sample potentially harboring the organism from which the nucleic acid was isolated is obtained and nucleic acids are obtained from the sample.
  • the nucleic acids are contacted with the probe under conditions which permit the probe to specifically hybridize to any complementary sequences present in the sample.
  • conditions which permit the probe to specifically hybridize to complementary sequences may be determined by placing the probe in contact with complementary sequences from samples known to contain the complementary sequence, as well as control sequences which do not contain the complementary sequence.
  • Hybridization conditions such as the salt concentration of the hybridization buffer, the formamide concentration of the hybridization buffer, or the hybridization temperature, may be varied to identify conditions which allow the probe to hybridize specifically to complementary nucleic acids (see discussion on specific hybridization conditions).
  • Hybridization may be detected by labeling the probe with a detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product.
  • detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product.
  • Many methods for using the labeled probes to detect the presence of complementary nucleic acids in a sample are familiar to those skilled in the art. These include Southern Blots, Northern Blots, colony hybridization procedures, and dot blots. Protocols for each of these procedures are provided in Ausubel and Sambrook.
  • more than one probe may be used in an amplification reaction to determine whether the sample contains an organism containing a nucleic acid sequence of the invention (e.g., an organism from which the nucleic acid was isolated).
  • the probes comprise oligonucleotides.
  • the amplification reaction may comprise a PCR reaction. PCR protocols are described in Ausubel and Sambrook (see discussion on amplification reactions). In such procedures, the nucleic acids in the sample are contacted with the probes, the amplification reaction is performed, and any resulting amplification product is detected.
  • the amplification product may be detected by performing gel electrophoresis on the reaction products and staining the gel with an intercalator such as ethidium bromide.
  • an intercalator such as ethidium bromide.
  • one or more of the probes may be labeled with a radioactive isotope and the presence of a radioactive amplification product may be detected by autoradiography after gel electrophoresis.
  • Probes derived from sequences near the 3' or 5' ends of a nucleic acid sequence of the invention can also be used in chromosome walking procedures to identify clones containing additional, e.g., genomic sequences. Such methods allow the isolation of genes which encode additional proteins of interest from the host organism.
  • nucleic acid sequences of the invention are used as probes to identify and isolate related nucleic acids.
  • the so-identified related nucleic acids may be cDNAs or genomic DNAs from organisms other than the one from which the nucleic acid of the invention was first isolated.
  • a nucleic acid sample is contacted with the probe under conditions which permit the probe to specifically hybridize to related sequences. Hybridization of the probe to nucleic acids from the related organism is then detected using any of the methods described above.
  • nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency.
  • a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45°C in a solution consisting of 0.9 M NaCl, 50 mM NaH 2 PO4, pH 7.0, 5.0 mM Na 2 EDTA, 0.5% SDS, 1OX Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 107 cpm (specific activity 4-9 X 108 cpm/ug) of 32P end-labeled oligonucleotide probe are then added to the solution.
  • the membrane is washed for 30 minutes at room temperature (RT) in IX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at Tm-IO 0 C for the oligonucleotide probe.
  • IX SET 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA
  • nucleic acids such as cDNAs or genomic DNAs, which hybridize to the detectable probe
  • nucleic acids having different levels of homology to the probe can be identified and isolated.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe. Very stringent conditions are selected to be equal to or about 5°C lower than the Tm for a particular probe.
  • Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm DNA, 50% formamide.
  • Formulas for SSC and Denhardt's and other solutions are listed, e.g., in Sambrook.
  • hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above.
  • the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution.
  • the filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto.
  • the hybridization may be carried out at 15-25 0 C below the Tm.
  • the hybridization may be conducted at 5-10 0 C below the Tm.
  • hybridizations in 6X SSC are conducted at approximately 68°C.
  • hybridizations in 50% formamide containing solutions are conducted at approximately 42°C. All of the foregoing hybridizations would be considered to be under conditions of high stringency.
  • the filter is washed to remove any non- specifically bound detectable probe.
  • the stringency used to wash the filters can also be varied depending on the nature of the nucleic acids being hybridized, the length of the nucleic acids being hybridized, the degree of complementarity, the nucleotide sequence composition (e.g., GC v. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examples of progressively higher stringency condition washes are as follows: 2X SSC, 0.1 % SDS at room temperature for 15 minutes (low stringency); 0. IX SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.
  • IX SSC 0.5% SDS for 15 to 30 minutes at between the hybridization temperature and 68°C (high stringency); and 0.15M NaCl for 15 minutes at 72°C (very high stringency).
  • a final low stringency wash can be conducted in 0. IX SSC at room temperature.
  • Nucleic acids which have hybridized to the probe can be identified by autoradiography or other conventional techniques. The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence.
  • the hybridization temperature may be decreased in increments of 5°C from 68 0 C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM.
  • the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization.
  • These conditions are considered to be "moderate” conditions above 50 0 C and "low” conditions below 50 0 C.
  • An example of “moderate” hybridization conditions is when the above hybridization is conducted at 55°C.
  • An example of "low stringency” hybridization conditions is when the above hybridization is conducted at 45°C.
  • the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
  • concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50 0 C.
  • 6X SSC 0.5% SDS at 50 0 C.
  • probes and methods of the invention can be used to isolate, or identify (e.g., using an array), nucleic acids having a sequence with at least about 950%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity to a nucleic acid sequence of the invention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900
  • homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein.
  • allelic variants may have a substitution, deletion or addition of one or more nucleotides when compared to a nucleic acid of the invention.
  • probes and methods of the invention may be used to isolate, or identify (e.g., using an array), nucleic acids which encode polypeptides having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity (homology) to a polypeptide of the invention comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive amino acids thereof as determined using a sequence alignment algorithm, e.g., such as the FASTA version
  • the invention further provides for nucleic acids complementary to (e.g., antisense sequences to) the nucleic acid sequences of the invention, e.g., enzyme-encoding sequences.
  • Antisense sequences are capable of inhibiting the transport, splicing or transcription of enzyme-encoding genes.
  • the inhibition can be effected through the targeting of genomic DNA or messenger RNA.
  • the inhibition can be effected using DNA, e.g., an inhibitory ribozyme, or an RNA, e.g., a double-stranded iRNA (e.g., siRNA, miRNA), comprising a sequence of the invention.
  • the transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage.
  • the invention provides a set of inhibitors comprising oligonucleotides capable of binding enzyme gene and/or message, in either case preventing or inhibiting the production or function of enzyme.
  • the association can be through sequence specific hybridization.
  • Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of enzyme message.
  • the oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes.
  • the oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One may screen a pool of many different such oligonucleotides for those with the desired activity.
  • Antisense Oligonucleotides capable of binding enzyme message which can inhibit enzyme activity by targeting mRNA or genomic DNA.
  • Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such enzyme oligonucleotides using the novel reagents of the invention.
  • gene walking/ RNA mapping protocols to screen for effective antisense oligonucleotides are well known in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183, describing an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection.
  • antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.
  • the antisense oligonucleotides can be single stranded or double-stranded RNA or DNA. The optimal length can be determined by routine screening.
  • the antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening.
  • nucleotide and nucleic acid analogues are known which can address this potential problem.
  • PNAs peptide nucleic acids
  • Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144: 189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N. J., 1996).
  • Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino carbamate nucleic acids, as described above.
  • Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense enzyme sequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
  • the invention provides for with ribozymes capable of binding enzyme message that can inhibit enzyme activity by targeting mRNA.
  • ribozymes capable of binding enzyme message that can inhibit enzyme activity by targeting mRNA.
  • Strategies for designing ribozymes and selecting the enzyme-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention.
  • Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA.
  • the ribozyme recognizes and binds a target RNA through complementary basepairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA.
  • Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
  • a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide.
  • antisense technology where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule
  • This potential advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted
  • RNA This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
  • the enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RnaseP-like RNA (in association with an RNA guide sequence).
  • hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.
  • RNA interference RNA interference
  • the invention provides an RNA inhibitory molecule, a so-called "RNAi" molecule, comprising an enzyme sequence of the invention.
  • RNAi or RNA interference molecules include small interfering RNA, or siRNAs, for inhibiting transcription, and microRNAs, or miRNAs, for inhibiting translation.
  • the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule.
  • RNAi can inhibit expression of an enzyme gene.
  • the RNAi is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length.
  • the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs.
  • ssRNA single-stranded RNA
  • dsRNA double-stranded RNA
  • RNAi RNA interference
  • RNAi double-stranded RNA
  • the RNAi's of the invention are used in gene- silencing therapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7: 1040-1046.
  • the invention provides methods to selectively degrade RNA using the RNAi's of the invention. The process may be practiced in vitro, ex vivo or in vivo.
  • the RNAi molecules of the invention can be used to generate a loss-of- function mutation in a cell, an organ or an animal. Methods for making and using RNAi molecules for selectively degrade KNA are well known in the art, see, e.g., U.S. Patent No. 6,506,559; 6,511,824; 6,515,109; 6,489,127.
  • the invention provides methods of generating variants of the nucleic acids and polypeptides of the invention, e.g., nucleic acids encoding an enzyme, peptide or an antibody of the invention. These methods can be repeated or used in various combinations to generate enzymes or antibodies having an altered or different activity or an altered or different stability from that of an enzyme or antibody encoded by the template nucleic acid. These methods also can be repeated or used in various combinations, e.g., to generate variations in gene/ message expression, message translation or message stability.
  • the genetic composition of a cell is altered by, e.g., modification of a homologous gene ex vivo, followed by its reinsertion into the cell.
  • variants can include polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of an enzyme of the invention.
  • Variants can be produced by any number of means included methods such as, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof.
  • GSSM Gene Site Saturation Mutagenesis
  • optimal directed evolution system or “optimized directed evolution” includes a method for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below.
  • synthetic ligation reassembly or “SLR” includes a method of ligating oligonucleotide fragments in a non-stochastic fashion, and explained in detail, below.
  • a nucleic acid of the invention can be altered by any means. For example, random or stochastic methods, or, non-stochastic, or "directed evolution," methods, see, e.g., U.S. Patent No. 6,361,974. Methods for random mutation of genes are well known in the art, see, e.g., U.S. Patent No. 5,830,696.
  • mutagens can be used to randomly mutate a gene. Mutagens include, e.g., ultraviolet light or gamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to induce DNA breaks amenable to repair by recombination.
  • Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
  • Other mutagens are analogues of nucleotide precursors, e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence.
  • Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.
  • nucleic acids e.g., genes
  • Stochastic fragmentation
  • modifications, additions or deletions are introduced by error-prone PCR, shuffling, oligonucleotide- directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM) synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic
  • GSSM Gene Site Saturation Mutagenesis
  • SLR synthetic ligation reassembly
  • Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al. (1996) “Oligonucleotide-directed random mutagenesis using the phosphorothioate method” Methods MoI. Biol. 57:369-374; Smith (1985) "In vitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985) "Strategies and applications of in vitro mutagenesis” Science 229: 1193-1201; Carter (1986) "Site-directed mutagenesis” Biochem. J.
  • Additional protocols used in the methods of the invention include point mismatch repair (Kramer (1984) "Point Mismatch Repair” Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al. (1985) "Improved oligonucleotide site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using Ml 3 vectors” Methods in Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) "Use of oligonucleotides to generate large deletions" Nucl. Acids Res.
  • Non-stochastic, or "directed evolution,” methods include, e.g., saturation mutagenesis, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof are used to modify the nucleic acids of the invention to generate enzymes with new or altered properties (e.g., activity under highly acidic or alkaline conditions, high temperatures, and the like).
  • Polypeptides encoded by the modified nucleic acids can be screened for an activity before testing for proteolytic or other activity. Any testing modality or protocol can be used, e.g., using a capillary array platform. See, e.g., U.S. Patent Nos. 6,361,974; 6,280,926; 5,939,250.
  • non-stochastic gene modification a "directed evolution process” is used to generate enzymes and antibodies with new or altered properties. Variations of this method have been termed “Gene Site Saturation MutagenesisTM, “site-saturation mutagenesis,” “saturation mutagenesis” or simply “GSSMTM.” It can be used in combination with other mutagenization processes. See, e.g., U.S. Patent Nos. 6,171,820; 6,238,884. Thus, the invention provides methods for making enzyme using Gene Site Saturation mutagenesis, or, GSSM, as described herein, and also in U.S. Patent Nos. 6,171,820; 6,238,884, and 6,579,258.
  • GSSM comprises providing a template polynucleotide and a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide with the oligonucleotides, thereby generating polynucleotides comprising homologous gene sequence variations.
  • codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amino acid residue in an enzyme active site or ligand binding site targeted to be modified.
  • These oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N,N,G/T sequence, and, optionally, a second homologous sequence.
  • the downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids.
  • one such degenerate oligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions.
  • At least two degenerate cassettes are used - either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.
  • more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site.
  • This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).
  • oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.
  • simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence.
  • degenerate cassettes having less degeneracy than the N,N,G/T sequence are used.
  • degenerate triplets allows for systematic and easy generation of a full range of possible natural amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide (in alternative aspects, the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated.
  • an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet 32 individual sequences can code for all 20 possible natural amino acids.
  • Nondegenerate oligonucleotides can optionally be used in combination with degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides one means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.
  • each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 natural amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations).
  • the 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening.
  • an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased proteolytic activity under alkaline or acidic conditions), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.
  • favorable amino acid changes may be identified at more than one amino acid position.
  • One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3 x 3 x 3 or 27 total possibilities, including 7 that were previously examined - 6 single point mutations (i.e. 2 at each of three positions) and no change at any position.
  • site-saturation mutagenesis can be used together with another stochastic or non-stochastic means to vary sequence, e.g., synthetic ligation reassembly (see below), shuffling, chimerization, recombination and other mutagenizing processes and mutagenizing agents.
  • This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.
  • the invention provides a non-stochastic gene modification system termed "synthetic ligation reassembly,” or simply “SLR,” a “directed evolution process,” to generate enzymes and antibodies with new or altered properties.
  • SLR is a method of ligating oligonucleotide fragments together non-stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non- stochastically. See, e.g., U.S. Patent Nos.
  • SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucle
  • SLR does not depend on the presence of high levels of homology between polynucleotides to be rearranged.
  • this method can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10 different chimeras.
  • SLR can be used to generate libraries comprised of over io 1000 different progeny chimeras.
  • aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecule shaving an overall assembly order that is chosen by design. This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
  • the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.
  • the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends. If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).
  • the annealed building pieces are treated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent bonding of the building pieces.
  • a ligase e.g. T4 DNA ligase
  • the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates that serve as a basis for producing a progeny set of finalized chimeric polynucleotides.
  • These parental oligonucleotide templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, e.g., chimerized or shuffled.
  • the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points.
  • the demarcation points can be located at an area of homology, and are comprised of one or more nucleotides.
  • demarcation points are preferably shared by at least two of the progenitor templates.
  • the demarcation points can thereby be used to delineate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides.
  • the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules.
  • a demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences.
  • a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences. Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences. In one aspect, a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.
  • a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides.
  • all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.
  • the assembly order i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
  • the assembly order is by design (or non- stochastic) as described above. Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.
  • the ligation reassembly method is performed systematically.
  • the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g. one by one.
  • this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups.
  • the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.
  • the saturation mutagenesis and optimized directed evolution methods also can be used to generate different progeny molecular species.
  • the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template. Alternatively, a codon can be altered such that the coding for an originally amino acid is altered.
  • This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecular homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.
  • the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g. by mutagenesis) or in an in vivo process (e.g. by utilizing the gene splicing ability of a host organism).
  • nucleotides e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences
  • a nucleic acid building block is used to introduce an intron.
  • functional introns are introduced into a man-made gene manufactured according to the methods described herein.
  • the artificially introduced intron(s) can be functional in a host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.
  • the invention provides a non-stochastic gene modification system termed "optimized directed evolution system" to generate enzymes and antibodies with new or altered properties.
  • Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination.
  • Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
  • a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence. This method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
  • this method provides a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.
  • Previously if one generated, for example, 10 13 chimeric molecules during a reaction, it would be extremely difficult to test such a high number of chimeric variants for a particular activity.
  • a significant portion of the progeny population would have a very high number of crossover events which resulted in proteins that were less likely to have increased levels of a particular activity.
  • the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events.
  • each of the molecules chosen for further analysis most likely has, for example, only three crossover events.
  • the boundaries on the functional variety between the chimeric molecules is reduced. This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.
  • One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides corresponding to fragments or portions of each parental sequence.
  • Each oligonucleotide in one aspect includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order.
  • protocols for practicing these methods of the invention can be found in U.S. Patent Nos. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.
  • the number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.
  • three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.
  • a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences. The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low.
  • each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
  • a probability density function can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides corresponding to each variant, and the concentrations of each variant during each step in the ligation reaction.
  • PDF probability density function
  • a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events.
  • These methods are directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of a nucleic acid encoding a polypeptide through recombination.
  • This system allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
  • a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.
  • the method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
  • aspects of the invention include a system and software that receive a desired crossover probability density function (PDF), the number of parent genes to be reassembled, and the number of fragments in the reassembly as inputs.
  • PDF crossover probability density function
  • the output of this program is a "fragment PDF" that can be used to determine a recipe for producing reassembled genes, and the estimated crossover PDF of those genes.
  • the processing described herein is preferably performed in MATLABTM (The Mathworks, Natick, Massachusetts) a programming language and development environment for technical computing.
  • these processes can be iteratively repeated. For example a nucleic acid (or, the nucleic acid) responsible for an altered enzyme or antibody phenotype is identified, re-isolated, again modified, re-tested for activity. This process can be iteratively repeated until a desired phenotype is engineered. For example, an entire biochemical anabolic or catabolic pathway can be engineered into a cell, including proteolytic activity. Similarly, if it is determined that a particular oligonucleotide has no affect at all on the desired trait (e.g., a new enzyme phenotype), it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed.
  • a nucleic acid or, the nucleic acid
  • In vivo shuffling of molecules is use in methods of the invention that provide variants of polypeptides of the invention, e.g., antibodies, enzymes, and the like.
  • In vivo shuffling can be performed utilizing the natural property of cells to recombine multimers. While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.
  • the invention provides a method for producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide.
  • the invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell.
  • the regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide.
  • hybrid polynucleotide is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences.
  • hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules.
  • hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.
  • the invention also provides methods of making sequence variants of the nucleic acid and enzyme and antibody sequences of the invention or isolating enzymes using the nucleic acids and polypeptides of the invention.
  • the invention provides for variants of a protein-encoding gene of the invention, which can be altered by any means, including, e.g., random or stochastic methods, or, non-stochastic, or "directed evolution," methods, as described above.
  • the isolated variants may be naturally occurring.
  • Variant can also be created in vitro. Variants may be created using genetic engineering techniques such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, and standard cloning techniques.
  • variants, fragments, analogs, or derivatives may be created using chemical synthesis or modification procedures.
  • Other methods of making variants are also familiar to those skilled in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids which encode polypeptides having characteristics which enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. These nucleotide differences can result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
  • variants may be created using error prone PCR.
  • error prone PCR PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product.
  • Error prone PCR is described, e.g., in Leung, D.W., et al., Technique, 1 :11-15, 1989) and Caldwell, R. C. & Joyce G.F., PCR Methods Applic, 2:28-33, 1992.
  • nucleic acids to be mutagenized are mixed with PCR primers, reaction buffer, MgCl 2 , MnCl 2 , Taq polymerase and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product.
  • the reaction may be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl 2 , 0.5 mM MnCl 2 , 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP.
  • PCR may be performed for 30 cycles of 94°C for 1 min, 45 0 C for 1 min, and 72°C for 1 min. However, it will be appreciated that these parameters may be varied as appropriate.
  • the mutagenized nucleic acids are cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids is evaluated. Variants may also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized. Clones containing the mutagenized DNA are recovered and the activities of the polypeptides they encode are assessed.
  • Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, e.g., U.S. Patent No. 5,965,408.
  • Still another method of generating variants is sexual PCR mutagenesis.
  • sexual PCR mutagenesis forced homologous recombination occurs between DNA molecules of different but highly related DNA sequence in vitro, as a result of random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction.
  • Sexual PCR mutagenesis is described, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751. Briefly, in such procedures a plurality of nucleic acids to be recombined are digested with DNase to generate fragments having an average size of 50-200 nucleotides.
  • Fragments of the desired average size are purified and resuspended in a PCR mixture.
  • PCR is conducted under conditions which facilitate recombination between the nucleic acid fragments.
  • PCR may be performed by resuspending the purified fragments at a concentration of 10-30 ng/:l in a solution of 0.2 mM of each dNTP, 2.2 mM MgCl 2 , 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100.
  • PCR 2.5 units of Taq polymerase per 100:1 of reaction mixture is added and PCR is performed using the following regime: 94°C for 60 seconds, 94°C for 30 seconds, 50-55 0 C for 30 seconds, 72°C for 30 seconds (30-45 times) and 72 0 C for 5 minutes.
  • oligonucleotides may be included in the PCR reactions.
  • the Klenow fragment of DNA polymerase I may be used in a first set of PCR reactions and Taq polymerase may be used in a subsequent set of PCR reactions. Recombinant sequences are isolated and the activities of the polypeptides they encode are assessed.
  • Variants may also be created by in vivo mutagenesis.
  • random mutations in a sequence of interest are generated by propagating the sequence of interest in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways.
  • a bacterial strain such as an E. coli strain
  • Such "mutator" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA.
  • Mutator strains suitable for use for in vivo mutagenesis are described, e.g., in PCT Publication No. WO 91/16427.
  • Variants may also be generated using cassette mutagenesis.
  • cassette mutagenesis a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide "cassette" that differs from the native sequence.
  • the oligonucleotide often contains completely and/or partially randomized native sequence.
  • Recursive ensemble mutagenesis may also be used to generate variants.
  • Recursive ensemble mutagenesis is an algorithm for protein engineering (protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.
  • variants are created using exponential ensemble mutagenesis.
  • Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins.
  • Exponential ensemble mutagenesis is described, e.g., in Delegrave (1993) Biotechnology Res. 11 : 1548-1552. Random and site-directed mutagenesis are described, e.g., in Arnold (1993) Current Opinion in Biotechnology 4:450-455.
  • the variants are created using shuffling procedures wherein portions of a plurality of nucleic acids which encode distinct polypeptides are fused together to create chimeric nucleic acid sequences which encode chimeric polypeptides as described in, e.g., U.S. Patent Nos. 5,965,408; 5,939,250.
  • the invention also provides variants of polypeptides of the invention comprising sequences in which one or more of the amino acid residues (e.g., of an exemplary polypeptide, such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, etc.) are substituted with a conserved or non-conserved amino acid residue (e.g., a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics.
  • polypeptides of the invention include those with conservative substitutions of sequences of the invention, e.g., the exemplary sequences of the invention, such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, etc., including but not limited to the following replacements: replacements of an aliphatic amino acid such as Alanine, Valine, Leucine and Iso leucine with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue such as Aspartic acid and Glutamic acid with another acidic residue; replacement of a residue bearing an amide group, such as Asparagine and Glutamine, with another residue bearing an amide group; exchange of a basic residue such as Lysine and Arginine with another basic residue; and replacement of an aromatic residue such as Phenylalanine, Tyrosine with another aromatic residue.
  • Other variants are those in which one or more of the amino acid residues of the polypeptides of the
  • variants within the scope of the invention are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide, for example, polyethylene glycol.
  • additional variants within the scope of the invention are those in which additional amino acids are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.
  • the variants, fragments, derivatives and analogs of the polypeptides of the invention retain the same biological function or activity as the exemplary polypeptides, e.g., a proteolytic activity, as described herein.
  • the variant, fragment, derivative, or analog includes a proprotein, such that the variant, fragment, derivative, or analog can be activated by cleavage of the proprotein portion to produce an active polypeptide.
  • the invention provides methods for modifying enzyme-encoding nucleic acids to modify codon usage.
  • the invention provides methods for modifying codons in a nucleic acid encoding an enzyme to increase or decrease its expression in a host cell, e.g., a bacterial, insect, mammalian, yeast or plant cell.
  • the invention also provides nucleic acids encoding an enzyme modified to increase its expression in a host cell, enzyme so modified, and methods of making the modified enzymes.
  • the method comprises identifying a "non-preferred” or a "less preferred” codon in enzyme-encoding nucleic acid and replacing one or more of these non-preferred or less preferred codons with a "preferred codon” encoding the same amino acid as the replaced codon and at least one non-preferred or less preferred codon in the nucleic acid has been replaced by a preferred codon encoding the same amino acid.
  • a preferred codon is a codon over- represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell.
  • Host cells for expressing the nucleic acids, expression cassettes and vectors of the invention include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Thus, the invention provides methods for optimizing codon usage in all of these cells, codon-altered nucleic acids and polypeptides made by the codon-altered nucleic acids.
  • Exemplary host cells include gram negative bacteria, such as Escherichia coli; gram positive bacteria, such as any Bacillus (e.g., B. cereus or B. subtilis) or Streptomyces, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris.
  • Exemplary host cells also include eukaryotic organisms, e.g., various yeast, such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines.
  • yeast such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines.
  • yeast such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lac
  • the codons of a nucleic acid encoding an enzyme isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell different from the bacteria from which the enzyme was derived, a yeast, a fungi, a plant cell, an insect cell or a mammalian cell.
  • Methods for optimizing codons are well known in the art, see, e.g., U.S. Patent No. 5,795,737; Baca (2000) Int. J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12: 185-188; Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect. Immun.
  • the invention provides transgenic non-human animals comprising a nucleic acid, a polypeptide (e.g., an enzyme or an antibody of the invention), an expression cassette, a vector, a transfected or a transformed cell of the invention.
  • the transgenic non-human animals can be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice, comprising a nucleic acid or protein of the invention as a heterologous or recombinant sequence. These animals can be used, e.g., as in vivo models to study enzyme activity, or, as models to screen for agents that change the enzyme activity in vivo.
  • the coding sequences for the polypeptides to be expressed in the transgenic non-human animals can be designed to be constitutive, or, under the control of tissue-specific, developmental-specific or inducible transcriptional regulatory factors.
  • Transgenic non-human animals can be designed and generated using any method known in the art; see, e.g., U.S. Patent Nos.
  • U.S. Patent No. 6,211,4208 describes making and using transgenic non-human mammals which express in their brains a nucleic acid construct comprising a DNA sequence.
  • U.S. Patent No. 5,387,742 describes injecting cloned recombinant or synthetic DNA sequences into fertilized mouse eggs, implanting the injected eggs in pseudo-pregnant females, and growing to term transgenic mice whose cells express proteins related to the pathology of Alzheimer's disease.
  • U.S. Patent No. 6,187,992 describes making and using a transgenic mouse whose genome comprises a disruption of the gene encoding amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • knockout animals can also be used to practice the methods of the invention.
  • the transgenic or modified animals of the invention comprise a "knockout animal,” e.g., a “knockout mouse,” engineered not to express an endogenous gene, which is replaced with a gene expressing an enzyme of the invention, or, a fusion protein comprising an enzyme of the invention.
  • functional knockouts can also be generated using antisense sequences of the invention, e.g., double-stranded RNAi molecules.
  • the invention provides transgenic plants and seeds comprising a nucleic acid, a polypeptide (e.g., an enzyme or an antibody of the invention), an expression cassette or vector or a transfected or transformed cell of the invention.
  • the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot).
  • the invention also provides methods of making and using these transgenic plants and seeds.
  • the transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with any method known in the art. See, for example, U.S. Patent No. 6,309,872.
  • Nucleic acids and expression constructs of the invention can be introduced into a plant cell by any means.
  • nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or expression constructs can be episomes.
  • Introduction into the genome of a desired plant can be such that the host's enzyme production is regulated by endogenous transcriptional or translational control elements.
  • the invention also provides "knockout plants” where insertion of gene sequence by, e.g., homologous recombination, has disrupted the expression of the endogenous gene. Means to generate "knockout" plants are well-known in the art, see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao (1995) Plant J 7:359-365. See discussion on transgenic plants, below.
  • the first step in production of a transgenic plant involves making an expression construct for expression in a plant cell.
  • These techniques are well known in the art. They can include selecting and cloning a promoter, a coding sequence for facilitating efficient binding of ribosomes to mRNA and selecting the appropriate gene terminator sequences.
  • a constitutive promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Other promoters are more specific and respond to cues in the plant's internal or external environment.
  • An exemplary light-inducible promoter is the promoter from the cab gene, encoding the major chlorophyll a/b binding protein.
  • the nucleic acid is modified to achieve greater expression in a plant cell.
  • a sequence of the invention is likely to have a higher percentage of A-
  • A-T nucleotides in the coding sequence can be substituted with G-C nucleotides without significantly changing the amino acid sequence to enhance production of the gene product in plant cells.
  • Selectable marker gene can be added to the gene construct in order to identify plant cells or tissues that have successfully integrated the transgene. This may be necessary because achieving incorporation and expression of genes in plant cells is a rare event, occurring in just a few percent of the targeted tissues or cells.
  • Selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides. Only plant cells that have integrated the selectable marker gene will survive when grown on a medium containing the appropriate antibiotic or herbicide. As for other inserted genes, marker genes also require promoter and termination sequences for proper function.
  • making transgenic plants or seeds comprises incorporating sequences of the invention and, optionally, marker genes into a target expression construct (e.g., a plasmid, a phage), along with positioning of the promoter and the terminator sequences.
  • a target expression construct e.g., a plasmid, a phage
  • This can involve transferring the modified gene into the plant through a suitable method.
  • a construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. For example, see, e.g., Christou (1997) Plant MoI. Biol. 35: 197-203; Pawlowski (1996) MoI. Biotechnol.
  • protoplasts can be immobilized and injected with a nucleic acids, e.g., an expression construct.
  • a nucleic acids e.g., an expression construct.
  • plant regeneration from protoplasts is not easy with cereals, plant regeneration is possible in legumes using somatic embryogenesis from protoplast derived callus.
  • Organized tissues can be transformed with naked DNA using gene gun technique, where DNA is coated on tungsten microprojectiles, shot 1/lOOth the size of cells, which carry the DNA deep into cells and organelles. Transformed tissue is then induced to regenerate, usually by somatic embryogenesis. This technique has been successful in several cereal species including maize and rice.
  • Nucleic acids can also be introduced in to plant cells using recombinant viruses.
  • Plant cells can be transformed using viral vectors, such as, e.g., tobacco mosaic virus derived vectors (Rouwendal (1997) Plant MoI. Biol. 33:989- 999), see Porta (1996) "Use of viral replicons for the expression of genes in plants," MoI. Biotechnol. 5:209-221.
  • nucleic acids e.g., an expression construct
  • suitable T-DNA flanking regions can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
  • Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl. Acad. Sci.
  • the DNA in an A. tumefaciens cell is contained in the bacterial chromosome as well as in another structure known as a Ti (tumor-inducing) plasmid.
  • the Ti plasmid contains a stretch of DNA termed T-DNA (-20 kb long) that is transferred to the plant cell in the infection process and a series of vir
  • A. tumefaciens can only infect a plant through wounds: when a plant root or stem is wounded it gives off certain chemical signals, in response to which, the vir genes of A. tumefaciens become activated and direct a series of events necessary for the transfer of the T-DNA from the Ti plasmid to the plant's chromosome. The T-DNA then enters the plant cell through the wound.
  • One speculation is that the T-DNA waits until the plant DNA is being replicated or transcribed, then inserts itself into the exposed plant DNA. In order to use A.
  • the tumor-inducing section of T-DNA have to be removed, while retaining the T-DNA border regions and the vir genes.
  • the transgene is then inserted between the T-DNA border regions, where it is transferred to the plant cell and becomes integrated into the plant's chromosomes.
  • the invention provides for the transformation of monocotyledonous plants using the nucleic acids of the invention, including important cereals, see Hiei (1997) Plant MoI. Biol. 35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803; Thykjaer (1997) supra; Park (1996) Plant MoI. Biol. 32:1135-1148, discussing T-DNA integration into genomic DNA. See also D ⁇ alluin, U.S. Patent No. 5,712,135, describing a process for the stable integration of a DNA comprising a gene that is functional in a cell of a cereal, or other monocotyledonous plant.
  • the third step can involve selection and regeneration of whole plants capable of transmitting the incorporated target gene to the next generation.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts , pp. 21-73, CRC Press, Boca Raton, 1985.
  • Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins.
  • the expression cassette After the expression cassette is stably incorporated in transgenic plants, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Since transgenic expression of the nucleic acids of the invention leads to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product.
  • the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a plant of the invention and another plant.
  • the desired effects can be enhanced when both parental plants express the polypeptides of the invention.
  • the desired effects can be passed to future plant generations by standard propagation means.
  • Transgenic plants of the invention can be dicotyledonous or monocotyledonous.
  • monocot transgenic plants of the invention are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
  • dicot transgenic plants of the invention are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • the transgenic plants and seeds of the invention include a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachio, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solatium, Sorghum, Theobromus, Trigonella,
  • the nucleic acids of the invention are expressed in plants which contain fiber cells, including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and flax.
  • the transgenic plants of the invention can be members of the genus Gossypium, including members of any Gossypium species, such as G. arboreum;. G. herbaceum, G. barbadense, and G. hirsutum.
  • the invention also provides for transgenic plants to be used for producing large amounts of the polypeptides (e.g., antibodies, enzymes) of the invention.
  • polypeptides e.g., antibodies, enzymes
  • transgenic plants to be used for producing large amounts of the polypeptides (e.g., antibodies, enzymes) of the invention.
  • polypeptides e.g., antibodies, enzymes
  • Palmgren 1997 Trends Genet. 13:348
  • Chong 1997) Transgenic Res. 6:289-296 (producing human milk protein beta-casein in transgenic potato plants using an auxin-inducible, bidirectional mannopine synthase (masl',2 1 ) promoter with Agrobacterium tumefaciens-mediated leaf disc transformation methods).
  • transgenic plants of the invention can screen for plants of the invention by detecting the increase or decrease of transgene mRNA or protein in transgenic plants.
  • Means for detecting and quantitation of mRNAs or proteins are well known in the art.
  • transgenic plants of the invention are produced by transformation of natural oleaginous plants.
  • the genetically transformed plants of the invention are then reproduced sexually so as to produce transgenic seeds of the invention. These seeds can be used to obtain transgenic plant progeny.
  • the enzyme gene is operably linked to an inducible promoter.
  • the invention provides isolated, synthetic or recombinant polypeptides having a sequence identity (e.g., at least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or complete sequence identity) to an exemplary sequence of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO
  • the identity can be over the full length of the polypeptide, or, the identity can be over a region of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues.
  • Polypeptides of the invention can also be shorter than the full length of exemplary polypeptides.
  • the invention provides a polypeptide comprising only a subsequence of a sequence of the invention
  • exemplary subsequences can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues.
  • the invention provides polypeptides (peptides, fragments) ranging in size between about 5 and the full length of a polypeptide, e.g., an enzyme, exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of an exemplary enzyme of the invention.
  • an enzyme exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of an exemplary enzyme of the invention.
  • Peptides of the invention can be useful as, e.g., labeling probes, antigens, toleragens, motifs, enzyme active sites.
  • Polypeptides of the invention also include antibodies capable of binding to an enzyme of the invention.
  • the invention provides enzymes having at least one of a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity.
  • a hydrolase activity e.g., an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidas
  • the invention provides enzymes having decontamination activity. In one aspect, the invention provides enzymes having non-heme chloroperoxidase (nhCPOs) activity.
  • nhCPOs non-heme chloroperoxidase
  • the invention provides methods for making an using these enzyme, e.g., methods for decontamination, bleaching and degradation of lignin, and the like. The following chart describes selected characteristics, including activity profiles
  • exemplary nucleic acids and polypeptides of the invention include sequence identity comparison of the exemplary sequences to public databases; the listed exemplary enzymatic activities were determined based on this sequence identity comparison to the to public databases.
  • Exemplary sequences described in this chart have been subject to a BLAST search (as described in detail, below) against two sets of databases. The first database set is available through NCBI (National Center for Biotechnology Information). All results from searches against these databases are found in the columns entitled “NR Description”, “NR Accession Code”, “NR Evalue” or “NR Organism”. "NR” refers to the Non-Redundant nucleotide database maintained by NCBI. This database is a composite of GenBank, GenBank updates, and EMBL updates.
  • the entries in the column "NR Description” refer to the definition line in any given NCBI record, which includes a description of the sequence, such as the source organism, gene name/protein name, or some description of the function of the sequence.
  • the entries in the column “NR Accession Code” refer to the unique identifier given to a sequence record.
  • the entries in the column “NR Evalue” refer to the Expect value (Evalue), which represents the probability that an alignment score as good as the one found between the query sequence (the sequences of the invention) and a database sequence would be found in the same number of comparisons between random sequences as was done in the present BLAST search.
  • the entries in the column “NR Organism” refer to the source organism of the sequence identified as the closest BLAST hit.
  • the second set of databases is collectively known as the GENESEQTM database, which is available through Thomson Derwent (Philadelphia, PA). All results from searches against this database are found in the columns entitled “Geneseq Protein Description”, “Geneseq Protein Accession Code”, “Geneseq Protein Evalue”, “Geneseq DNA Description”, “Geneseq DNA Accession Code” or “Geneseq DNA Evalue”. The information found in these columns is comparable to the information found in the NR columns described above, except that it was derived from BLAST searches against the GENESEQTM database instead of the NCBI databases. In addition, this table includes the column "Predicted EC No.”.
  • An EC number is the number assigned to a type of enzyme according to a scheme of standardized enzyme nomenclature developed by the Enzyme Commission of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB).
  • the results in the "Predicted EC No.” column are determined by a BLAST search against the Kegg (Kyoto Encyclopedia of Genes and Genomes) database. If the top BLAST match has an Evalue equal to or less than e "6 , the EC number assigned to the top match is entered into the table. The EC number of the top hit is used as a guide to what the EC number of the sequence of the invention might be.
  • the columns "Query DNA Length” and “Query Protein Length” refer to the number of nucleotides or the number amino acids, respectively, in the sequence of the invention that was searched or queried against either the NCBI or Geneseq databases. The columns
  • Geneseq or NR DNA Length and “Geneseq or NR Protein Length” refer to the number of nucleotides or the number amino acids, respectively, in the sequence of the top match from the BLAST search. The results provided in these columns are from the search that returned the lower Evalue, either from the NCBI databases or the GENESEQTM database.
  • the columns “GENESEQTM or NR %ID Protein” and “GENESEQTM or NR %ID DNA” refer to the percent sequence identity between the sequence of the invention and the sequence of the top BLAST match. The results provided in these columns are from the search that returned the lower Evalue, either from the NCBI databases or the GENESEQTM database.
  • the polypeptide having a sequence as set forth in SEQ ID NO:2, encoded e.g., by SEQ ID NO:1, based on, inter alia, sequence identity comparison to public databases has esterase activity based on similarity to : [Solibacter usitatus Ellin6076] Zi
  • Pseudomonas probable hydrolase [Pseudomonas Pseudomonas aeruginosa polypeptide 107, 108 aeruginosa PAOl] 9946723 3.00E-54 aeruginosa PAOl #3. ABO81098 5.00E-55
  • Amidohydrolase [Solibacter usitatus Ellin6076] gi
  • Amidohydrolase [Solibacter usitatus Solibacter usitatus M. xanthus protein 113, 114 Ellin6076] 67929705 5.00E-89 Ellin6076 sequence, seq id 9726. ABM95911 2.00E-29
  • COG0657 Esterase/lipase Rubrivivax Anti-biofilm 121, 122 [Rubrivivax gelatinosus PMl] 47575591 1.00E-108 gelatinosus PMl polypeptide #7. ADR51221 9.00E-53
  • Beta-lactamase [Parvularcula bermudensis HTCC2503] gi
  • COG0657 Esterase/lipase [Burkholderia pseudomallei S 13] gij67757144
  • carboxylesterase [Oceanobacillus Oceanobacillus DNA encoding 139, 140 iheyensis HTE831] 22778115 9 00E-93 iheyensis HTE831 hydrolase BD423 ABG31303 4 00E-89
  • acyl-CoA thioesterase 1 Pseudomonas Prokaryotic essential 141, 142 [Pseudomonas fluorescens Pf-5] 68345928 7 00E-52 fluorescens Pf-5 gene #34740.
  • COG0400 Predicted esterase Burkholde ⁇ a Prokaryotic essential 143, 144 [Burkholde ⁇ a fungorum LB400] 48785748 1 00E- 108 fungorum LB400 gene #34740 ABU21945 1 00E- 109
  • Geobacillus carboxylesterase [Geobacillus kaustophilus Sequence of new 145, 146 kaustophilus HTA426] 56381422 1 00E- 142 HTA426 lipase AAP95375 1 00E- 142
  • Arylesterase [Methylobacillus flagellatus KT] gi
  • Arylesterase [Methylobacillus Methylobacillus Anti-biofilm 149, 150 flagellatus KT] 68189034 2 00E-51 flagellatus KT polypeptide #7 ADR51243 1 00E-118 uncultured Anti-biofilm
  • Patatin [Thiomicrospira denitrificans ATCC venificus esterase 177, 178 denitrificans ATCC 33889] 78777519 1.00E-20 33889 SNP6-24LC. AAW23079 1.00E- 145
  • Symbiobacterium putative lipase [Symbiobacterium thermophilum IAM Anti-biofilm 181, 182 thermophilum IAM 14863] 51856952 1.00E-IOl 14863 polypeptide #7. ADR51213 1.00E- 180
  • lipase/esterase [uncultured uncultured DNA encoding 185, 186 bacterium] 45775279 8.00E-56 bacterium hydrolase BD423.
  • COG2021 Homoserine glutamicum MP protein acetyltransferase [Rubrivivax Rubrivivax sequence SEQ ID 89, 90 gel atinosus PMl] 47572957 3.00E-32 gelatinosus PMl NO:1148. AAB79726 2.00E-28
  • SEQ ID NO: 190 has aminopeptidase and/or metallopeptidase activity
  • SEQ ID NO: 192 has enzymatic activity against P-F and P-S bonds/G,V agents, and/or can hydrolytically degrade VX
  • SEQ ID NO: 194 has OPAA activity.
  • Amino acid or amino acid sequence can include an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.
  • polypeptide and protein can include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids.
  • polypeptide also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides.
  • the peptides and polypeptides of the invention also include all "mimetic” and “peptidomimetic” forms, as described in further detail, below.
  • the invention provides polypeptides, nucleic acids, infective vehicles (e.g., viruses, phages comprising organophosphoesterase-encoding nucleic acids of the invention), transduced or infected cells or plants and/or transgenic plants to, and methods of using them, e.g., provide self-protecting, pesticide-resistant cells or plants, where in one aspect the polypeptides of the invention act to hydrolyze P-S or P-F bonds and detoxify acetylcholinesterase- or butyrylcholinesterase- inhibitors.
  • the polypeptides of the invention include enzymes in an active or inactive form.
  • the polypeptides of the invention include proproteins before “maturation” or processing of prepro sequences, e.g., by a proprotein-processing enzyme, such as a proprotein convertase to generate an "active" mature protein.
  • the polypeptides of the invention include enzymes inactive for other reasons, e.g., before “activation” by a post- translational processing event, e.g., an endo- or exo-peptidase or proteinase action, a phosphorylation event, an amidation, a glycosylation or a sulfation, a dimerization event, and the like.
  • prepro domain sequences and signal sequences are well known in the art, see, e.g., Van de Ven (1993) Crit. Rev..Oncog. 4(2): 115-136.
  • the protein is purified from the extracellular space and the N-terminal protein sequence is determined and compared to the unprocessed form.
  • the polypeptides of the invention include all active forms, including active subsequences, e.g., catalytic domains or active sites, of an enzyme of the invention.
  • the invention provides catalytic domains or active sites as set forth below.
  • the invention provides a peptide or polypeptide comprising or consisting of an active site domain as predicted through use of a database such as Pfam (which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, The Pfam protein families database, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S.R. Eddy, S. Griffiths-Jones, K.L. Howe, M.
  • Pfam which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families
  • the invention includes polypeptides with or without a signal sequence and/or a prepro sequence.
  • the invention includes polypeptides with heterologous signal sequences and/or prepro sequences.
  • the prepro sequence (including a sequence of the invention used as a heterologous prepro domain) can be located on the amino terminal or the carboxy terminal end of the protein.
  • the invention also includes isolated or recombinant signal sequences, prepro sequences and catalytic domains (e.g., "active sites") comprising sequences of the invention.
  • Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo.
  • the peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.
  • peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrif ⁇ eld (1997) Methods Enzymol. 289:3-13) and automated synthesis may be achieved, e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • the peptides and polypeptides of the invention can also be glycosylated.
  • the glycosylation can be added post-translationally either chemically or by cellular biosynthetic mechanisms, wherein the later incorporates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence.
  • the glycosylation can be O-linked or N-linked.
  • the peptides and polypeptides of the invention include all “mimetic” and “peptidomimetic” forms.
  • the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention.
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
  • a mimetic composition is within the scope of the invention if it has an enzyme activity.
  • Polypeptide mimetic compositions of the invention can contain any combination of non-natural structural components.
  • mimetic compositions of the invention include one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a polypeptide of the invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
  • peptide bonds can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC).
  • DCC N,N'-dicyclohexylcarbodiimide
  • DIC N,N'-diisopropylcarbodiimide
  • a polypeptide of the invention can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues.
  • Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
  • Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L- phenylglycine; D- or L- 2 thieneylalanine; D- or L-I, -2, 3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D- (trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p- biphenylphenylalanine; D- or L-p-methoxy-biphenylpheny
  • Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
  • Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
  • Carboxyl side groups e.g., aspartyl or glutamyl
  • Carboxyl side groups can also be selectively modified by reaction with carbodiimides (R' -N-C-N-R') such as, e.g., 1- cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia- 4,4- dimetholpentyl) carbodiimide.
  • Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.
  • Nitrile derivative e.g., containing the CN-moiety in place of COOH
  • Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
  • Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclo- hexanedione, or ninhydrin, preferably under alkaline conditions.
  • Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O- acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
  • alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and corresponding amines
  • Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-V-nitrobenzo-oxa-l ⁇ -diazole.
  • cysteinyl residues e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid
  • chloroacetyl phosphate N-alkylmaleimides
  • 3-nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
  • Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro- benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide.
  • Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4- hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,- dimethylproline.
  • Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
  • mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
  • a residue, e.g., an amino acid, of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality.
  • any amino acid naturally occurring in the L-conf ⁇ guration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D- amino acid, but also can be referred to as the R- or S- form.
  • the invention also provides methods for modifying the polypeptides of the invention by either natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc), or by chemical modification techniques, and the resulting modified polypeptides. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation.
  • Solid-phase chemical peptide synthesis methods can also be used to synthesize the polypeptides, or fragments thereof, of the invention. Such method have been known in the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc, 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J.
  • a plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the pin's or rod's tips.
  • a process step i.e., inverting and inserting the rod's and pin's tips into appropriate solutions, amino acids are built into desired peptides.
  • FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an
  • Enzymes of the invention provides novel enzymes, e.g., proteins comprising at least about
  • polypeptides of the invention e.g., a protein having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, etc., (all even numbered sequences from SEQ ID NO: 2 through SEQ ID NO: 194), antibodies that bind them, and methods for making and using them.
  • the polypeptides of the invention can have any enzyme activity
  • the enzymes of the invention can have modified or new activities as compared to the exemplary enzymes or the activities described herein.
  • the invention includes enzymes with and without signal sequences and the signal sequences themselves.
  • the invention includes immobilized enzymes, anti-enzyme antibodies and fragments thereof.
  • the invention provides proteins for inhibiting enzyme activity, e.g., antibodies that bind to the enzyme active site.
  • the invention includes homodimers and heterocomplexes, e.g., fusion proteins, heterodimers, etc., comprising the enzymes of the invention.
  • the invention includes enzymes having activity over a broad range of high and low temperatures and pH's (e.g., acidic and basic aqueous conditions).
  • the invention provides methods of generating enzymes having altered (higher or lower) K cat /K m .
  • site-directed mutagenesis is used to create additional enzyme enzymes with alternative substrate specificities. The can be done, for example, by redesigning the substrate binding region or the active site of the enzyme.
  • enzymes of the invention are more stable at high temperatures, such as 8O 0 C to 85°C to 9O 0 C to 95 0 C, as compared to enzymes from conventional or moderate organisms.
  • Various proteins of the invention have an enzyme activity, under various conditions.
  • the invention provides methods of making enzymes with different catalytic efficiency and stabilities towards temperature, oxidizing agents and pH conditions. These methods can use, e.g., the techniques of site-directed mutagenesis and/or random mutagenesis. In one aspect, directed evolution can be used to produce enzymes with alternative specificities and stability.
  • the proteins of the invention are used in methods of the invention that can identify enzyme modulators, e.g., activators or inhibitors. Briefly, test samples (e.g., compounds, such as members of peptide or combinatorial libraries, broths, extracts, and the like) are added to enzyme assays to determine their ability to modulate, e.g., inhibit or activate, substrate cleavage. These inhibitors can be used in industry and research to reduce or prevent undesired isomerization. Modulators found using the methods of the invention can be used to alter (e.g., decrease or increase) the spectrum of activity of an enzyme.
  • test samples e.g., compounds, such as members of peptide or combinatorial libraries, broths, extracts, and the like
  • enzyme assays e.g., compounds, such as members of peptide or combinatorial libraries, broths, extracts, and the like
  • Modulators found using the methods of the invention can be used to alter (e.g., decrease or increase) the spectrum of
  • the invention also provides methods of discovering enzymes using the nucleic acids, polypeptides and antibodies of the invention.
  • lambda phage libraries are screened for expression-based discovery of enzymes.
  • the invention uses lambda phage libraries in screening to allow detection of toxic clones; improved access to substrate; reduced need for engineering a host, by-passing the potential for any bias resulting from mass excision of the library; and, faster growth at low clone densities.
  • Screening of lambda phage libraries can be in liquid phase or in solid phase.
  • the invention provides screening in liquid phase. This gives a greater flexibility in assay conditions; additional substrate flexibility; higher sensitivity for weak clones; and ease of automation over solid phase screening.
  • the invention provides screening methods using the proteins and nucleic acids of the invention involving robotic automation. This enables the execution of many thousands of biocatalytic reactions and screening assays in a short period of time, e.g., per day, as well as ensuring a high level of accuracy and reproducibility (see discussion of arrays, below). As a result, a library of derivative compounds can be produced in a matter of weeks.
  • the invention includes enzyme enzymes which are non-naturally occurring enzymes having a different enzyme activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the non-naturally occurring enzyme.
  • These enzymes have an amino acid sequence not found in nature. They can be derived by substitution of a plurality of amino acid residues of a precursor enzyme with different amino acids.
  • the precursor enzyme may be a naturally-occurring enzyme or a recombinant enzyme.
  • the enzyme variants encompass the substitution of any of the naturally occurring L-amino acids at the designated amino acid residue positions.
  • the invention provides signal sequences (e.g., signal peptides (SPs)), prepro domains and catalytic domains (CDs).
  • SPs signal peptides
  • CDs catalytic domains
  • the SPs, prepro domains and/or CDs of the invention can be isolated or recombinant peptides or can be part of a fusion protein, e.g., as a heterologous domain in a chimeric protein.
  • the invention provides nucleic acids encoding these catalytic domains (CDs), prepro domains and signal sequences (SPs, e.g., a peptide having a sequence comprising/ consisting of amino terminal residues of a polypeptide of the invention).
  • the invention provides a signal sequence comprising a peptide comprising/ consisting of a sequence as set forth in residues 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44 (or a longer peptide) of a polypeptide of the invention.
  • exemplary signal sequences consist of (or, comprise):
  • SEQ ID NO: 104 MKKLFAGFVLFISFYSHA;
  • SEQ ID NO: 106 MQSAASILALALALLAGGVWL
  • SEQ ID NO: 108 MRKKLVRLLAWIAGLCLLGLVLLVAAFW APSRSV;
  • SEQ ID NO: 150 MLRTLYLILVLMGLFPLS STVMA;
  • SEQ ID NO: 154 MNKRSLRKCLSAVGVVAILFSVQQVLA;
  • SEQ ID NO: 158 MATMMRGASKLLAGMALAVSALTATGEAFA;
  • SEQ ID NO: 188 MKHYVIALTTAFLLYT ALPATA;
  • SEQ ID NO:78 MNAFWNRLAGATLMLALMSTAWA
  • SEQ ID NO: 84 MNAFWNRLAGATLMLALMSTAWA
  • SEQ ID NO:90 MVIYRQLLAGILSLSLSLAALA;
  • SEQ ID NO:94 MMKPLIA VLALLW ACQDPGFTQA
  • SEQ ID NO:96 MNTSLCAAALAAWVLMPTAMA.
  • the enzyme signal sequences (SPs), CDs, and/or prepro sequences of the invention can be isolated peptides, or, sequences joined to another enzyme or a nonenzyme polypeptide, e.g., as a fusion (chimeric) protein.
  • the invention provides polypeptides comprising enzyme signal sequences of the invention.
  • polypeptides comprising enzyme signal sequences SPs, CDs, and/or prepro of the invention comprise sequences heterologous to enzymes of the invention (e.g., a fusion protein comprising an SP, CD, and/or prepro of the invention and sequences from another enzyme or a non-enzyme protein).
  • the invention provides enzymes of the invention with heterologous SPs, CDs, and/or prepro sequences, e.g., sequences with a yeast signal sequence.
  • a protein of the invention can comprise a heterologous SP and/or prepro in a vector, e.g., a pPIC series vector (Invitrogen, Carlsbad, CA).
  • SPs, CDs, and/or prepro sequences of the invention are identified following identification of novel enzyme polypeptides.
  • the pathways by which proteins are sorted and transported to their proper cellular location are often referred to as protein targeting pathways.
  • One of the most important elements in all of these targeting systems is a short amino acid sequence at the amino terminus of a newly synthesized polypeptide called the signal sequence.
  • This signal sequence directs a protein to its appropriate location in the cell and is removed during transport or when the protein reaches its final destination.
  • Most lysosomal, membrane, or secreted proteins have an amino-terminal signal sequence that marks them for translocation into the lumen of the endoplasmic reticulum.
  • the signal sequences can vary in length from 13 to 45 or more amino acid residues.
  • Various methods of recognition of signal sequences are known to those of skill in the art. For example, in one aspect, novel enzyme signal peptides are identified by a method referred to as SignalP.
  • SignalP uses a combined neural network which recognizes both signal peptides and their cleavage sites. (Nielsen, et al., "Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.” Protein Engineering, vol. 10, no. 1, p. 1-6 (1997).
  • enzymes of the invention may not have SPs and/or prepro sequences, and/or catalytic domains (CDs).
  • the invention provides polypeptides (e.g., enzymes) lacking all or part of an SP, a CD and/or a prepro domain.
  • the invention provides a nucleic acid sequence encoding a signal sequence (SP), a CD, and/or prepro from one enzyme operably linked to a nucleic acid sequence of a different enzyme or, optionally, a signal sequence (SPs) and/or prepro domain from a non-enzyme protein may be desired.
  • the invention also provides isolated or recombinant polypeptides comprising signal sequences (SPs), prepro domain and/or catalytic domains (CDs) of the invention and heterologous sequences.
  • SPs signal sequences
  • CDs catalytic domains
  • heterologous sequences are sequences not naturally associated with an SP, prepro domain and/or CD.
  • the sequence to which the SP, prepro domain and/or CD are not naturally associated can be on the SP 's, prepro domain and/or CD's amino terminal end, carboxy terminal end, and/or on both ends of the SP and/or CD.
  • the invention provides an isolated or recombinant polypeptide comprising (or consisting of) a polypeptide comprising a signal sequence (SP), prepro domain and/or catalytic domain (CD) of the invention with the proviso that it is not associated with any sequence to which it is naturally associated (e.g., enzyme sequence).
  • SP signal sequence
  • CD catalytic domain
  • the invention provides isolated or recombinant nucleic acids encoding these polypeptides.
  • the isolated or recombinant nucleic acid of the invention comprises coding sequence for a signal sequence (SP), prepro domain and/or catalytic domain (CD) of the invention and a heterologous sequence (i.e., a sequence not naturally associated with the a signal sequence (SP), prepro domain and/or catalytic domain (CD) of the invention).
  • the heterologous sequence can be on the 3' terminal end, 5' terminal end, and/or on both ends of the SP, prepro domain and/or CD coding sequence.
  • the invention provides fusion of N-terminal or C-terminal subsequences of enzymes of the invention (e.g., signal sequences, prepro sequences) with other polypeptides, active proteins or protein fragments.
  • enzymes of the invention e.g., signal sequences, prepro sequences
  • the production of an enzyme of the invention may also be accomplished by expressing the enzyme as an inactive fusion protein that is later activated by a proteolytic cleavage event (using either an endogenous or exogenous protease activity, e.g. trypsin) that results in the separation of the fusion protein partner and the mature enzyme, e.g., enzyme of the invention.
  • the fusion protein of the invention is expressed from a hybrid nucleotide construct that encodes a single open reading frame containing the following elements: the nucleotide sequence for the fusion protein, a linker sequence (defined as a nucleotide sequence that encodes a flexible amino acid sequence that joins two less flexible protein domains), protease cleavage recognition site, and the mature enzyme (e.g., any enzyme of the invention) sequence.
  • a linker sequence defined as a nucleotide sequence that encodes a flexible amino acid sequence that joins two less flexible protein domains
  • protease cleavage recognition site e.g., any enzyme of the invention
  • the fusion protein can comprise a pectate lyase sequence, a xylanase sequence, a phosphatidic acid phosphatase sequence, or another sequence, e.g., a sequence that has previously been shown to be over-expressed in a host system of interest.
  • Any host system can be used (see discussion, above), for example, E. coli or Pichia pastoris .
  • the arrangement of the nucleotide sequences in the chimeric nucleotide construction can be determined based on the protein expression levels achieved with each fusion construct.
  • the nucleotide sequences is assembled as follows: Signal sequence/fusion protein/linker sequence/protease cleavage recognition site/ mature enzyme (e.g., any enzyme of the invention) or Signal sequence/pro sequence/mature enzyme/linker sequence/fusion protein.
  • the expression of enzyme (e.g., any enzyme of the invention) as an inactive fusion protein may improve the overall expression of the enzyme's sequence, may reduce any potential toxicity associated with the overproduction of active enzyme and/or may increase the shelf life of enzyme prior to use because enzyme would be inactive until the fusion protein e.g. pectate lyase is separated from the enzyme, e.g., hydrolase or oxidoreductases, or enzymes with decontamination activity.
  • the invention provides specific formulations for the activation of an enzyme of the invention expressed as a fusion protein.
  • the activation of the enzyme activity initially expressed as an inactive fusion protein is accomplished using a proteolytic activity or potentially a proteolytic activity in combination with an amino-terminal or carboxyl-terminal peptidase.
  • the peptides and polypeptides of the invention can also be glycosylated, for example, in one aspect, comprising at least one glycosylation site, e.g., an N-linked or O-linked glycosylation.
  • the polypeptide can be glycosylated after being expressed in a P. pastoris or a S. pombe.
  • the glycosylation can be added post-translationally either chemically or by cellular biosynthetic mechanisms, wherein the later incorporates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence.
  • the invention provides hybrid enzymes and fusion proteins, including peptide libraries, comprising sequences of the invention.
  • the peptide libraries of the invention can be used to isolate peptide modulators (e.g., activators or inhibitors) of targets.
  • the peptide libraries of the invention can be used to identify formal binding partners of targets, such as ligands, e.g., cytokines, hormones and the like.
  • the fusion proteins of the invention are conformationally stabilized (relative to linear peptides) to allow a higher binding affinity for targets.
  • the invention provides fusions of enzymes of the invention and other peptides, including known and random peptides. They can be fused in such a manner that the structure of the enzymes are not significantly perturbed and the peptide is metabolically or structurally conformationally stabilized. This allows the creation of a peptide library that is easily monitored both for its presence within cells and its quantity.
  • Amino acid sequence variants of the invention can be characterized by a predetermined nature of the variation, a feature that sets them apart from a naturally occurring form, e.g, an allelic or interspecies variation of an enzyme sequence.
  • the variants of the invention exhibit the same qualitative biological activity as the naturally occurring analogue.
  • the variants can be selected for having modified characteristics.
  • the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed enzyme variants screened for the optimal combination of desired activity.
  • amino acid substitutions can be single residues; insertions can be on the order of from about 1 to 20 amino acids, although considerably larger insertions can be done.
  • Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70 residues or more.
  • substitutions, deletions, insertions or any combination thereof may be used. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.
  • the invention provides enzymes where the structure of the polypeptide backbone, the secondary or the tertiary structure, e.g., an alpha-helical or beta-sheet structure, has been modified.
  • the charge or hydrophobicity has been modified.
  • the bulk of a side chain has been modified.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative. For example, substitutions can be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example an alpha-helical or a beta- sheet structure; a charge or a hydrophobic site of the molecule, which can be at an active site; or a side chain.
  • the invention provides substitutions in polypeptide of the invention where (a) a hydrophilic residues, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g.
  • variants can exhibit the same qualitative biological activity (i.e. enzyme activity) although variants can be selected to modify the characteristics of the enzymes as needed.
  • enzymes of the invention comprise epitopes or purification tags, signal sequences or other fusion sequences, etc.
  • the enzymes of the invention can be fused to a random peptide to form a fusion polypeptide.
  • fused or “operably linked” herein is meant that the random peptide and the enzyme are linked together, in such a manner as to minimize the disruption to the stability of the enzyme structure, e.g., it retains enzyme activity.
  • the fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) can comprise further components as well, including multiple peptides at multiple loops.
  • the peptides e.g., enzyme subsequences
  • nucleic acids encoding them are randomized, either fully randomized or they are biased in their randomization, e.g. in nucleotide/residue frequency generally or per position.
  • Randomized means that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively.
  • the nucleic acids which give rise to the peptides can be chemically synthesized, and thus may incorporate any nucleotide at any position. Thus, when the nucleic acids are expressed to form peptides, any amino acid residue may be incorporated at any position.
  • the synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized nucleic acids.
  • the library can provide a sufficiently structurally diverse population of randomized expression products to affect a probabilistically sufficient range of cellular responses to provide one or more cells exhibiting a desired response.
  • the invention provides an interaction library large enough so that at least one of its members will have a structure that gives it affinity for some molecule, protein, or other factor.
  • a variety of apparatus and methodologies can be used to in conjunction with the polypeptides and nucleic acids of the invention, e.g., to screen polypeptides for enzyme activity, to screen compounds as potential activators or inhibitors of an enzyme activity (e.g., for potential drug screening), for antibodies that bind to a polypeptide of the invention, for nucleic acids that hybridize to a nucleic acid of the invention, to screen for cells expressing a polypeptide of the invention and the like. See, e.g., U.S. Patent No. 6,337,187. Capillary Arrays
  • Capillary arrays such as the GIGAMATRIXTM, Diversa Corporation, San Diego, CA, can be used to in the methods of the invention.
  • Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array, including capillary arrays.
  • Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention.
  • Capillary arrays provide another system for holding and screening samples.
  • a sample screening apparatus can include a plurality of capillaries formed into an array of adjacent capillaries, wherein each capillary comprises at least one wall defining a lumen for retaining a sample.
  • the apparatus can further include interstitial material disposed between adjacent capillaries in the array, and one or more reference indicia formed within of the interstitial material.
  • a capillary for screening a sample wherein the capillary is adapted for being bound in an array of capillaries, can include a first wall defining a lumen for retaining the sample, and a second wall formed of a filtering material, for filtering excitation energy provided to the lumen to excite the sample.
  • a polypeptide or nucleic acid e.g., a ligand or a substrate, can be introduced into a first component into at least a portion of a capillary of a capillary array.
  • Each capillary of the capillary array can comprise at least one wall defining a lumen for retaining the first component.
  • An air bubble can be introduced into the capillary behind the first component.
  • a second component can be introduced into the capillary, wherein the second component is separated from the first component by the air bubble.
  • a sample of interest can be introduced as a first liquid labeled with a detectable particle into a capillary of a capillary array, wherein each capillary of the capillary array comprises at least one wall defining a lumen for retaining the first liquid and the detectable particle, and wherein the at least one wall is coated with a binding material for binding the detectable particle to the at least one wall.
  • the method can further include removing the first liquid from the capillary tube, wherein the bound detectable particle is maintained within the capillary, and introducing a second liquid into the capillary tube.
  • the capillary array can include a plurality of individual capillaries comprising at least one outer wall defining a lumen.
  • the outer wall of the capillary can be one or more walls fused together.
  • the wall can define a lumen that is cylindrical, square, hexagonal or any other geometric shape so long as the walls form a lumen for retention of a liquid or sample.
  • the capillaries of the capillary array can be held together in close proximity to form a planar structure.
  • the capillaries can be bound together, by being fused (e.g., where the capillaries are made of glass), glued, bonded, or clamped side-by- side.
  • the capillary array can be formed of any number of individual capillaries, for example, a range from 100 to 4,000,000 capillaries
  • a capillary array can form a micro titer plate having about 100,000 or more individual capillaries bound together
  • Nucleic acids or polypeptides (including en2ymes, peptides or antibodies) of the invention can be immobilized to or applied to an array
  • Arrays can be used to screen for or monitor libraries of compositions (e g , small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention
  • a monitored parameter is transcript expression of an enzyme gene
  • One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or "biochip.”
  • arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by
  • Polypeptide arrays can also be used to simultaneously quantify a plurality of proteins
  • the present invention can be practiced with any known “array,” also referred to as a “microarray” or “nucleic acid array” or “polypeptide array” or “antibody array” or “biochip,” or variation thereof
  • Arrays are gene ⁇ cally a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e g , oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e g , niRNA transc ⁇ pts
  • array or “microarray” or “biochip” or “chip” as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface, as discussed in further detail, below
  • the enzymes are used as immobilized forms Any immobilization method can be used, e g , immobilization upon an inert support such as diethylaminoethyl-cellulose, porous glass, chitin or cells Cells that express enzymes of the invention can be immobilized by cross-linking, e g with glutaraldehyde to a substrate surface
  • any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as desc ⁇ bed, for example, in U S Patent Nos 6,277,628, 6,277,489, 6,261,776, 6,258,606, 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr.
  • the invention provides isolated, synthetic or recombinant antibodies that specifically bind to an enzyme of the invention. These antibodies can be used to isolate, identify or quantify the enzyme of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention or other related enzymes.
  • the invention also provides antibodies, or "antibody-like” structures (discussed below), having an enzymatic activity of a polypeptide of the invention, e.g., an antibody or "antibody-like” structure of the invention having haloperoxidase activity (e.g., a chloroperoxidase activity), a dehalogenase activity, an oxidoreductase activity, and/or a prolidase or imidodipeptidase activity.
  • haloperoxidase activity e.g., a chloroperoxidase activity
  • dehalogenase activity e.g., a dehalogenase activity
  • an oxidoreductase activity e.g., an oxidoreductase activity
  • prolidase or imidodipeptidase activity e.g., imidodipeptidase activity.
  • antibody includes a peptide or polypeptide
  • antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • Single chain antibodies are also included by reference in the term "antibody
  • the antibodies can be used in immunoprecipitation, staining, immunoaff ⁇ nity columns, and the like.
  • nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of the invention.
  • the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased.
  • the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.
  • Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
  • Polypeptides or peptides can be used to generate antibodies, which bind specifically to the polypeptides of the invention.
  • the resulting antibodies may be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample.
  • a protein preparation such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the polypeptides of the invention.
  • the antibody is attached to a solid support, such as a bead or other column matrix.
  • the protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of the invention.
  • binding may be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding may be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the sample may be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassays, and Western Blots.
  • Polyclonal antibodies generated against the polypeptides of the invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to a non-human animal. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies which may bind to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used.
  • Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Techniques described for the production of single chain antibodies (see, e.g., U.S.
  • Patent No. 4,946,778 can be adapted to produce single chain antibodies to the polypeptides of the invention.
  • transgenic mice may be used to express humanized antibodies to these polypeptides or fragments thereof.
  • Antibodies generated against the polypeptides of the invention may be used in screening for similar polypeptides from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody and those polypeptides which specifically bind the antibody are detected. Any of the procedures described above may be used to detect antibody binding. Immobilized polypeptides
  • polypeptides including peptides, enzymes and antibodies of the invention, are used as immobilized forms.
  • the immobilized peptides, enzymes and antibodies of the invention can be used, e.g., for decontamination. Any immobilization method or form of support can be used, e.g., arrays, beads, capillary supports and the like, as described above.
  • peptide/ enzyme/ antibody immobilization can occur upon an inert support such as diethylaminoethyl-cellulose, porous glass, chitin or cells.
  • cells that express peptides, enzymes and antibodies of the invention can be immobilized by cross-linking, e.g. with glutaraldehyde to a substrate surface.
  • Immobilized enzymes of the invention can be prepared containing enzyme bound to a dry, porous particulate hydrophobic support, with a surfactant, such as a polyoxyethylene sorbitan fatty acid ester or a polyglycerol fatty acid ester.
  • the support can be an aliphatic olef ⁇ nic polymer, such as a polyethylene or a polypropylene, a homo- or copolymer of styrene or a blend thereof or a pre-treated inorganic support.
  • These supports can be selected from aliphatic olef ⁇ nic polymers, oxidation polymers, blends of these polymers or pre-treated inorganic supports in order to make these supports hydrophobic.
  • This pre- treatment can comprise silanization with an organic silicon compound.
  • the inorganic material can be a silica, an alumina, a glass or a ceramic.
  • Supports can be made from polystyrene, copolymers of styrene, polyethylene, polypropylene or from co-polymers derived from (meth)acrylates. See, e.g., U.S. Patent No. 5,773,266.
  • kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, transgenic seeds or plants or plant parts and/or polypeptides (e.g., peptides, enzymes and antibodies) of the invention.
  • the kits can also comprise the decontaminating, neutralizing or detoxifying compositions of the invention.
  • the kits also can contain instructional material teaching/ informing protocols for using the methodologies and the uses of the invention, e.g., for industrial, military, homeland security uses of the invention, as described herein.
  • the methods of the invention provide whole cell evolution, or whole cell engineering, of a cell to develop a new cell strain having a new phenotype by modifying the genetic composition of the cell, where the genetic composition is modified by addition to the cell of a nucleic acid, e.g., a enzyme-encoding nucleic acid of the invention.
  • a nucleic acid e.g., a enzyme-encoding nucleic acid of the invention.
  • At least one metabolic parameter of a modified cell is monitored in the cell in a "real time" or "on-line” time frame.
  • a plurality of cells such as a cell culture, is monitored in "real time” or “on-line.”
  • a plurality of metabolic parameters is monitored in "real time” or “on-line.”
  • Metabolic parameters can be monitored using the fluorescent polypeptides of the invention (e.g., enzymes of the invention comprising a fluorescent moiety).
  • Metabolic flux analysis is based on a known biochemistry framework.
  • a linearly independent metabolic matrix is constructed based on the law of mass conservation and on the pseudo-steady state hypothesis (PSSH) on the intracellular metabolites.
  • PSSH pseudo-steady state hypothesis
  • Metabolic phenotype relies on the changes of the whole metabolic network within a cell. Metabolic phenotype relies on the change of pathway utilization with respect to environmental conditions, genetic regulation, developmental state and the genotype, etc.
  • the dynamic behavior of the cells are analyzed by investigating the pathway utilization. For example, if the glucose supply is increased and the oxygen decreased during the yeast fermentation, the utilization of respiratory pathways will be reduced and/or stopped, and the utilization of the fermentative pathways will dominate.
  • the methods of the invention can help determine how to manipulate the fermentation by determining how to change the substrate supply, temperature, use of inducers, etc. to control the physiological state of cells to move along desirable direction.
  • the MFA results can also be compared with transcriptome and proteome data to design experiments and protocols for metabolic engineering or gene shuffling, etc.
  • any modified or new phenotype can be conferred and detected, including new or improved characteristics in the cell. Any aspect of metabolism or growth can be monitored.
  • the engineered phenotype comprises increasing or decreasing the expression of an mRNA transcript or generating new transcripts in a cell. This increased or decreased expression can be traced by use of a enzyme-encoding nucleic acid of the invention.
  • mRNA transcripts, or messages also can be detected and quantified by any method known in the art, including, e.g., Northern blots, quantitative amplification reactions, hybridization to arrays, and the like.
  • Quantitative amplification reactions include, e.g., quantitative PCR, including, e.g., quantitative reverse transcription polymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or "real-time kinetic RT-PCR" (see, e.g., Kreuzer (2001) Br. J. Haematol. 114:313-318; Xia (2001) Transplantation 72:907-914).
  • the engineered phenotype is generated by knocking out expression of a homologous gene.
  • the gene's coding sequence or one or more transcriptional control elements can be knocked out, e.g., promoters or enhancers.
  • the expression of a transcript can be completely ablated or only decreased.
  • the engineered phenotype comprises increasing the expression of a homologous gene. This can be effected by knocking out of a negative control element, including a transcriptional regulatory element acting in cis- or trans-, or, mutagenizing a positive control element.
  • a negative control element including a transcriptional regulatory element acting in cis- or trans-, or, mutagenizing a positive control element.
  • One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array.
  • the engineered phenotype comprises increasing or decreasing the expression of a polypeptide or generating new polypeptides in a cell. This increased or decreased expression can be traced by use of a enzyme or an antibody of the invention.
  • Polypeptides, peptides and amino acids also can be detected and quantified by any method known in the art, including, e.g., nuclear magnetic resonance (NMR), spectrophotometry, radiography (protein radiolabeling), electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffi ⁇ sion chromatography, various immunological methods, e.g.
  • polypeptides including enzymes, peptides, antibodies
  • polypeptides having a hydrolase activity, an esterase activity e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non- heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, including other enzymes with decontamination activity, e.g., for toxin or poison decontamination, including for example nerve
  • the invention provides compositions and methods for decontamination, e.g., of toxins such as nerve agents, e.g., V agents (VX agent), G agents (sarin, soman, cyclosarin) or H agents (e.g., mustard gases) and/or biological agents, for civilian, military and/or homeland security purposes.
  • toxins such as nerve agents, e.g., V agents (VX agent), G agents (sarin, soman, cyclosarin) or H agents (e.g., mustard gases) and/or biological agents, for civilian, military and/or homeland security purposes.
  • Non-heme chloroperoxidases nhCPOs
  • the invention provides polypeptides having chloroperoxidase activity, including non-heme chloroperoxidase (nhCPOs) activity, or heme-based chloroperoxidase activity, for e.g., bleaching and degradation of lignin or other natural products, s
  • nhCPOs non-heme chloroperoxidase
  • heme-based chloroperoxidase activity for e.g., bleaching and degradation of lignin or other natural products, s
  • these enzymes have hypohalite forming, oxygen transfer and perhydro lysis mechanisms of action.
  • Conventional or high throughput assays can be used to determine the activity of an enzyme, e.g. , if it is a heme or non-heme CPO of the invention, e.g., if a polypeptide is within the scope of the invention, as described, for example, in Pelletier (1995) Biochim. Biophys. Acta 1250:149-157 (describes halogenation assay by non-heme chloroperoxidase); Hofmann (1998) J. MoI. Biol.
  • the electrophiles formed by this mechanism can halogenate organic substrates.
  • the enzymes can also catalyze the transfer of oxygen from hydrogen peroxide to organic substrates.
  • Chemical bleaching of lignin in pulp and paper manufacture uses hypohalites at large excess and creates significant manufacturing and waste removal costs.
  • These novel enzymes can act to catalytically and locally form hypohalite in the paper making process and can substitute for expensive and environmentally burdensome chemical generation of hypohalite.
  • the assay was based on tetriso inhibitory activity toward butyrylcholinesterase (BChE) and uses resorufin butyrate as a detection substrate. Enzymatic hydrolysis of resoruf ⁇ n butyrate by BChE forms resorufin anion that is detected by its fluorescence emission. Enzyme hits were screened on the basis of fluorescence emission intensity detected in the host cultures containing tetriso degrading activity.
  • BChE butyrylcholinesterase
  • Three discrete enzymes (the exemplary polypeptides having a sequence as set forth in SEQ ID NO:76 (encoded, e.g., by SEQ ID NO:75), SEQ ID NO:78 (encoded, e.g., by SEQ ID NO:77) and SEQ ID NO:86 (encoded, e.g., by SEQ ID NO:85)) were ultimately identified with P-F bond hydrolysis activity.
  • the enzyme sequences were found to be homologous to a family of nhCPO enzymes.
  • the invention provides polypeptides (including enzymes, peptides, antibodies) of the invention having activity that can be separated into two classes of organophosphoesterases - which, in one aspect, can rapidly detoxify acetylcholinesterase- or butyrylcholinesterase- inhibitors by hydrolyzing P-S or P-F bonds (e.g., inhibitors applied as pesticides).
  • organophosphoesterases include enzymes, peptides, antibodies
  • the invention provides polypeptides (including enzymes, peptides, antibodies) of the invention having activity that can be separated into two classes of organophosphoesterases - which, in one aspect, can rapidly detoxify acetylcholinesterase- or butyrylcholinesterase- inhibitors by hydrolyzing P-S or P-F bonds (e.g., inhibitors applied as pesticides).
  • nucleic acids that encode them including vectors and transgenic cells and plants comprising these organophosphoesterase-encoding nucleic acids.
  • the invention provides transgenic cells and plants (and seeds) capable of expressing these enzyme sequences, e.g., in a crop plant, to provide a self- protecting, pesticide-resistant plant, e.g., a self-protecting, pesticide-resistant crop plant.
  • the transgenic cell and plant system of the invention provides the plant or cell protection under conditions of pesticide application to maintain robust growth and development of the cell or plant, e.g., a plant crop.
  • polypeptides having organophosphoesterase activity of the invention can also be directly applied to a cell or a plant, or alternatively, nucleic acids, vectors or infective entities (e.g., phages, viruses, fungi, etc.) can be used to infect or transduce a cell, plant or crop to effect expression of the organophosphoesterase in the cell, plant or crop, thereby generating a self-protecting, pesticide-resistant cell, plant or crop.
  • this expression e.g., from a vector, phage, virus, fungi, etc.
  • the expression is constitutive.
  • the invention provides polypeptides, nucleic acids, infective vehicles (e.g., viruses, phages comprising organophosphoesterase-encoding nucleic acids of the invention), transduced or infected cells or plants and/or transgenic plants to, and methods of using them, e.g., provide a self-protecting, pesticide-resistant cell or a plant.
  • organophosphoesterases of the invention can act to hydro lyze P-S or P-F bonds in inhibitors of acetyl- cholinesterases or butyrylcholinesterases.
  • the compositions and methods of the invention can be used to detoxify pesticide inhibitors.
  • compositions and methods of the invention are non- toxic to the plants they are applied to as they primarily detoxify pesticides.
  • these compositions and methods of the invention are used to detoxify the following pesticides, and analogs and equivalents thereof: Demeton-S, Demeton-S-methyl, Demeton-S-methylsulphon, Demeton-methyl, Parathion, Phosmet, Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos-ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate- methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Proth
  • the invention provides polypeptides (including enzymes, peptides, antibodies of the invention), and novel mixture/ formulation/ combinations of enzymes (including polypeptides of the invention, known enzymes, or a mixture thereof) for decontamination, neutralization or detoxification (which includes neutralization), e.g., of toxins such as nerve agents, e.g., V agents (VX agent), G agents (sarin, soman, cyclosarin, tabun) or H agents (e.g., mustard gases) and/or biological agents (e.g., anthrax), for civilian, military and/or homeland security purposes.
  • V agents VX agent
  • G agents sarin, soman, cyclosarin, tabun
  • H agents e.g., mustard gases
  • biological agents e.g., anthrax
  • H agents (mustard) and the persistent nerve agent VX contain sulfur molecules that are readily subject to oxidation reactions; thus, the invention provides polypeptides and methods for catalyzing the oxidation of sulfur-containing toxins, such as H agents (e.g., mustard gas).
  • V agents, e.g., VX and the G agents (G nerve agents) contain phosphorus groups that can be hydrolyzed; thus, the invention provides polypeptides and methods for catalyzing the oxidation of phosphorus-containing toxins, such as VX.
  • Enzymes used to practice the decontamination, neutralization or detoxification compositions e.g., enzyme mixtures/ formulations/ combinations of the invention
  • methods of the invention including the exemplary combinations described herein for decontamination, neutralization or detoxification of V agents, H agents, G agents and biological agents, also include use of hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, in addition to the described haloperoxidases, e.g., heme-based (hCPO) or non-heme chloroperoxidases, dehalogenases, prolidases, imidodipeptidases and organophosphoric acid anhydrolases.
  • hydrolases such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoeste
  • Toxic agents that can be decontaminated, neutralized and/or detoxified by practicing the compositions and/or methods of the invention include: G- H- V
  • the invention provides enzymes, e g , haloperoxidases, including chloroperoxidases (CPOs), and mixtures/ formulations/ combinations of enzymes, for decontamination, neutralization or detoxification of V agents, such as VX, Russian V-gas (Russian VX), Tet ⁇ so or related compounds (see table, below), e g , any phosphorylthiocholine compound Haloperoxidases, e.g , CPOs, degrade all VX stereoisomers by oxidation
  • CPOs chloroperoxidases
  • Activity of chloroperoxidases of the invention, and of enzymes used to practice the decontamination, neutralization or detoxification formulations and methods of the invention also can include the activity comp ⁇ sing (the ability to) catalyze the hydrolysis of methylphosphono fluoridates and/or thiophospho ⁇ c esters
  • the compositions (e g., mixtures/ formulations/ combinations of enzymes) and/or methods of the invention can be used agamst.
  • V-Senes Agents decontaminated by compositions and methods of the invention are V-Senes Agents decontaminated by compositions and methods of the invention.
  • VX also is called by the chemical name methylphosphonothioic acid, S-[2-[bis(l- methylethyl)amino]ethyl]- O-ethyl ester, and has the molecular formula CnH 26 NO 2 PS and formula weight 267 37 CAS registry number 50782-69-9
  • VR is a liquid organophosphate nerve agent with an "oily" consistency which is colorless when pure, and is also called O -isobutyl S-(2-diethylammoethyl)methyl phosphothioate, O-isobutyl S-(2-diethylaminoethyl)methyl thiophosphonate, Oisobutyl S-(N,N-diethylammoethyl) methylphosphonothioate, Russian V-gas, Russian VX, RVX Tet ⁇ so, also detoxified by the enzymes of the invention, is a VX analogue, with the chemical name O,0-dns
  • Toxicity of nerve agents is typically desc ⁇ bed in 2 ways LCt50 and LD50 LCt50 refers to the inhalational toxicity of the vapor form.
  • Ct refers to the concentration of the vapor or aerosol in the air (measured as mg/m ) multiplied by the time the individual is exposed (measured in mmutes)
  • VX is the most toxic of the nerve agents (see Table 2)
  • VX also is the least volatile of the nerve agents This characte ⁇ stic makes VX a hazard by percutaneous and dermal routes
  • G agents tend to volatilize instead of penetrating the skin
  • the decontamination, detoxification (neutralization) methods of the invention can be practiced in conjunction with any known decontamination, detoxification (neutralization) protocol known in the art, e g , as desc ⁇ bed by Yang (1999) Chemical detoxification of nerve agent VX, Ace Chem Res 32 109-115, Szafraniec (1990) On the Stoichiometry of Phosphonothiolate Ester Hydrolysis, CRDEC-TR-212, July 1990, AD-A250773, Epstein (1974) The kinetics and mechanisms of hydrolysis of phosphonothiolates, Phosphorus, 1974, 4, 157-163, Ketelaar (1956) Metal-catalyzed hydrolysis of thiophospho ⁇ c esters, Nature 177 392-393, Albizo, et al , Hydrolysis of GD and VX, in Proceedmgs, Army Science Conference (16th), Volume 1, 25-27 October 1988, pp.
  • the invention provides mixtures/ formulations/ combinations of at least a haloperoxidase and/or a prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA) for decontamination, neutralization or detoxification of an V agent.
  • OPAA organophosphoric acid anhydrolase
  • the prolidase is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO: 194.
  • the haloperoxidase is a heme-based peroxidase activity
  • an enzyme having heme-based peroxidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:3; SEQ
  • the enzyme can be a heme- based chloroperoxidase activity encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 1 , or the enzyme can have an amino acid sequence as set forth in SEQ ID NO:2.
  • the enzymatic activity comprises a non-heme-based peroxidase activity
  • the enzyme having non-heme-based chloroperoxidase activity can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID
  • any known prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA) can be used in combination with any enzyme of this invention (see, e.g., U.S.
  • Patent No:s 6,080,566; 5,928,927; 6,524,834; 6,469,145; 6,838,277) including a prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA), or a haloperoxidase of the invention.
  • OPAA organophosphoric acid anhydrolase
  • any known haloperoxidase e.g., chloroperoxidase
  • 6,251,386 can be used in combination with any enzyme of this invention, including a prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA) or a haloperoxidase of the invention.
  • OPAA organophosphoric acid anhydrolase
  • hydrolases such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of V agents, such as VX, Soviet V-gas (Russian VX), Tetriso or related compounds, e.g., any phosphorylthiocho line compound.
  • V agents such as VX, Soviet V-gas (Russian VX), Tetriso or related compounds, e.g., any phosphorylthiocho line compound.
  • the invention provides enzymes, e.g., haloperoxidases, including chloroperoxidases, and/or dehalogenases, and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having haloperoxidase and/or dehalogenase activity, and/or known haloperoxidases and dehalogenases), for decontamination or detoxification (which includes neutralization) of H agents, such as mustard gas, which in one form has the formula 1, 1' thiobis [2 chloroethane] bis-(2-chloroethyl) sulphide; or, beta, beta' dichloroethyl sulphide; also known as 2, 2' dichloroethyl sulphide; or, bis (beta- chloroethyl) sulphide; or l-chloro-2 (beta-chlorodiethylthio) ethane; or sulphide,
  • the invention provides mixtures/ formulations/ combinations of at least a haloperoxidase and/or a dehalogenase for decontamination, neutralization or detoxification of an H agent.
  • the dehalogenase has a sequence as set forth in SEQ ID NO:70 (encoded, e.g., by SEQ ID NO:69) and/or SEQ ID NO:92 (encoded, e.g., by SEQ ID NO:91).
  • the dehalogenases of the invention and the dehalogenases used to practice the decontamination/ neutralization / detoxification compositions (e.g., formulations) and methods of the invention, act by de-chlorinating an H agent, e.g., a mustard gas.
  • an H agent e.g., a mustard gas.
  • the haloperoxidase is a heme-based peroxidase activity, e.g., an enzyme having heme-based peroxidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9;
  • the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:
  • the enzyme can be a heme- based chloroperoxidase activity encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 1 , or the enzyme can have an amino acid sequence as set forth in SEQ ID NO:2.
  • the enzymatic activity comprises a non-heme-based peroxidase activity
  • the enzyme having non-heme-based chloroperoxidase activity can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO: 65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
  • any known dehalogenase can be used in combination with any enzyme of this invention, including a dehalogenase or a haloperoxidase of the invention.
  • any known haloperoxidase e.g., chloroperoxidase
  • any enzyme of this invention including a dehalogenase or a haloperoxidase of the invention.
  • hydrolases such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of H agents.
  • the invention provides enzymes, e.g., prolidases, organophosphoric acid anhydrolases (OPAAs), and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having prolidase and/or OPAA activity, and/or known prolidases or OPAAs), for decontamination, neutralization or detoxification (which includes neutralization) of G agents - organophosphate nerve agents - such as tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF) (also, see above).
  • enzymes e.g., prolidases, organophosphoric acid anhydrolases (OPAAs), and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having prolidase and/or OPAA activity, and/or known prolidases or OPAAs), for decontamination, neutralization or detoxification (which includes neutralization) of G
  • Polypeptides having diisopropyl- fluorophosphatase (DFPase) activity can also be used in the compositions (e.g., mixtures/ formulations/ combinations of enzymes) and methods of the invention for decontamination, neutralization or detoxification of G agents.
  • DFPase diisopropyl- fluorophosphatase
  • prolidases and organophosphoric acid anhydrolases (OPAA) of the invention catalyze the hydrolysis of organophosphorous compound soman, and in one aspect, also catalyze the hydrolysis of the dipeptide Gly-Pro.
  • Sarin (GB) has the chemical name methylphosphonofluoridic acid, (1- methylethyl) ester, and has the molecular formula C 4 Hi O FO 2 P and formula weight 140.09. Its Chemical Abstracts Service registry number is 107-44-8.
  • Sarin and other G agents are rapidly hydrolyzed in basic solutions, e.g., Na 2 CO 3 , NaOH, or KOH; 1 GB has a half-life of 0.5 minutes at pH 11 at 25EC.
  • the invention can be practiced in conjunction with known means for hydrolyzing G agents, including e.g., known catalysts for GB hydrolysis such as hypochlorite anion (OCl " ), several metal ions and their complexes (Cu +2 , UO 2 +2 , ZrO +2 , Mo ⁇ 2 +2 , Th +4 ), and iodosobenzoic acid derivatives.
  • Decontamination systems based on this chemistry that can be incorporated into the protocols of this invention include: • solids, powders and solutions containing various types of bleach (NaOCl " or
  • G-series nerve agents share a number of common physical and chemical properties. At room temperature, the G-series nerve agents are volatile liquids, making them a serious risk for exposure by: dermal contact with liquid nerve agent or inhalation of nerve agent vapor. GB is the most volatile of these agents and evaporates at the same rate as water; GD is the next most volatile. Dispersal devices or an explosive blast also can aerosolize nerve agents. Nerve agent vapors are denser than air, making them particularly hazardous for persons in low areas or underground shelters. GB and GD are colorless, while GA ranges from colorless to brown. GB is odorless, while GA and GD smell fruity.
  • hydrolases such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of G agents.
  • the invention provides enzymes, e.g., haloperoxidases, including chloroperoxidases, dehalogenases, prolidases, organophosphoric acid anhydrolases (OPAAs), and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having similar activity), for decontamination or detoxification (which includes neutralization) of biological agents, e.g., biological warfare agents, including naturally occurring biological agents, e.g., spores and toxins from bacteria, such as Bacillus anthracis.
  • enzymes e.g., haloperoxidases, including chloroperoxidases, dehalogenases, prolidases, organophosphoric acid anhydrolases (OPAAs), and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having similar activity)
  • biological agents e.g., biological warfare agents, including naturally occurring biological agents, e.g.,
  • the toxic biological agents neutralized, ameliorated, sequestered or killed by practicing the invention include, e.g., spores from Bacillus anthracis, the bacterium that causes anthrax. Spores of Bacillus anthracis, can survive drought, bitter cold and other harsh conditions for decades, yet still germinate almost instantly to infect and kill once inside an animal or human host. Because anthrax spores may survive traditional drinking water disinfection methods and can attach themselves to the inside surface of water pipes, the compositions of the invention can be used to disinfect water treatment facilities in the unlikely event of the release of anthrax in a water supply.
  • the invention provides mixtures/ formulations/ combinations of at least a haloperoxidase for decontamination, neutralization or detoxification of a biological agent, e.g., spores and toxins from bacteria, such as Bacillus anthracis.
  • a biological agent e.g., spores and toxins from bacteria, such as Bacillus anthracis.
  • the haloperoxidase has a heme-based peroxidase activity
  • an enzyme having heme-based peroxidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:3; SEQ
  • the enzyme can be a heme- based chloroperoxidase activity encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 1 , or the enzyme can have an amino acid sequence as set forth in SEQ ID NO:2.
  • the enzymatic activity comprises a non-heme-based peroxidase activity
  • the enzyme having non-heme-based chloroperoxidase activity can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
  • any known haloperoxidase e.g., chloroperoxidase
  • any enzyme of this invention including a haloperoxidase of the invention.
  • hydrolases such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of biological agents, e.g., spores and toxins from bacteria, such as Bacillus anthracis.
  • biological agents e.g., spores and toxins from bacteria, such as Bacillus anthracis.
  • mixtures/ formulations/ combinations of the invention comprising at least a haloperoxidase also comprise a halite component (e.g., a solution comprising a halite component), e.g. a halite component comprising a chlorite component, such as a sodium chlorite or a sodium iodite (this is an alternative embodiment for all aspects of the invention comprising use of a haloperoxidase, e.g., a CPO, as a decontaminating reagent, whether for biological reagents or for other toxic agents).
  • the enzyme and chlorite component form ClO 2 (chlorine dioxide).
  • polypeptides of the invention can be formulated to achieve a desired effect in practicing the decontamination, detoxification (neutralization) methods of the invention.
  • the desired effect can be a therapeutic (e.g., including a detoxifying, neutralizing and/or a decontaminating effect) or a prophylactic (preventive) effect.
  • the polypeptides of the invention are formulated with emulsifiers, stabilizing agents, foaming agents, surfactants, foams, propellants, liposomes, nanostructures and the like, or can be formulated with appropriate compounds to generate emulsions.
  • any known application device can be used, for example, any spray or foaming device (e.g., for topical skin application), or use of any inhaler or nebulizer for direct inhalation of an enzyme or a formulation/ combination/ mixture of the invention, e.g., an inhaler or nebulizer as described in U.S. patent publication no. 20060039870.
  • Any pulmonary delivery device e.g., an inhaler (e.g., a dry powder inhaler) or nebulizer, which are well known in the art, can be used.
  • Pulmonary delivery devices are well known to provide local effects in the lungs and pulmonary system by delivering active agents, including chemical compounds, antibodies, polypeptides, and proteins.
  • Pulmonary delivery devices allow for higher bioavailability of an active agent due to the large surface area of the pulmonary epithelium, resulting in lower doses and fewer side effects. Furthermore, pulmonary delivery devices are cost-effective, easy to use, and are non-invasive.
  • a pulmonary delivery device for practicing the invention is activatable by inhalation; it can automatically dispense active agent (e.g., an enzyme, or a combination formulation, of the invention) upon inhalation.
  • active agent e.g., an enzyme, or a combination formulation, of the invention
  • Aerosol containers which contain an enzyme, or a combination formulation, of the invention can be used.
  • these devices can comprise use of propellants.
  • Devices used to practice the invention can administer a plurality of metered doses in a controlled manner, allowing controlled and consistent dosing of active agents into the subject's skin, mouth, bronchial passages, pulmonary epithelium.
  • Devices used to practice the invention can operate by utilizing a propellant to eject a constant volume of an active agent, which is adsorbed and/or inhaled by the subject.
  • the formulations of the invention, and/or a device used to practice the invention can include a surfactant to prevent aggregation of the "active agent” (e.g., comprising an enzyme, or a combination formulation, of the invention).
  • the "active agent” can be dissolved or suspended in solution.
  • Devices used to practice the invention can utilize propellants for simultaneous inhalation (for pulmonary inhalation devices) or surface application (for skin or mouth delivery devices) and activation of active agent.
  • holding chambers e.g., spacers, are used to store the aerosolized "active agent", eliminating the need for simultaneous activation and inhalation.
  • a device used to practice the invention provides a constant, metered dosage of the active agent to allow for consistent dosing.
  • nebulizers are used to deliver "active agents", e.g., compositions comprising an enzyme, or a combination formulation, of the invention.
  • Nebulizers can operate by creating a mist, i.e., nebulizing or atomizing, a formulation of the invention in solution, which is inhaled by the subject.
  • the active agent can be dissolved or suspended in solution.
  • the droplets can be created by any method known in the art, including the use of a fan, a vibrating member, or ultrasonic apparatus.
  • Nebulizers can be more "gentle" than inhalers and powered propellant devices used to practice this invention, and are appropriate for individuals unable to use inhalers, such as infants, young children, and individuals that are seriously ill or incapacitated. Examples of nebulizers are described in, e.g., U.S. patent nos. 6,029,661; 6,748,945; 6,530,370; 6,598,602; 6,009,869.
  • Devices used to practice the invention can administer dry powders which is "eaten” (mouth administered) or inhaled by the subject.
  • any method known in the art can be used to propel the active agent, including pneumatic systems, powered fans, or mechanical propulsion, e.g., squeezing of the container.
  • An exemplary device can simply upon the inhalation by the subject.
  • active agent are blended with propellants.
  • there is no blending thus allowing delivery of larger payloads of active agent; see e.g., U.S. patent no. 6,029,661.
  • devices used to practice the invention generate an aerosolization of a liquid or a dry powder formulation of the invention, e.g., for skin application, mouth administration, or for inhalation into the lung; and in one aspect, a propellant is used.
  • a propellant can be used, e.g., a chloroflourocarbon, a hydrofluorocarbon, a hydochlorofluorocarbon, or a hydrocarbon, including triflouromethane, dichlorodiflouromethane, dichlorotetrafiioroethanol, and 1,1,1,2-tetraflouroethane, or combinations thereof; see, e.g., propellant formulations as described in U.S. patent no.5,672,581.
  • the present invention also encompasses any methods known in the art for skin, mouth and/or pulmonary administration; e.g., delivery by intradermal or topical administration, intratracheal inhalation, insufflation, or intubation.
  • the present invention can encompass delivery of a formulation of the invention by solution, a powder, or a mist into the mouth, lungs, or onto or into the skin, by a syringe, tube, or similar device.
  • the invention also provides compositions, e.g., pharmaceutical compositions, comprising an enzyme, or a combination formulation, of the invention.
  • a pharmaceutical composition of the invention can comprise an enzyme or a combination formulation of the invention in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.
  • compositions can also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline, PBS), carbohydrates (e.g., glucose, mannose, sucrose or dextrose), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline, PBS
  • carbohydrates e.g., glucose, mannose, sucrose or dextrose
  • mannitol proteins
  • proteins polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., antioxidants, chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g.,
  • particles are suspended or dissolved within a propellant.
  • Dry powders suitable can include amorphous active agents, crystalline active agents and mixtures of both amorphous and crystalline active agents.
  • Dry powder active agents can have a particle size selected to prevent penetration into the alveoli of the lungs, e.g., from about 0.01 ⁇ m to about 4 ⁇ m, or less than 3 ⁇ m, or from about 0.5 ⁇ m to about 1 ⁇ m, or about 1 ⁇ m in diameter.
  • dry powder active agents have a moisture content below about 10% by weight, or below about 5% by weight, or below about 3% by weight.
  • a lyophilized compound of the invention (including only one, or a combination of enzymes)xomprises (is comprised of) 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more of enzyme.
  • Dry powders used to deliver enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention can be prepared by spray drying under conditions which result in a substantially amorphous powder.
  • Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention can be dissolved in a physiologically acceptable aqueous buffer, e.g., a citrate buffer having a pH range from about pH 2 to pH 9.
  • Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention can be dissolved at a concentration from 0.01% by weight to 1% by weight, e.g., at about 0.1% to 0.2%.
  • solutions of the invention are spray dried, and in one aspect, a substantially amorphous powder is generated, e.g., in a conventional spray drier, e.g., as available from a commercial suppliers such as Niro A/S (Denmark), Buchi (Switzerland) and the like.
  • spray dried powders e.g., amorphous powders
  • this formulation of the invention is ground or milled to produce particles within a desired size range.
  • Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention also can be in a crystalline form. Crystalline dry powders can be prepared by grinding or jet milling the bulk crystalline active agent.
  • Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention also can be with a powder dispersion device to provide more efficient and reproducible delivery of the active agent and to improve handling characteristics of the formulation of the invention.
  • Exemplary excipients used in formulations of the invention include carbohydrates, e.g., monosaccharides such as fructose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, cellobiose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; amino acids, such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, and the like; organic salts prepared from organic acids and bases, such as sodium cit
  • compositions of the invention can also comprise lactose, trehalose, raffinose, maitodextrins, glycine, sodium citrate, human serum albumin and mannitol for purposes of which they are well known in the art.
  • the amount of enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention, to be administered is an amount necessary to deliver a therapeutically effective or prophylactically effective amount to achieve a desired result, whether that be, in alternative aspects, only partial amelioration of symptoms or a complete abatement of symptoms.
  • amounts and types of formulations will vary widely depending upon the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention, used; the severity of the condition; the weight of the subject; desired therapeutic effect; nature and amount of toxic agent exposure.
  • the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention e.g., comprising a pharmaceutical composition of the invention, is delivered in one or more doses.
  • the enzyme of the invention or enzyme formulation/ mixture/ combination of the invention (e.g., comprising a pharmaceutical composition of the invention), is delivered in aerosol form.
  • a liquid aerosol formulation of the invention comprises compounds of the invention and a dispersing agent in a physiologically acceptable diluent.
  • the dry powder aerosol formulations are a finely divided solid form of enzymes of the invention, or enzyme formulations/ mixtures combinations of the invention and a dispersing agent.
  • the enzyme of the invention or enzyme formulation/ mixture/ combination of the invention (e.g., comprising a pharmaceutical composition of the invention) can be suspended, dispersed, or dissolved in solution, e.g., comprising a solvent or liquid dispersive medium that contains, for example, water, ethanol, a polyol (e.g. glycerol, propylene glycol or the like), vegetable oils, non-toxic glycerine esters and suitable mixtures thereof. Suitable flowability may be maintained, by generation of liposomes, administration of a suitable particle size in the case of dispersions, or by the addition of surfactants.
  • a solvent or liquid dispersive medium that contains, for example, water, ethanol, a polyol (e.g. glycerol, propylene glycol or the like), vegetable oils, non-toxic glycerine esters and suitable mixtures thereof.
  • Suitable flowability may be maintained, by generation of liposomes, administration of a suitable particle size in the case of dispers
  • the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention e.g., comprising a pharmaceutical composition of the invention
  • further comprise antibacterial and/or antifungal agents e.g., antibiotics, antibodies, nucleic acids, or a chemical, e.g., a paraben, chlorobutanol, or sorbic acid.
  • the compositions of the invention further comprise an isotonic substances, e.g. sugars, buffers and sodium chloride to assure osmotic pressure similar to those of body fluids, e.g., blood.
  • Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention also can be in the form of a sterile solution.
  • a sterile solution This can be prepared by using an appropriate solvent and /or an excipient (see above), followed by sterile filtering.
  • sterile powders suitable for use in the preparation of sterile injectable solutions Alternative preparation methods include drying in vacuum and lyophilization, which provide powdery mixtures of enzymes of the invention, or formulations/ mixtures of the invention, and desired excipients for subsequent preparation of sterile solutions.
  • Enzymes of the invention also can be formulated as appropriate to be compatible with the contaminated surface to be detoxified or decontaminated (or for prophylactic/ preventive application), e.g., a formulation and/or dosage compatible with skin, mucus membranes, eyes, mouth, nasal passages, lungs and the like.
  • enzymes of the invention are also formulated to be compatible with inanimate objects onto which they can be applied when practicing the compositions and/or methods of the invention, for example, when they are applied to, or are a part of, any application device as described herein, or any product of manufacture, e.g., a gas mask, an air or water filter, a piece of clothing, textile or fabric, a lens, wood, steel, glass, polymers and the like.
  • Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention also can be formulated as appropriate in time release formulations, e.g., pills, geltabs, nanostructures, hydrogels, capsules, liposomes, e.g., for ingestion, implantation (e.g., subdermal or intradermal), topical application (e.g., as a gel), as a suppository, or injection (e.g., I.V., parenteral, and the like).
  • the time release can be for treatment (including neutralization, detoxification or decontamination purposes) or for prophylactic purposes (e.g., to anticipate, yet neutralize, detoxify or decontaminate exposure).
  • Enzymes of the invention also can be formulated as appropriate for any living or inanimate object; e.g., the compositions and/or methods of the invention can be practiced on humans or animal, including domestic (e.g., pets, zoo or farm animals), experimental or wild animals, including, e.g., mammals, reptiles, fish or fowl, and the like.
  • domestic e.g., pets, zoo or farm animals
  • experimental or wild animals including, e.g., mammals, reptiles, fish or fowl, and the like.
  • Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention also can be formulated in appropriate dosages (e.g., in a tablet, a gel, a liposome, a pill, a powder, an aerosol, a capsule, a geltab, a hydrogel, a lotion, an injectable formulation, a nanostructure, and the like), where the duration and frequency of administration is determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration.
  • An appropriate dosage and treatment regimen can provide a therapeutic and/or a prophylactic benefit - an amelioration of symptoms or damage to the exposed individual.
  • Appropriate dosages can be determined using experimental models and/or clinical trials, e.g., using a minimum dosage that is sufficient to provide effective therapy. Patients can be monitored for therapeutic effectiveness using physical examination, imaging studies, or assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art. Dose adjustments can be made based on the monitoring findings. For example, an individual with exposure to nerve agent is administered a composition of the invention and cessation of symptoms is monitored to determine appropriate timing and amount of dosages.
  • apparatus for sampling for the presence of toxins or agents can be used to determine when to use a composition or method of the invention, or to determine the effectiveness of a composition or method of the invention.
  • a device for detecting the chemical warfare agent VX as described in U.S. patent application publication no. 20040161856, can be used in the course of practicing the invention.
  • Enzymes of the invention also can be formulated for use as anthrax decontamination/ neutralization solutions, e.g., decontaminating a surface material.
  • an anthrax decontamination solution of the invention is applied by foam, spray, mist, fog, or steam..
  • the compositions of the invention are enhanced by use of ultraviolet light application.
  • decontamination/ neutralization solutions/ formulations/ mixtures of the invention e.g., for V agents, G agents, H agents and/or biological agents (e.g., anthrax) or pesticides are used in military defense applications, or, alternatively, for any civilian application, including decontamination (including neutralization of toxicity) of buildings, post offices, ventilation ducts, carpet, clothes and electronic equipment.
  • the decontamination/ neutralization solutions of the invention can be formulated for compatibility with firefighting foams.
  • the invention also provides equipment comprising the decontamination/ neutralization solutions/ formulations/ mixtures of the invention, for the decontamination and/or neutralization of any piece of equipment, clothing, building, ship, airplane, truck, car, train, container and the like, from any V agents, G agents, H agents, biological agents and/or pesticides, herbicides, insecticides. Because in one aspect the invention provides coating and paints comprising the decontamination/ neutralization solutions/ formulations/ mixtures of the invention that can be applied to any object, the invention also provides any piece of equipment, clothing, building, ship, airplane, truck, car, train, container and the like comprising an enzyme of the invention, or a solution/ formulation/ mixture of the invention.
  • the invention provides decontamination/ neutralization solutions/ formulations/ mixtures comprising mixtures of different classes of enzymes, wherein these enzymes can be the invention, or enzymes of the invention and known enzymes, or novel combinations of known enzymes.
  • the invention provides decontamination/ neutralization solutions/ formulations/ mixtures comprising one, two, three or four or more enzymes of the invention, or enzymes of the invention and known enzymes, or novel combinations of known enzymes.
  • These enzyme combination solutions/ formulations/ mixtures can be used in methods and products of manufacture of the invention for the decontamination, neutralization or detoxification of biological agents, e.g., spores and toxins from bacteria, such as Bacillus anthracis.
  • an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) three enzymes, each in a different class: a dehalogenase, a haloperoxidase, such as a chloroperoxidase, and an organophosphoric acid anhydrolase (OPAA) or a prolidase.
  • a dehalogenase such as a chloroperoxidase
  • a haloperoxidase such as a chloroperoxidase
  • OPAA organophosphoric acid anhydrolase
  • the dehalogenase is a polypeptide having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
  • the haloperoxidase is a chloroperoxidase
  • the chloroperoxidase is a heme-based chloroperoxidase, e.g., a polypeptide encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:1, or having an amino acid sequence as set forth in SEQ ID NO:2.
  • the haloperoxidase is a heme-based peroxidase encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:11
  • the haloperoxidase is a non-heme- based chloroperoxidase encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:6l; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID
  • the organophosphoric acid anhydrolase (OPAA) is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or having a sequence as set forth in SEQ ID NO: 194.
  • the DFPase can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71, or having an amino acid sequence as set forth in SEQ ID NO:72.
  • this combination/ mixture/ formulation further comprises at least one diisopropylfluorophosphatase (DFPase).
  • DFPase diisopropylfluorophosphatase
  • the DFPase is exchangeable with (can be used as a substitute for) the OPAA/ prolidase enzyme.
  • additional enzymes can be added to this exemplary "triple combination, e.g., hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having any one of these activities.
  • hydrolases such as esterases, e.g., cholinesterases
  • organophosphohydrolases such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having any one of these activities.
  • the "triple combination" dehalogenase, haloperoxidase (e.g., chloroperoxidase) and organophosphoric acid anhydrolase (OPAA) solution/ formulation/ mixture of the invention has the advantage to the user because this composition detoxifies multiple toxic agents in one application: dehalogenase - H agents, such as mustard gas; haloperoxidase - V agents (e.g., VX gas) and biological agents (e.g., bacillus spores); OPAA - G agents (e.g., Sarin).
  • dehalogenase - H agents such as mustard gas
  • haloperoxidase - V agents e.g., VX gas
  • biological agents e.g., bacillus spores
  • OPAA - G agents e.g., Sarin
  • the composition when the "multiple combination" enzyme mixture of the invention comprises a haloperoxidase enzyme, the composition also comprises a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
  • a halite component e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
  • an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) two enzymes, each in a different class.
  • these “double enzyme combinations of the invention” can detoxify, neutralize or decontaminate multiple toxic agents in one application.
  • a “double enzyme combination of the invention” comprises at least one diisopropylfluorophosphatase (DFPase) or organophosphoric acid anhydrolase (OPAA) or a prolidase, and at least one haloperoxidase (e.g., chloroperoxidase, or CPO).
  • DFPase diisopropylfluorophosphatase
  • OPAA organophosphoric acid anhydrolase
  • prolidase e.g., chloroperoxidase, or CPO
  • haloperoxidase e.g., chloroperoxidase, or CPO
  • the DFPase and/or OPAA or prolidase are used to decontaminate, neutralize or detoxify G agents.
  • the haloperoxidase e.g., CPO
  • V agents H agents and/or biological agents (e.g., anthrax).
  • a dehalogenase is also added to this decontamination/ neutralization solution/ formulation/ mixture of the invention for added effect against H agents (e.g., mustard gas) (e.g., to supplement the effect of CPO on H agents).
  • H agents e.g., mustard gas
  • a cholinesterase is also added to this decontamination/ neutralization solution/ formulation/ mixture of the invention for added effect against on G agents (e.g., to supplement the effect of OPAA or prolidase and/or DFPase on G agents).
  • the polypeptide having diisopropyl-fluorophosphatase (DFPase) activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72.
  • haloperoxidases that can be used in this exemplary "double enzyme combination of the invention" are the same as discussed above for the dehalogenase/ haloperoxidase/ OPAA "triple combination” solution/ formulation/ mixture of the invention.
  • the enzyme having organophosphoric acid anhydrolase (OPAA) or prolidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
  • the composition can also comprise a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
  • a halite component e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
  • an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) two enzymes: at least one dehalogenase (DH) enzyme and at least one haloperoxidase (e.g., a chloroperoxidase, or CPO).
  • dehalogenases and haloperoxidases are used to decontaminate, neutralize, detoxify H agents.
  • Exemplary haloperoxidases that can be used in this exemplary "double enzyme combination of the invention” are the same as discussed above for the dehalogenase/ haloperoxidase/ OPAA "triple combination" solution/ formulation/ mixture of the invention.
  • Exemplary dehalogenases can have a sequence as set forth in SEQ ID NO: 69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
  • At least one diisopropylfluorophosphatase (DFPase), organophosphoric acid anhydrolase (OPAA) or prolidase and/or cholinesterase (or two, or all three) are also added to the DH and CPO mixture to also decontaminate, neutralize, detoxify G agents.
  • DFPase diisopropylfluorophosphatase
  • OPAA organophosphoric acid anhydrolase
  • prolidase and/or cholinesterase or two, or all three
  • the polypeptide having diisopropylfluorophosphatase (DFPase) activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO: 72.
  • the enzyme having organophosphoric acid anhydrolase (OPAA) or prolidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
  • the composition can also comprise a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
  • a halite component e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
  • an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) two enzymes: at least one dehalogenase (DH) enzyme and at least one organophosphoric acid anhydrolase (OPAA) or prolidase.
  • dehalogenases and haloperoxidases are used to decontaminate, neutralize, detoxify H agents (e.g., mustard gas); organophosphoric acid anhydrolases are used to decontaminate, neutralize, detoxify G agents (e.g., Sarin).
  • this exemplary mixture of the invention can also comprise at least one haloperoxidase to augment the decontamination, neutralization, detoxification of H agents.
  • the composition can also comprise a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
  • this exemplary mixture of the invention can also comprise at least one diisopropylfluorophosphatase (DFPase) and/or cholinesterase enzyme to supplement the OPAA or prolidase decontamination, neutralization, detoxification of G agent.
  • DFPase diisopropylfluorophosphatase
  • cholinesterase enzyme to supplement the OPAA or prolidase decontamination, neutralization, detoxification of G agent.
  • enzyme combinations of the invention can be formulated individually or collectively in one or more formulations.
  • enzymes used in these decontamination, neutralization, detoxification combinations of the invention can be individually or collectively formulated in any manner, e.g., as described herein, for example, as an edible delivery agent, an injectable liquid, a spray, a tablet, a pill, a gel, a hydrogel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, an aerosol, a powder, a lyophilized compound, a propellant, a foam, an emulsion, a nanostructure or a combination thereof.
  • a lyophilized compound of the invention (including only one, or a combination of enzymes) comprises (is comprised of) 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or more of enzyme.
  • Eyes The most common effects of nerve agents on the eyes are conjunctival injection and pupillary constriction, known as miosis.
  • the patient complains of eye pain, dim vision, and blurred vision. This is most likely from direct contact between the agent and eye.
  • Miosis may persist for long periods and may be unilateral. Severe miosis results in the complaint of dim vision. Ciliary spasm also may cause eye pain. Patients exposed to VX may not have miosis. This is most likely because the eye usually is not exposed directly to the agent, unlike with G agents. Miosis may be a delayed sign of VX exposure.
  • Nose Rhinorrhea is most common after a vapor exposure but also can be observed with exposures by other routes.
  • Lungs Shortness of breath is an important complaint. Patients may describe chest tightness, respiratory distress, or gasping and even may present in apnea. Broncho- constriction and excessive bronchial secretions cause these important life-threatening symptoms. With severe exposures, death may result from central respiratory depression and/or complete paralysis of the muscles of respiration. Respiratory failure is the major cause of death in nerve agent poisoning.
  • Skeletal muscle Fasciculations are the most specific identifiable manifestations of intoxication with these agents. Upon initial exposure they can be localized, but they then spread to cause generalized involvement of the entire musculature (after severe exposures). Myoclonic jerks (twitches) may be observed. Eventually, muscles fatigue and a flaccid paralysis ensues.
  • Heart The patient may present with either bradycardia or tachycardia. Heart rate depends on the predominance of adrenergic stimulation (resulting in tachycardia) or of the parasympathetic tone (causing bradycardia via vagal stimulation). Heart rate is an unreliable sign of nerve agent poisoning. Many disturbances in cardiac rhythm have been reported after both organophosphate and nerve agent poisonings. Heart blocks and premature ventricular contractions can be observed. The 2 arrhythmias of greatest concern that have been reported include torsade de pointes and ventricular fibrillation.
  • Central nervous system Smaller exposures to nerve agents may result in behavioral changes such as anxiety, psychomotor depression, intellectual impairment, and unusual dreams. Large exposures to nerve agents result in loss of consciousness and seizures. Most signs and symptoms are related to the excessive activation and subsequent fatigue at the cholinergic receptor. Some authors have divided exposures into minimal, moderate, and severe toxicity. Signs and symptoms associated with each exposure are
  • the invention provides enzymes, e.g., haloperoxidases, including chloroperoxidases, and/or dehalogenases, and mixtures/ formulations/ combinations of 15 enzymes, for decontamination or detoxification (which includes neutralization) of H agents, such as mustard gas.
  • haloperoxidases including chloroperoxidases, and/or dehalogenases
  • mixtures/ formulations/ combinations of 15 enzymes for decontamination or detoxification (which includes neutralization) of H agents, such as mustard gas.
  • H agents such as mustard gas.
  • mustard gas vapour or liquid In evaluating acute poisoning by mustard gas: the effects of exposure to mustard gas vapour or liquid are typically delayed for several hours. The delay is shorter in case of liquid contamination. In the first hour after exposure to mustard gas vapour or liquid no signs or symptoms are usually produced, but nausea, 20 retching, vomiting and eye smarting have been occasionally reported.
  • Exposure to superlethal concentrations may induce convulsions, coma and death within one hour after exposure.
  • Nausea, fatigue, headache, eye inflammation with intense eye pain, lachrymation, blepharospasm, photophobia and rhinorrhoea, followed by reddening of face and neck, soreness of throat and increased pulse and respiratory rate develop at two to six hours post exposure.
  • Six to twenty four hours post exposure the above symptoms are generally increased in severity and are accompanied by skin inflammation followed by blister formation in the warmest areas such as genito-perineal area, buttocks, axillae and on the inner aspects of thighs. In the next twenty four hours the condition generally worsens, blistering becomes more marked, coughing appears.
  • Mucus, pus and necrotic slough may be expectorated. Intense itching of skin and increased skin pigmentation occur. The blood count may reveal anemia and neutropenia four days post exposure. In general, initial leukocytosis on the first 2 to 3 days after exposure is followed by leukopenia in severe intoxicated patients.
  • the following exemplary enzymes of the invention hydrolyze P-F bonds (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO: 102, encoded, e.g., by SEQ ID NO: 101, etc.): SEQ ID NOS:
  • the following exemplary enzymes of the invention hydrolyze P-S bonds (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO:76, encoded, e.g., by SEQ ID NO:79, etc.): SEQ ID NOS: The following exemplary enzymes of the invention hydrolyze both P-S and P-F bonds (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO: 118, encoded, e.g., by SEQ ID NO:117, etc.):
  • the following exemplary enzymes of the invention can be used for decontamination, including by not limited to crop plant protection and/or for nerve agent detoxification; (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO: 118, encoded, e.g., by SEQ ID NO: 117, which include enzymes active against P-F and P-S bonds and/or G,V agents; etc.):

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Abstract

L'invention concerne de nouvelles enzymes et de nouveaux procédés de décontamination active à base d'enzymes, par exemple la désintoxication d'agents neurotoxiques. Dans un aspect, l'invention concerne des enzymes possédant une activité chloroperoxydase non hème (nhCPO) et des procédés de blanchiment et de dégradation de lignine. Cette invention concerne des polypeptides, des acides nucléiques, des véhicules infectieux, des cellules ou des plantes infectées ou transduites, et/ou des plantes transgéniques et des procédés utilisant celles-ci afin d'obtenir par exemple des plantes ou des cellules résistant aux herbicides et/ou aux pesticides, à protection autonome. Dans un aspect, les polypeptides de l'invention agissent de façon à hydrolyser des liaisons P-S ou P-F et désintoxiquer des acétylcholinestérases ou des butyrylcholinestérases. Dans un aspect, l'invention concerne un mélange d'enzymes ou une composition qui comprend des activités d'oxydation ou d'hydrolyse spécifique de l'agent à large spectre ainsi qu'une génération d'hypohalogénite pour l'oxydation ou l'hydrolyse d'agents V, d'agents G et d'agents H et qui génère des oxydants tels que la dioxine de chlorure et des radicaux réactifs destinés à tuer des agents biologiques, notamment des spores d'anthrax.
PCT/US2006/013031 2005-04-06 2006-04-06 Enzymes et préparations de décontamination d'agents de guerre chimiques et biologiques WO2008036061A2 (fr)

Priority Applications (4)

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CA002602185A CA2602185A1 (fr) 2005-04-06 2006-04-06 Enzymes et formulations pour decontamination a large specificite des agents de guerre chimique et biologique
EP06851338A EP1928560A2 (fr) 2005-04-06 2006-04-06 Enzymes et préparations de décontamination d'agents de guerre chimiques et biologiques
JP2008535508A JP2009517002A (ja) 2005-04-06 2006-04-06 化学及び生物兵器の広域特異性除染のための酵素及び処方物
IL186399A IL186399A0 (en) 2005-04-06 2007-10-07 Enzymes and formulations for broad-specificity decontamination of chemical and biological warfare agents

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US66933005P 2005-04-06 2005-04-06
US60/669,330 2005-04-06
US68713005P 2005-06-02 2005-06-02
US60/687,130 2005-06-02

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Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2248893A1 (fr) * 2009-05-06 2010-11-10 Novozymes A/S Enzymes DFPase issus d'Octopus Vulgaris
WO2010128116A1 (fr) * 2009-05-06 2010-11-11 Novozymes A/S Enzymes dfpase à partir d'aplysia californica
CN101451144B (zh) * 2008-06-11 2011-06-01 贵州大学 一种能够自发消除农残的果实种质培育方法
WO2011146629A3 (fr) * 2010-05-19 2012-05-31 Qiagen Gaithersburg Inc. Procédés et compositions pour purification séquence-spécifique et analyse multiplex d'acides nucléiques
WO2012087789A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
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WO2012087793A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
WO2012087790A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
WO2012087788A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
US8288520B2 (en) 2008-10-27 2012-10-16 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and system
WO2013021065A1 (fr) * 2011-08-10 2013-02-14 Novozymes A/S Polypeptides ayant une activité peroxygénase et polynucléotides codant pour ceux-ci
WO2013021061A1 (fr) * 2011-08-10 2013-02-14 Novozymes A/S Polypeptides ayant une activité peroxygénase et polynucléotides codant pour ceux-ci
WO2013021064A1 (fr) * 2011-08-10 2013-02-14 Novozymes A/S Polypeptides ayant une activité peroxygénase et polynucléotides codant pour ceux-ci
US8389219B2 (en) 2000-06-15 2013-03-05 Qiagen Gaithersburg, Inc. Detection of nucleic acids by type-specific hybrid capture method
WO2013079533A1 (fr) * 2011-12-02 2013-06-06 Novozymes A/S Polypeptides présentant une activité peroxygénase et polynucléotides codant pour ceux-ci
WO2013144105A1 (fr) * 2012-03-31 2013-10-03 Novozymes A/S Epoxydation utilisant la peroxygénase
WO2014056924A2 (fr) * 2012-10-12 2014-04-17 Novozymes A/S Polypeptides à activité peroxygénase
US8722328B2 (en) 2010-01-04 2014-05-13 Qiagen Gaithersburg, Inc. Methods, compositions, and kits for recovery of nucleic acids or proteins from fixed tissue samples
WO2014056917A3 (fr) * 2012-10-12 2014-06-19 Novozymes A/S Polypeptides à activité peroxygénase
WO2014056922A3 (fr) * 2012-10-12 2014-07-17 Novozymes A/S Polypeptides à activité peroxygénase
US8901287B2 (en) 2004-10-20 2014-12-02 Qiagen Gaithersburg, Inc. Detection of nucleic acids by target-specific hybrid capture method
US20150232816A1 (en) * 2012-10-12 2015-08-20 Novozymes A/S Polypeptides Having Peroxygenase Activity
US20160053239A1 (en) * 2013-03-22 2016-02-25 American University Of Cairo (Auc) Heavy metal resistant esterase
US9376727B2 (en) 2010-05-25 2016-06-28 Qiagen Gaithersburg, Inc. Fast results hybrid capture assay and associated strategically truncated probes
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WO2023030374A1 (fr) * 2021-09-03 2023-03-09 中国人民解放军军事科学院军事医学研究院 Composition de décontamination et son application
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372465B2 (en) * 1998-03-18 2002-04-16 Novozymes A/S Haloperoxidases with altered pH profiles
US6455751B1 (en) * 1999-03-03 2002-09-24 The Regents Of The University Of California Oxidizer gels for detoxification of chemical and biological agents
US20020165113A1 (en) * 2000-09-07 2002-11-07 Unilever Home & Personal Care Usa, Division Of Conopco, Inc. Detergent compositions comprising novel phenol oxidizing enzymes
US6703540B1 (en) * 2000-03-09 2004-03-09 The United States Of America As Represented By The Secretary Of Agriculture Transformation of plants with a chloroperoxidase gene to enhance disease resistance
US20040048249A1 (en) * 2000-01-21 2004-03-11 Tang Y. Tom Novel nucleic acids and secreted polypeptides
US20040109853A1 (en) * 2002-09-09 2004-06-10 Reactive Surfaces, Ltd. Biological active coating components, coatings, and coated surfaces

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372465B2 (en) * 1998-03-18 2002-04-16 Novozymes A/S Haloperoxidases with altered pH profiles
US6455751B1 (en) * 1999-03-03 2002-09-24 The Regents Of The University Of California Oxidizer gels for detoxification of chemical and biological agents
US20040048249A1 (en) * 2000-01-21 2004-03-11 Tang Y. Tom Novel nucleic acids and secreted polypeptides
US6703540B1 (en) * 2000-03-09 2004-03-09 The United States Of America As Represented By The Secretary Of Agriculture Transformation of plants with a chloroperoxidase gene to enhance disease resistance
US20020165113A1 (en) * 2000-09-07 2002-11-07 Unilever Home & Personal Care Usa, Division Of Conopco, Inc. Detergent compositions comprising novel phenol oxidizing enzymes
US20040109853A1 (en) * 2002-09-09 2004-06-10 Reactive Surfaces, Ltd. Biological active coating components, coatings, and coated surfaces

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CONESA ET AL.: 'Expression of the Caldariomyces fumago Chloroperoxidase in Aspergillus niger and Characterization of the Recombinant Enzyme' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 276, no. 21, 25 May 2001, pages 17635 - 17640 *
NUELL ET AL.: 'Isolation and Nucleotide Sequence of the Chloroperoxidase Gene from Caldariomyces fumago' JOURNAL OF BACTERIOLOGY vol. 170, no. 2, February 1988, pages 1007 - 1011 *

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WO2008036061A3 (fr) 2008-12-18
JP2009517002A (ja) 2009-04-30

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