US 2011/0296543 A1 · NUCLEIC ACIDS AND PROTEINS AND METHODS FOR MAKING AND USING THEM

Abstract

The invention provides polypeptides, including enzymes, structural proteins and binding proteins, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. Polypeptides, including enzymes and antibodies, and nucleic acids of the invention can be used in industrial, experimental, food and feed processing, nutritional and pharmaceutical applications, e.g., for food and feed supplements, colorants, neutraceuticals, cosmetic and pharmaceutical needs. Polypeptides of the invention can be used in food processing, brewing, bath additives, alcohol production, peptide synthesis, enantioselectivity, hide preparation in the leather industry, waste management and animal degradation, silver recovery in the photographic industry, medical treatment, silk degumming, biofilm degradation, biomass conversion to a biofuel (e.g., a bioethanol, biomethanol, biopropanol or biobutanol, a biodiesel, etc.), biodefense, antimicrobial agents and disinfectants, personal care and cosmetics, biotech reagents, in corn wet milling and pharmaceuticals such as digestive aids and anti-inflammatory (anti-phlogistic) agents.

An isolated, synthetic or recombinant nucleic acid comprising

(A) (a) 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 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, and all nucleic acids (polynucleotide sequences) disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:1 through SEQ ID NO:108,699, over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues, or over the full length of a protein coding sequence (transcript) or gene;
wherein the nucleic acid encodes at least one polypeptide having an enzymatic activity, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered (amino acid) SEQ ID NO:s in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO:108,699;
(b) a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid (a polynucleotide sequence) comprising 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, and all nucleic acids (polynucleotide sequences) disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:1 through SEQ ID NO:108,699,
wherein the nucleic acid encodes a polypeptide having an activity as set forth in Table 3, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered SEQ ID NO:s in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO:108,699,
and the stringent conditions include a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes,
and optionally the nucleic acid is at least about 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more residues in length or the full length of the gene or transcript;
(c) a nucleic acid (polynucleotide) encoding at least one polypeptide having enzyme activity, binding activity and/or structural activity, wherein the polypeptide comprises the sequence of 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, etc. and all polypeptides (amino acid sequences) disclosed in the SEQ ID listing, which include all even numbered (amino acid) SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:108,699, or enzymatically active fragments thereof;
(d) (i) the nucleic acid (polynucleotide) of (a), (b) or (c) and encoding a polypeptide having at least one conservative amino acid substitution and retaining its enzyme activity, binding activity and/or structural activity;
or, (ii) the nucleic acid of (d)(i), wherein the at least one conservative amino acid substitution comprises substituting an amino acid with another amino acid of like characteristics;
or, a conservative substitution comprises:
replacement of an aliphatic amino acid with another aliphatic amino acid;
replacement of a Serine with a Threonine or vice versa;
replacement of an acidic residue with another acidic residue;
replacement of a residue bearing an amide group with another residue bearing an amide group;
exchange of a basic residue with another basic residue;
or replacement of an aromatic residue with another aromatic residue;
(e) the nucleic acid (polynucleotide) of (a), (b), (c) or (d) encoding a polypeptide having an enzyme activity, binding activity and/or structural activity but lacking a signal sequence, a prepro domain, a dockerin domain, and/or a carbohydrate binding module (CBM),
wherein optionally the carbohydrate binding module (CBM) comprises, or consists of, a xylan binding module, a cellulose binding module, a lignin binding module, a xylose binding module, a mannanse binding module, a xyloglucan-specific module and/or a arabinofuranosidase binding module;
(f) the nucleic acid (polynucleotide) of (a), (b), (c), (d) or (e) encoding a polypeptide having an enzyme activity, binding activity and/or structural activity further comprising a heterologous sequence;
(g) the nucleic acid (polynucleotide) of (f), wherein the heterologous sequence comprises, or consists of a sequence encoding:
(i) a heterologous signal sequence, a heterologous carbohydrate binding module, a heterologous dockerin domain, a heterologous catalytic domain (CD), or a combination thereof;
(ii) the sequence of (ii), wherein the heterologous signal sequence, carbohydrate binding module or catalytic domain (CD) is derived from a heterologous enzyme;
or, (iii) a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme;
(h) the nucleic acid (polynucleotide) of (g), wherein the heterologous carbohydrate binding module (CBM) comprises, or consists of, a xylan binding module, a cellulose binding module, a lignin binding module, a xylose binding module, a mannanse binding module, a xyloglucan-specific module and/or a arabinofuranosidase binding module;
(i) the nucleic acid (polynucleotide) of (g), wherein the heterologous signal sequence targets the encoded protein to a vacuole, the endoplasmic reticulum, a chloroplast or a starch granule;
or
(j) a nucleic acid sequence (polynucleotide) fully (completely) complementary to (a), (b), (c), (d), (e), (f), (g), (h) or (i);
(B) the isolated, synthetic or recombinant nucleic acid of (A), wherein the sequence identity is 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%, 99%, or more sequence identity to 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, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:1 through SEQ ID NO:108,699;
(C) the isolated, synthetic or recombinant nucleic acid of (A) or (B), wherein the sequence identity is over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues, or the full length of a gene or a transcript;
(D) the isolated, synthetic or recombinant nucleic acid of any of (A) to (C), wherein the nucleic acid sequence comprises the sequence of 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, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:1 through SEQ ID NO:108,699;
(E) the isolated, synthetic or recombinant nucleic acid of any of (A) to (D), wherein the nucleic acid sequence encodes a polypeptide having the sequence of 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, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:1 through SEQ ID NO:108,699;
(F) the isolated, synthetic or recombinant nucleic acid of any of (A) to (E), wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection;
(G) the isolated, synthetic or recombinant nucleic acid of (F), wherein 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;
(H) the isolated, synthetic or recombinant nucleic acid of any of (A) to (G), wherein at least one enzymatic activity is one of the enzymatic activities described in Table 1, Table 3 and/or Table 4;
(I) the isolated, synthetic or recombinant nucleic acid of any of (A) to (H), wherein the protein is a structural or binding protein;
(J) the isolated, synthetic or recombinant nucleic acid of any of (A) to (I), wherein the at least one enzymatic activity has aldolase, alpha-galactosidase, amidase, secondary amidase, amylase, catalase, dehalogenase, endoglucanase, epoxide hydrolase, or esterase activity, or has hydrolase, protease, peptidase, glucosidase, glucosidase, intein, isomerase, laccase, lipase, or monooxygenase activity;
or has nitroreductase, nitrilase, P450 enzyme, pectate lyase, phosphatase, phospholipase, phytase, polymerase or xylanase activity;
or has at least one enzymatic activity in the carotenoid pathway;
(K) the isolated, synthetic or recombinant nucleic acid of any of (A) to (J), wherein the enzyme or protein is thermostable or thermotolerant, or the polypeptide retains enzyme activity, binding activity and/or structural activity under conditions comprising a temperature range from about −100° C. to about −80° C., about −80° C. to about −40° C., about −40° C. to about −20° C., about −20° C. to about 0° C., about 0° C. to about 37° C., about 0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. to about 25° C., about 25° C. to about 37° C., about 37° C. to about 45° C., about 45° C. to about 55° C., about 55° C. to about 70° C., about 70° C. to about 75° C., about 75° C. to about 85° C., about 85° C. to about 90° C., about 90° C. to about 95° C., about 95° C. to about 100° C., about 100° C. to about 105° C., about 105° C. to about 110° C., about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115° C. or more;
or
(L) the isolated, synthetic or recombinant nucleic acid of any of (A) to (K), where the isolated, synthetic or recombinant nucleic acid encodes a polypeptide having an enzyme activity, binding activity and/or structural activity, wherein the nucleic acid comprises a sequence that hybridizes under stringent conditions to a nucleic acid comprising 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, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:1 through SEQ ID NO:108,699, and the nucleic acid encodes a polypeptide having enzyme activity, binding activity and/or structural activity.

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Nucleic acids and proteins and methods for making and using them

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Nucleic acids and proteins and methods for making and using them

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Abstract

Claims32