ZA200503555B - Proteases, nucleic acids encoding them and methods for making and using them - Google Patents

Proteases, nucleic acids encoding them and methods for making and using them Download PDF

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ZA200503555B
ZA200503555B ZA200503555A ZA200503555A ZA200503555B ZA 200503555 B ZA200503555 B ZA 200503555B ZA 200503555 A ZA200503555 A ZA 200503555A ZA 200503555 A ZA200503555 A ZA 200503555A ZA 200503555 B ZA200503555 B ZA 200503555B
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South Africa
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seq
polypeptide
nucleic acid
sequence
set forth
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ZA200503555A
Inventor
Cayouette Michelle
Mcclure Amy
Gramatikova Svetlana
Barton Nelson
Aboushadi Nahla
Hansen Connie Jo
Sun May
Dycaico Mark
Stege Justin
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Diversa Corp
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Description

{ @® + WO 2004/4033668 PCT/US20()3/032819
Co PROTEASES, NUCLEIC ACIDS ENCODING THEM
AND METHODS FOR MAKING AND USING THEM
~ TECHNICAL FIELD :
This invention relates to molecular and cellular biology and biochemistry.
In particwlar, the invention relates to protease enzymes, polynucleotides encodirag the enzymes , methods of making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of diagnostic, therapeutic. and industrial contexts. The polypeptides of the invention can be used as, e.g., an aclditive for a deterge=nt, for processing foods and for chemical synthesis utilizing a reverse reaction.
Additionally, the polypeptides of the invention can be used in food processing, brewing, } bath addi tives, alcohol production, peptide synthesis, enantioselectivity, hide pre=paration in the lea-ther industry, waste management and animal degradation, silver recove=ry in the } photogragphic industry, medical treatment, silk degumming, biofilm degradation, . biomass conversion to ethanol, biodefense, antimicrobial agents and disinfectants, personal care and cosmeetics, biotech reagents, in increasing starch yield from corn wet millingz and pharmace=uticals such as digestive aids and anti-inflammatory (anti-phlogistic) aggents.
BACKGROUND
Enzymes are used within a wide range of applications in industr=y, research, and medicine. Through the use of enzymes, industrial processes can bes carried out at reduced temperatures and pressures and with less dependence on the use o=f corrosive or toxic substances. The use of enzymes can thus reduce production costs, energy co nsumption, and pollution as compared to non-enzymatic products and processes_ An important group of enzymes is the proteases. Proteases are carborayl hydrolase s which generally act to cleave peptide bonds of proteins or peptides.
Proteolytic enzymes are ubiquitous in occurrence, found in all living organisms, and are essential £or cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Inadustrial applicatioms of proteases include food processing, brewing, alcohol production, poeptide synthesis, enantioselectivity, hide preparation in the leather industry, waste management and anima | degradation, silver recovery in the photographic industry, medical trezatment, silk degumming, biofilm degradation, biomass conversion to ethanol, biodefense, antimicrobial agents and disinfectants, personal care and cosmetics, biotech reage=nts and in increasiang starch yield from corn wet milling. Additionally, proteases are impeortant compaenents of laundry detergents and other products. Within biological research, proteas=ses are used in purification processes to degrade unwanted proteins. It is often desirable to employ proteases of low specificity or mixtures of more specific proteases to obtain the necessary degree of degradation. . :
Proteases are classified according to their catalytic mechanisums. The
International Union of Biochemistry and Molecular Biology (IUBMB) recognizes four mecha_nistic classes: (1) the serine proteases; (2) the cysteine proteases; (3) thhe-aspartic proteas=ses; and (4) the metalloproteases. In addition, the [UBMB recognizes a class of endopeptidases (oligopeptidases) of unknown catalytic mechanism. Classification by catalytic types has been suggested to be extended by a classification by families based on the evolutionary relationships of proteases (see, e.g., Rawlings, N.D. and Barrett, A.J. (1993)e, Biochem. J., 290, 205-218). The serine proteases have alkaline pH optima, the metalleoproteases are optimally active around neutrality, and the cysteine and aspartic enzymes have acidic pH optima (Biotechnology Handbooks. Bacilluis. vol. 2. edited by 16 Harwawod, 1989 Plenum Press, New York). Aspartic proteases are rare for bacteria and to date noone have been reported for bacterial pathogens. Metalloproteases, on the other hand, sseem to be a common feature in most bacterial pathogens. Thus, basic two classes of bacterial proteases are serine proteases and metalloproteases.
Serine proteases are characterized by a catalytic triad of serirae, histidine, and aspartic acid residues. They include a diverse class of enzymes having a wide range of specificities and biological functions. The serine proteases class comprisess two distinc=t families: the chymotrypsin family, which includes the mammalian exazymes such as chy amotrypsin, trypsin, elastase, or kallikrein, and the subtilisin family, wiaich include the bacterial enzymes such as subtilisin. The general 3D structure is differerzt in two families, but they have the same active site geometry and catalysis proceeds ~via the same - mecha nism. Serine proteases are used for a variety of industrial purposes. F or example, the ser—ine protease subtilisin is used in laundry detergents to aid in the removal of proteiraceous stains (e.g., Crabb, ACS Symposium Series 460:82-94, 1991). In the food proces -sing industry, serine proteases are used to produce protein-rich conceratrates from fishamd livestock, and in the preparation of dairy products (Kida et al., Jourraal of
Ferme:-ntation and Bioengineering 80:478-484, 1995; Haard and Simpson, in Martin, A.
Co M., ed ., Fisheries Processing: Biotechnological Applications, Chapman and Hall, London, 1994, 132-154; Bos et al., European Patent Office Publication 494 149 Al).
Metalloproteases (MPs) and serine proteases form the most diverse of the catalytic types of proteases. They can be found in bacteria, fungi, as well as in higher organisms. They differ widely in their sequences and structures, but the gre=at majority of enzymes contain a zinc atom which is catalytically active. In some cases, zi nc may be replaced by another metal such as cobalt or nickel without loss of activity. Whe catalytic mechanism leads to the formation of a non-covalent tetrahedral intermediate= after the attack of” a zinc-bound water molecule on the carbonyl group of the scissile yond. This : intermed3ate is further decomposed by transfer of the glutamic acid portion to the leaving group.
In general, enzymes, including proteases, are active over a narrow range of environomental conditions (temperature, pH, etc.), and many are highly specizfic for particular substrates. The narrow range of activity for a given enzyme limits its applicability and creates a need for a selection of enzymes that (a) have similar activities but are active under different conditions or (b) have different substrates. For instance, an enzyme c apable of catalyzing a reaction at 50°C may be so inefficient at 35°CC, that its use at the low=er temperature will not be feasible. For this reason, laundry detergeents generally contain a selection of proteolytic enzymes, allowing the detergent t-o be used over a broad range of wash temperature and PH. In view of the specificity of proteolytic enzymes &and the growing use of proteases in industry, research, and medicines, there is an ongoing meed in the art for new enzymes and new enzyme inhibitors.
SUMMARY
The invention provides isolated or recombinant nucleic acids comprising a nucleic ac id sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%-, 56%, 57%, 58%, 59%m, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 7 1%, 72%, 73%, 74%, 75%, 76%, 71%, 18%, 19%, 80%, 81%, 82%, 83%, 84%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more=, or complete C100%) sequence identity to an exemplary nucleic acid of the invention, e.g,
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; SEEQID
NO:21; SEEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEZQID
NO:31; SEEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQID Co
NO:41; SEEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49; SE-QID
NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SE QID
NO:61; STEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID
NO:71; SZEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID
NO:81; STEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID
NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ ID N0:97; SEQ ID NO:99; SIEQ 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:L 19; SEQ ID
NO:121; SEQ IDNO:123; SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO: 129; SEQ ID
NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO: 139; SEQ ID
NO:141; SSEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO: X 50; SEQ ID
NO:158; SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO: 1 87; SEQ ID
NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:2218; SEQ ID
NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:2248 and/or
SEQ ID NJO:254, over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 105 O, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 175 0, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 250-0, or more residues, encodes at least one polypeptide having a protease activity, and the se=-quence identities are determined by analysis with a sequence compsarison algorithm or by a visual inspection.
Exemplary nucleic acids of the invention also include isolated or . recombinant nucleic acids encoding a polypeptide having a sequence as set feorth in SEQ
ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; oo
SEQ ID NNO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NI0:22; 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 NO0:40; SEQ ID NO:42; SEEQ ID
NO:44; SEEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:52; S.EEQ ID
NO:54; SEEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEEQ ID
NO:64; SEEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID
NO:74; SEEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82; SEEQ ID
NO:84; SEEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92; SEEQ ID
NO:94; SEEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQID
NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:L 12; SEQ ID
NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:L 22; SEQ ID
NO:124; SEQ 1D NO:126; SEQ ID NO:128; SEQ ID NOQ:130; SEQ ID NO: 1. 32; SEQ ID
Cr ‘ @® WO 2004/033668 PCT/US2003/032819 Co
NO:134; SEQ IID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID
NO:144; SEQ IID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID
NO:172; SEQ IID NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID
NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID
NO:235; SEQ IID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or a polypeptide encoded by SEQ» ID NO:145, and subsequences thereof and variants thereof. In one aspect, the polypeptide has a protease activity. —
The following list summarizes polypeptide sequence and nucleic acid coding sequence relationships between exemplary sequences of the invention; for example, SEQ IID NO:2 is encoded by SEQ ID NO:1, SEQ ID NO:255 is encoded by
SEQ ID NO:254 , etc.).
DNA SEQ Protein SEQ ID .
ID NOS: NOS: : 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 16 17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 . 33 34 36 37 38 39 40 41 42 43 44 ) 45 46 . .
© 47 48 49 50 51 52 53 54 : 55 56 57 58 59 60 61 62 —-- 63 84 : 65 66 67 68 69 70 7 72 : 73 74 : a 75 76 . 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 9% 97 08 99 100 101 102 103 104 105 106 107 108 109 110 111 112 : 113 114 115 116 117 118 119 120 121 122 . 6 :
A as . _ @® WO 2004/0323668 PCT/US -2003/032819 123 124 1258 126 1277 128 - 120 130 131 132 : 133 134 135- 136 137 138 - 139 140 141 142 143 144 145 N/A 146 147 . 150 151 :
Co 158 169 164 165 171 172 179 180 ’ 187 188 ’ 193 194 199 200 204 205 210 211 218 219 222 223 229 230 234 . 235 241 242 248 249 254 255
In one aspect, the invention also provides proteases, and protease-encoding nucleic acid s, with a common novelty in that they were initially isolated/ deriwved from mixed cultures. The invention provides protease-encoding nucleic acids isola_ted from mixed cultures comprising a nucleic acid sequence having at least about 10, 1_5, 20, 25, 30, 35, 40, BS, 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%, 16%, 77%, ,
78%, 7924, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 942%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequmence identity to an exemplary nucleic acid of the invention, e.g., SEQ ID NO:1; SEQ ID NO:3; SEQ ID
NO:5; SE2Q ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQQ ID NO:15;
SEQ IDIMO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID N0:23; SEQ ID» NO:25; SEQ
ID NO:2°7; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID N0:35; SEQ ID
NO:37; S-EQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID
NO:47; S-EQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ ID
NO:57; S-EQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID
NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID
NO:77; S EQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQID
NO:87; S EQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQID
NO:97; S-EQ ID NO:99; SEQ ID NO:101; SEQ ID N0:103; SEQ ID NO:1 05; SEQ ID
NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID N(O:115; SEQ ID
NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID N(O:125; SEQ ID
NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID N(O:135; SEQ ID
NO:137, SEQ ID NO:139; SEQ ID NO:141; SEQ ID NOQ:143; SEQ ID N(O:145; SEQ ID
NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NQ:164; SEQ ID N(O:171; SEQ ID
NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID N(:204; SEQ ID
NO:210; SEQ ID NO:218; SEQ ID N0:222; SEQ ID NO:229; SEQ ID N(O:234; SEQ ID
NO:241; SEQ ID NO:248 and/or SEQ ID NO:254, over a region of at leas& about 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 10550, 1100, 1150, or more.
In one aspect, the invention also provides proteases, and prcotease-encoding nucleic acids, with a common novelty in that were initially derived from a «common source, e. g., an archeal source, a bacterial source, a fungal source (e.g., fila_mentous ascomyce=tes, such as Cochliobolus heterostrophus, e.g., C. heterostrophus strain C4, having A" TCC accession no. 48331), or an environmental source, e.g., a mixed environmental source, e.g., as set forth below. . ) SEQ ID NOD: Source : 1,2 Archea 17,18 Archea 63, 64 Archea :
co SE : o WO 2004033668 PCT/US2003/0328 M9 16, 16 Bacteria i ) 13, 14 Bacteria ~ 5 6 Bacteria : 3,4 Bacteria 57,58 Bacteria 7.8 Bacteria : 187, 188 Cochlicbolus heterostrophus strain C4 (ATCC 48331) 210, 21 Cochliobolus heterostrophus strain C4 (ATCC 48331) = 234, 235 Cochliobolus heterostrophus strain C4 (ATCC 48331) 164, 165 Cochliobolus heterostrophus strain C4 (ATCC 48331) 189, 200 Cochliobolus heterostrophus strain C4 (ATCC 48331) 229, 230 Cochliobolus heterostrophus strain C4 (ATCC 48331) 158, 159 Cochliobolus heterostrophus strain C4 (ATCC 48331) 193, 194 Cochliobolus heterostrophus strain C4 (ATCC 48331) . 222, 223 Cochliobolus heterostrophus strain C4 (ATCC 48331) 179, 180» Cochliobolus heterostrophus strain C4 (ATCC 48331) 218, 219 Cochliobolus heterastrophus strain C4 (ATCC 48331) 150, 151 Cochliobolus heterostrophus strain C4 (ATCC 48331) : 171, 172 Cochliobolus heterostrophus strain C4 (ATCC 48331) 204, 205 Cochiiobolus heterostrophus strain C4 (ATCC 48331) 254, 255 Cochliobolus heterostrophus strain C4 (ATCC 48331) 248, 249 Cochliobolus heterostrophus strain C4 (ATCC 48331) 241, 242 Cochliobolus heterostrophus strain C4 (ATCC 48331) 85, 86 Environmental 11,12 Environmental 121, 122 Environmental 117, 118 Environmental 119, 120 Environmental 83, 84 Environmental : 9,10 Environmental 93, 94 Environmental 101, 102 Environmental 127, 128 . Environmental 129, 130 Environmental 139, 140 Environmental 146, 147 Environmental - 33, 34 Environmental 113, 114 Environmental . .
: NO 2004/033668 P«CT/US2003/032819 ® 39, 40 Environmental 71,72 Environmental 133, 134 Environmental 45, 46 Environmental 77.78 Environmental } 19, 20 Environmental © 89,60 ‘Environmental 41, 42 Environmental —— 111, 112 Environmental 123, 124 Environmental 125, 126 Environmental 107, 108 Environmental 109, 110 Environmental 79, 80 Environmental 23,24 Environmental 27,28 Environmental 143, 144 Environmental . 69, 70 Environmental 141, 142 Environmental : 55, 56 Environmental 61, 62 Environmental 73,74 Environmental 87, 88 Environmental 37, 38 Environmental 47, 48 Environmental 51, 52 Environmental 65, 66 Environmental ) 29, 30 Environmental 67, 68 Environmental 25, 26 Environmental 75,76 Environmental 81, 82 Environmental 31, 32 Environmental 35, 36 Environmental 43, 44 Environmental 49, 50 Environmental 137,138 Environmental 131, 132 Environmental :
( WOR 2004/033668 PCT/US2003/032819 . 95, 98 Environmental = 103, 104 Environmental 135, 136 Environmental : 145 Environmental | . 105, 106 Environmental 99, 100 Environmental . 97,98 Environmental 89, 90 Environmental - . 91,92 Environmental : 21,22 Environmental 116, 116 Environmental 63,54 Environmental
For example (referring to the above list), the proteases, and protease- . enc-oding nucleic acids, as set forth in SEQ ID NO:2 (encoded by SEQ ID NO:1), SEQ ID
NO :18 (encoded by SEQ ID NO: 17), SEQ ID NO:64 (encoded by SEQ ID NO:63) and
SEQQID NO:16 (encoded by SEQ ID NO:15) have a common novelty in that were init& ally derived from an archeal source, etc. with polypepticles and nucleic acids initially derived from bacterial, fungal (Cochliobolus heterostrophus=), or environmental sources.
In one aspect, the invention provides protease=s, and protease-encoding nucBeic acids, initially isolated/ derived from environmental sources, e.g., mixed envilronmental sources, comprising a nucleic acid sequence Bhaving at least about 10, 15, 20, 225, 30, 35, 40, 45, 50%, 51%, 52%, 53%, 54%, 55%, 564, 57%, 58%, 59%, 60%, : 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%om, 17%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 868%, 87%, 88%, 89%, 90%, 91%m, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequ_ence identity to an exemplary nucleic acid of the inventi_on 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, 1 150, 1200 or more, residu_es, wherein the nucleic acid enco-~des at least one polypeptide having a protease activity, a_nd the sequence identities are d_etermined by analysis with a sequence comparison algorithm or by a visual inspe=ction.
Regarding proteases, and protease-encoding n—ucleic acids, of the invention - with a common novelty in that they were initially derived frozm the filamentous ascormycetes Cochliobolus heterostrophus, in one aspect, thesse polypeptides and nucleic acids were initially isolated by growing Cochliobolus on either chicken feed or corn fiber, which was the sole nitrogemn source. Supernatant of the media was concentrated arad run ona gel. The resulting pro-teins isolated from the gel bands were analyzed by mas=s spectrometry. These prote#ins were sequenced and compared to the Cochliobolus g=enomic sequence. The proteases, amnd protease-encoding nucleic acids, of the invention in%_tially isolated from Cochliobolus= heterostrophus are summarized as follows: . SEQ ID MNO: of protein seqguence
SEQ ID NO: of faull SEQ ID NO: of DNA of codimng
Protease gene (exons and SEQ ID NOS: of sequence of coding sequence exons
ID introns) exon sequences __ sequence (exons only) only)»
A 181 182-186 187 188
B 206 207-209 210 21 c 231 232-233 234 235
D 160 161-163 164 165 .
E 195 196-198 199 =200
F 224 225-228 229 =30
G 162 163-157 158 “159
H 189 190-192 183 194 . I 220 221 222 223
J 173 174-178 179 -180
K 212 213-217 218 219
L 148 149 160 -161
M 166 167-170 171 —172
N 201 202-203 204 =05 o 250 251-253 254 =255
P 243 244-247 248 249
Q 236 237-240 241 2242
In one aspec=t, the sequence comparison algorithm is a BLAST versi_on 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 Hefault.
Another aspe=ct of the invention is an isolated or recombinant nuclei «c acid including at least 10 consec=utive bases of a nucleic acid sequence of the invention, : sequences substantially iderntical thereto, and the sequences complementary theretca.
In one aspect, protease activity of the invention comprises catalyzing hydrolysis of peptide bonds. The term “protease activity” includes hydrolysis of ary 12 a peptide bond, including protease activity, peptidase activity and/or proteinase activity.
The protease activity can comprise an endoprotease activity and/or an exoprotease activity. The protease activity can comprise= a carboxypeptidase activity, an : aminopeptidase activity, a serine protease ac=tivity, a metalloprotease activity (e.g., matrix metalloprotease or collagenase activity), a cysteine protease activity and/or an aspartic protease activity. In one aspect, protease actiivity can comprise activity the same or similar to a chymotrypsin, a trypsin, an elastase, a kallikrein and/or a subtilisiactivity.
The protease activity can comprise a peptidase activity, such as a dipeptidylpeptidase or a carboxypeptidase activity. In alternative asp-ects, the protease activity can comprise a . acrocylindropepsin activity, acrosin activity, actinidain activity, acylaminoacyl-peptidase activity, ADAM 17 endopeptidase activity, MA DAMI10 endopeptidase activity, adamalysin activity, ADAMTS-4 endopeptidase activity, adenain activity, aeromonolysin activity, oo alanine carboxypeptidase activity, alpha-lytic= endopeptidase activity, alternative- complement pathway C3/C5 convertase activ=ity, aminopeptidase B activity, aminopeptidase Ey activity, aminopeptidase WM activity, ananain activity, anthrax lethal factor endopeptidase activity, asclepain activity, aspartyl aminopeptidase activity, aspergillopepsin I activity, aspergillopepsin IN activity, assemblin activity, astacin activity, atrolysin A activity, atrolysin B activ=ity, atrolysin C activity, atrolysin E activity, atrolysin F activity, atroxase activity, aureolyssin activity, bacillolysin activity, bacterial leucyl aminopeptidase activity, barrierpepsin activity, Beta-Ala-His dipeptidase activity,
Beta-aspartyl-peptidase, beta-lytic metalloendllopeptidase activity, bleomycin hydrolase activity, bontoxilysin activity, bothrolysin activity, bothropasin activity, brachyurin activity, calpain-1 activity, calpain-2 activity, cancer procoagulant activity, candidapepsin activity, carboxypeptidase A activity, carboxy -peptidase A2 activity, carboxypeptidase B activity, carboxypeptidase C activity, carboxygpeptidase D activity, carboxypeptidase H activity, carboxypeptidase M activity, carboxypeptidase T activity, carboxypeptidase U activity, caricain activity, caspase-1 activity, cathepsin B activity, cathepsin D activity, cathepsin E activity, cathepsin F activity, cathepsin G activity, cathepsin H activity, cathepsin K activity, cathepsin L activity, cathepsin O activity, cathepsin S activity, cathepsin T activity, cathepsin V activity, cerewvisin activity, choriolysin H activity, choriolysin L activity, chymase activity, chymopapain activity, chymosin activity, chymotrypsin activity (e.g., chymotrypsin C ac=tivity), classical-complement pathway
C3/Cs5 convertase, clostridial aminopeptidase a_ctivity, clostripain activity, coagulation factor [Xa activity, coagulation factor VIIa acti vity, coagulation factor Xa activity,
@P coagulation factor Xa activity, coagulat=ion factor XIIa activity, coccolysin activity, complement component Clr activity, co-mplement component Cls activity, complement factor D activity, complement factor I activity, cruzipain activity, cucumisin activity, cysteine-type carboxypeptidase activity, cystinyl aminopeptidase activity, cytosol alanyl 6 aminopeptidase activity, cytosol nonspecific dipeptidase activity, dactylysin activity, deuterolysin activity, dipeptidase E activity, dipeptidyl-dipeptidase activity, dipeptidy!- peptidase I activity, dipeptidyl-peptidase= II activity, dipeptidyl-peptidase III aetivity, dipeptidyl-peptidase IV activity, D-stere-ospecific aminopeptidase activity, endopeptidase
Clp activity, endopeptidase La activity, endopeptidase So activity, endothelin-converting enzyme 1 activity, endothiapepsin activity, enteropeptidase activity, envelysin activity, fibrolase activity, ficain activity, flavast=acin activity, flavirin activity, fragilysin activity, fruit bromelain activity, furin activity, gzametolysin activity, gamma-D-glutamyl-meso- diaminopimelate peptidase I activity, gamnma-glutamyl hydrolase activity, gamma-renin activity, gastricsin activity, gelatinase A activity, gelatinase B activity, gingipain K activity, gingipain R activity, Glu-Glu d& peptidase activity, glutamate carboxypeptidase IT activity, Glutamate carboxypeptidase ac®ivity Glutamyl aminopeptidase activity,
Glutamyl endopeptidase II activity, Glut-amyl endopeptidase activity, Glycyl endopeptidase activity, Gly-X carboxype=ptidase activity, GPR endopeptidase activity,
Granzyme A activity, Granzyme B activity, Helper-component proteinase activity,
Hepacivirin activity, Histolysain activity~, HIV-1 retropepsin activity, HIV-2 retropepsin activity, Horrilysin activity, Hypodermira C activity, IgA-specific metalloendopeptidase activity, IgA-specific serine endopeptidase activity, Insulysin activity, Interstitial collagenase activity, Jararhagin activity, Kexin activity, Lactocepin activity, Legumain activity, Leishmanolysin activity, Leuco ysin activity, Leucyl aminopeptidase activity,
Leucyl endopeptidase activity, Leukocyt=e elastase activity, Limulus clotting enzyme activity, Limulus clotting factor B activisty, Limulus clotting factor C activity, L-peptidase activity, Lysine(arginine) carboxypeptidase activity, Lysosomal Pro-X carboxypeptidase activity, Lysostaphin activity, Lysyl amimopeptidase activity, Lysyl endopeptidase activity, Macrophage elastase activity, MTagnolysin activity, Matrilysin activity,
Memapsin 1, Memapsin 2, Membrane al -anine aminopeptidase, Membrane dipeptidase,
Membrane Pro-X carboxypeptidase, Mermnbrane-type matrix metalloproteinase-1, Meprin
A, Meprin B, Metallocarboxypeptidase ID, Methionyl aminopeptidase, Metridin, Met-Xaa dipeptidase, Microbial collagenase, Mito- chondrial intermediate peptidase, Mitochondrial processing peptidase, Mucoropepsin, Muacrolysin, Muramoylpentapeptide
( { @® WO 2004/033668 PCT/US2003/032819 carboxypeptidase, Muramoyltetrapeptide carbeoxypeptidase, Mycolysin, Myeloblastin,
Nardilysin, Neopenthesin, Neprilysin, Neurolysin, Neutrophil collagenase, N- formylmethionyl-peptidase, Nodavirus endope=ptidase, Non-stereospecific dipeptidase,
Nuclear-inclusion-a endopeptidase, OligopeptEdase A, Oligopeptidase B, Omptin, Ophiolysin, Oryzin, O-sialoglycoprotein endopeptidase, Pancreatic elastase II, Pancreatic elastase, Pancreatic endopeptidase E, Papain, Pappalysin-1, Penicillopepsin, PepB aminopeptidase, Pepsin A, Pepsin B, Peptidyl-_Asp metalloendopeptidase, Peptidyl- dipeptidase A, Peptidyl-dipeptidase B, Peptidy-1-dipeptidase Dcp, Peptidyl-glycinamidase,
Peptidyl-Lys metalloendopeptidase, Phytepsin,. Picornain 2A, Picornain 3G, Pitrilysin,
Plasma kallikrein, Plasmepsin I, Plasmepsin II,. Plasmin, Plasminogen activator Pla,
Polyporopepsin, Prepilin peptidase, Procollagem C-endopeptidase, Procollagen N- endopeptidase, Prolyl aminopeptidase, Prolyl o-ligopeptidase, Pro-opiomelanocortin converting enzyme, Proprotein convertase 1, Pxoprotein convertase 2, Proteasome endopeptidase complex, Protein C (activated), Proteinase K, Pseudolysin,
Pycnoporopepsin, Pyroglutamyl-peptidase I, Py/roglutamyl-peptidase II, Renin, Repressor lexA, Rhizopuspepsin, Rhodotorulapepsin, Ruberlysin, Russellysin, S2P endopeptidase,
Saccharolysin, Saccharopepsin, Scutelarin, Scytalidopepsin A activity, Scytalidopepsin B, . Semenogelase, Separase, Serine-type D-Ala-D—Ala carboxypeptidase, Serralysin, Signal peptidase I, Signal peptidase II, Snake venom factor V activator, Snapalysin, Spermosin, Staphopain, Ste24 endopeptidase, Stem bromelaain, Streptogrisin A, Streptogrisin B,
Streptopain, Stromelysin 1, Stromelysin 2, Subtilisin, Tentoxilysin, Thermitase,
Thermolysin, Thermomycolin, Thermopsin, Thesrmostable carboxypeptidase 1, Thimet : oligopeptidase, Thrombin activity, Tissue kallikrein activity, Togavirin activity, T- plasminogen activator activity, Trimerelysin I axctivity, Trimerelysin II activity, Tripeptide aminopeptidase activity, Tripeptidyl-peptidase LT activity, Tripeptidyl-peptidase IT activity,
Trypsin activity, Tryptase activity, Tryptophany-1 aminopeptidase activity, Tubulinyl-Tyr carboxypeptidase activity, Ubiquitinyl hydrolase 1 activity, U-plasminogen activator activity, V-cath endopeptidase activity, Venombein A activity, Venombin AB activity,
Xaa-Arg dipeptidase activity, Xaa-His dipeptida se, activity Xaa-methyl-His dipeptidase activity, Xaa-Pro aminopeptidase activity, Xaa-Fro dipeptidase activity, Xaa-Pro dipeptidyl-peptidase activity, Xaa-Trp aminopepwtidase activity, Yapsin 1 activity, Zinc D-
Ala-D-Ala carboxypeptidase activity or a combimation thereof.
Some alternative activities of exemplary polypeptides of the invention (for example, as listed above) were determined by ex_perimental data, by homology (sequence comparison) to other sequences, or by b-oth sequence comparison and experimental results. However, an exemplary speciess of the invention, or a genus of polypeptides based on an exemplary sequence, is not limited to any specific protease activity, Thus, in alternative, but not limiting aspects, a polypeptide having a sequence as set forth in SEQ
ID NO:2 (encoded by SEQ ID NO:1), can have an alkaline protease activity; a polypeptide having a sequence as set fomth in SEQ ID NO:4 (encoded by SEQ ID NO:3), can have a serine protease activity; a po lypeptide having a sequence as set forth-in SEQ "ID NO:6 (encoded by SEQ ID NO:5), can have a peptidase activity; a polypeptide having a sequence as set forth in SEQ ID NO:22 (encoded by SEQ ID NO:21, can have a serine protease activity; a polypeptide having za sequence as set forth in SEQ ID NO:26 (encoded by SEQ ID NO:25, can have a subtilisimm-like secreted protease activity; a polypeptide having a sequence as set forth in SEQ IBD NO:28 (encoded by SEQ ID NO:27), can have a : serine protease activity (e.g., an alkalines serine protease activity); a polypeptide having a sequence as set forth in SEQ ID NO:36 (encoded by SEQ ID NO:35), can have a serine : protease activity (e.g., an alkaline serine protease activity); a polypeptide having a sequence as set forth in SEQ ID NO:38 (encoded by SEQ ID NO:37), can have a serine protease activity; a polypeptide having & sequence as set forth in SEQ ID NO:42 (encoded by SEQ ID NO:41), can have a serine protease activity (e.g., an extracellular alkaline serine protease 2 activity); a polypeptides having a sequence as set forth in SEQ ID NO:50 (encoded by SEQ ID NO:49), can have a serine protease activity (e.g., an alkaline serine protease activity); a polypeptide having a sequence as set forth in SEQ ID NO:58 (encoded by SEQ ID NO:57), can have a serine protease activity; a polypeptide having a sequence as set forth in SEQ ID NO:68 (encoded by SEQ ID NO:67), can have a serine protease activity (e.g., an alkaline serine protease activity); a polypeptide having a sequence as set forth in SEQ ID NO:74 (encoded by SEQ ID NO:73), can have a serine protease activity (e.g., an alkaline serine protease activity); a polypeptide having a sequence as set forth in SEQ ID NO:76 (encoded by SEQ ID NO:75), can have a serine protease activity (e.g., a cold-active seri ne alkaline protease activity); a polypeptide having a sequence as set forth in SEQ [ID NO:82 (encoded by SEQ ID NO:81), can have a serine protease activity; a polypeptide having a sequence as set forth in SEQ ID NO:86 (encoded by SEQ ID NO:85), can have a protease II activity; a polypeptide having a sequence as set forth in SEQ ID NO:90 (encoded by SEQ ID NO:89), can have a serine metalloprotease activity; a polypeptide Enaving a sequence as set forth in SEQ ID NO:92 (encoded by SEQ ID NO:91), can have a metalloprotease activity; a polypeptide having a
@® WO 2004/033668 PCT/US2003/032819 sequence as set forth in SEQ ID NO:96 (encoded by SEQ ID NO:95), can have a serine protease activity (c.g., a cold-active serine alkaline protease activity); a polypeptide having a sequence as set forth in SEQ ID NO:98 (encoded by- SEQID N0:97), can have a peptidase activity; a polypeptide having a sequence as set for€h in SEQ ID NO:100 (encoded by SEQ ID NO:99), can have a probormone convertase activity; a polypeptide having a sequence as set forth in SEQ ID NO:106 (encoded b-y SEQ ID NO:105), can have a collagenase activity; a polypeptide having a sequence as set forth in SEQ ID
NO:112 (encoded by SEQ ID NO:1 11), can have an alkaline serine protease Il activity; a polypeptide having a sequence as set forth in SEQ ID NO:114# (encoded by SEQ ID
NO:113), can have a serine proteinase activity; a polypeptide “having a sequence as set forth in SEQ ID NO:120 (encoded by SEQ ID NO:119), can nave a subtilisin-like proteinase activity; a polypeptide having a sequence as set forth in SEQ ID NO:128 (encoded by SEQ ID NO:127), can have a serine proteinase activity (e.g., serine protease
A activity); a polypeptide having a sequence as set forth in SEEQ ID NO:134 (encoded by
SEQ ID NO:133), can have a leucine aminopeptidase activity = a polypeptide having a sequence as set forth in SEQ ID NO:136 (encoded by SEQ ID NO:135), can have a collagenase activity; a polypeptide having a sequence as set foerth in SEQ ID NO:142 (encoded by SEQ ID NO:142), can have a neutral proteinase activity; a polypeptide having a sequence as set forth in SEQ ID NO:146 (encoded byw SEQ ID NO:147), can have a serine protease activity; a polypeptide having a sequence as set forth in SEQID
NO:151 (encoded by SEQ ID NO:150), can have a metallopro-teinase activity or an aspartyl proteinase (aspartyl protease) activity; a polypeptide knaving a sequence as set forth in SEQ ID NO:159 (encoded by SEQ ID NO:158), can hzave a metalloproteinase activity or an carboxypeptidase activity (e.g., a serine-type carboxypeptidase activity); a sequence as set forth in SEQ ID NO:165 (encoded by SEQ ID NO:164), can have a peptidase activity, such as an aminopeptidase activity (e.g., a le2ucine aminopeptidase activity); a polypeptide having a sequence as set forth in SEQ MD NO:172 (encoded by
SEQ ID NO:171), can have a peptidase or a CaaX prenyl prote=ase activity (e.g., a CaaX processing activity); a polypeptide having a sequence as set for—th in SEQ ID NO:180 (encoded by SEQ ID NO:179), can have a carboxypeptidase activity (e.g., a zinc carboxypeptidase activity); a polypeptide having a sequence as. set forth in SEQ ID
NO:188 (encoded by SEQ ID NO:187), can have a serine prote=inase activity or a subtilase-like activity; a polypeptide having a sequence as set faorth in SEQ ID NO:194 (encoded by SEQ ID NO:193), can have a metalloproteinase activity or a peptidase activity (e.g., an aminopeptidase activity); a polypeptide having a sequence as set forth in
SEQ ID NO:200 (encode=d by SEQ ID NO:199), can have a carboxypeptidase activity ] (e.g., a carboxypeptidase= A activity or a zinc carboxypeptidase activity); a polypeptide having a sequence as set forth in SEQ ID NO:205 (encoded by SEQ ID NO:204), can have a carboxypeptidase activity (e.g., a zinc carboxypeptidase activity); a polypeptide having a sequence as set forth in SEQ ID NO:211 (encoded by SEQ ID NO:210), can : have a carboxypeptidase activity (e.g., a carboxypeptidase S1 activity or a serine carboxypeptidase activitsy); a polypeptide having a sequence as set forth in SEQ ID
NO:218 (encoded by SEQ ID NO:219), can have a zinc carboxypeptidase activity; a polypeptide having a sequence as set forth in SEQ ID NO:223 (encoded by SEQID
NO:222), can have a peptidase activity; a polypeptide having a sequence as set forth in
SEQ ID NO:230 (encode=d by SEQ ID NO:229), can have an alkaline or serine proteinase activity or a subtilase actIivity; a polypeptide having a sequence as set forth in SEQ ID
NO:235 (encoded by SEQQ ID NO:234), can have a metalloproteinase activity or an acylaminoacyl peptidase activity (e.g., a carboxypeptidase S1 activity); a polypeptide having a sequence as set forth in SEQ ID NO:242 (encoded by SEQ ID NO:241), can have a carboxypeptidase activity (e.g., a zinc carboxypeptidase activity); a polypeptide having a sequence as set forth in SEQ ID NO:248 (encoded by SEQ ID NO:249), can have an aspartyl protease activity; a polypeptide having a sequence as set forth in SEQ ID
NO:255 (encoded by SEQQ ID NO:254), can have a metalloproteinase activity or an carboxypeptidase activity (e.g., a serine-type carboxypeptidase activity). Any polypeptide of the invention, including polypeptides having the above-listed exemplary activities, may need proceessing (e.g., processing of a prepro form, phosphorylation, prenylation, dimerization, etc.) to generate the enzymatically active form of the enzyme.
In one asp ect, the isolated or recombinant nucleic acid encodes a polypeptide having a pro®ease activity which is thermostable. The polypeptide can retain a protease activity under econditions comprising a temperature range of between about 37°C to about 95°C; between about 55°C to about 85°C, between about 70°C to about 95°C, or, between about 20°C to about 95°C.
In another aspect, the isolated or recombinant nucleic acid encodes a ’ polypeptide having a prot=ease activity which is thermotolerant. The polypeptide can retain a protease activity after exposure to a temperature in the range from greater than 37°C to about 95°C or anywhere in the range from greater than 55°C to about 85°C. The - polypeptide can retain a protease activity after exposure to a temperature in the range between about 1°C to about 5°C, between about 5°C to aborut 15°C, between about 15°C to about 25°C, between about 25°C to about 37°C, between about 37°C to about 95°C, between about 55°C to about 85°C, between about 70°C to about 75°C, or between about oo 90°C to about 95°C, or more. In one aspect, the polypeptide retains a protease activity after exposure to a temperature in the range from greater thean 90°C to about 95°C at pH 4.5. : The invention provides isolated or recombirrant nucleic acids comprising a sequence that hybridizes under stringent conditions to a nucleic acid comprising a sequence as set forth in SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7;
SEQIDNO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID N-O:1 5; SEQ ID NO:17; SEQ
ID NO:19; SEQ ID NO:21; SEQ ID NO0:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID
NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35;'S EQ ID NO:37; SEQ ID
NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; S EQ ID NO:47; SEQ ID
NO:49; SEQ ID NO:51; 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; SEQ ID NO:67; SEQ ID
NO:69; SEQ ID NO: 71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQID
NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEEQ ID NO:87; SEQID
NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SIEQ 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:11 1; SEQ ID NO:113; SEQ ID NO:11 5; SEQID NO:117; SEQ ID
NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127; SEQ ID
NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:13 5; SEQ ID NO:137; SEQID
NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:14 5; SEQ ID NO:146; SEQ ID
NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQIDNO:17 1; SEQ ID NO:179; SEQID
NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID
NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241 ; SEQID
NO:248 and/or SEQ ID NO:254, or fragments or subsequences thereof. In one aspect, the nucleic acid encodes a polypeptide having a protease actiwity. The nucleic acid can be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 2200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more residues in length or the full length of the gene or transcript. In one aspect, the stringent conditions include a wash step comprising a wash in. 0.2X SSC at a temperature of about 65°C for about 15 minutes,
The invention provides a nucleic acid probe for identifying a nucleic acid encoding a polypeptide hawing a protease 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 sequaence comprising a sequence of the invention, or fragments or subsequences thereof, wherein the probe identifies the nucleic acid by binding or hybridization. The probe can comprise an oligonucleotide comprising at least-about 10 to 50, about 20 to 60, about 3 OQ to 70, about 40 to 80, or about 60 to 100 consecutive bases of a sequence comprising a seequence of the invention, or fragments or subsequences thereof.
The invention provides a nucleic acid probe for identifying a nucleic acid encoding a polypeptide hawing a protease activity, wherein the probe comprises a nucleic acid comprising a sequence at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, - 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 632%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, } 74%, 715%, 76%, 717%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 932%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to a nucleic acid of the invention, wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection. ‘
The probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 3 0 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 inventi on provides an amplification primer pair for amplifying a nucleic acid encoding a polypeptide having a protease activity, wherein the primer pair is capable of amplifying a mucleic 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, or about 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more consecutive bases of the sequence.
The invention provides amplification primer pairs, 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, 221, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of a nucleic acid of the invention, and a second member having a sequence as set forth by a ( . C WO 2004/033668 PCT/US20-03/032819 about the first (the 5%) 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27,28,29, 30 or more residues of the complementary strand of the first member.
The invention provides protease-encoding nucleic acids generated by amplification, ¢.g., polymerase chain reaction (PCR), using an amplification pmximer pair 6 of the inveention. The invention provides proteases generated by amplification. ¢.g., . polymerase chain reaction (PCR), using an amplification primer pair of the inv—ention.
The inven. tion provides methods of making a protease by amplification, e.g., polymerase chain reac-tion (PCR), using an amplification primer pair of the invention. In o-ne aspect, the amplification primer pair amplifies a nucleic acid from a library, e.g., a gen_e library, suchasan environmental library.
The invention provides methods of amplifying a nucleic acid enecoding a polypeptide having a protease activity comprising amplification of a template maucleic acid with an amplification primer sequence pair capable of amplifying a nucleic acicq sequence . of the inve ntion, or fragments or subsequences thereof,
The invention provides expression cassettes comprising a nucleiec acid of the invention or a subsequence thereof. In one aspect, the expression cassette can comprise the nucleic acid that is operably linked to a promoter. The promoter c-an be a viral, bacte rial, mammalian or plant promoter. In one aspect, the plant promotes can be a potato, rice=, corn, wheat, tobacco or barley promoter. The promoter can be a co nstitutive promoter. “The constitutive promoter can comprise CaMV358. In another aspect, the promoter can be an inducible promoter. In one aspect, the promoter can be a tis_sue- specific prosmoter or an environmentally regulated or a developmentally regulated promoter. "Thus, the promoter can be, e.g., a seed-specific, a leaf-specific, a root-specific, a stem-spec ific or an abscission-induced promoter. In one aspect, the expressior cassette can further €omprise a plant or plant virus expression vector.
The invention provides cloning vehicles comprising an €xpressior cassette (e.g., a vector) of the invention or a nucleic acid of the invention. The cloning ve=hicle can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artifici al chromosome. The viral vector can comprise an adenovirus vector-, a i retroviral vector or an adeno-associated viral vector, The cloning vehicle can comprise a bacterial arti ficial chromosome (BAC), a plasmid, a bacteriophage P1-derived ve=ctor (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
The invention provides transformed cell comprising a nuclei < acid of the invention or an expression cassette (e.g., a vector) of the invention, ora cloming vehicle of the inveention. In one aspect, the transformed cell can be a bacterial cell, a mammalian cell, a Fungal cell, a yeast cell, an insect cell or a plant cell. In one aspect, 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. In one aspect, the animal is a mouse. :
The invention provides transgenic plants comprising a nucle ic acid of the invention or an expression cassette (e.g., a vector) of the invention. The tra_nsgenic plant can be .a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapese=d plant, a soybean plant, a rice plant, a barley plant or a tobacco plant.
The invention provides transgenic seeds comprising a nuclei _c acid of the inventi-on or an expression cassette (e.g., a vector) of the invention. The tra nsgenic seed canbe 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 seequence complementary to or capable of hybridizing under stringent conditions to a : nucleic= acid of the invention. The invention provides methods of inhibitings the transla€ion of a protease message in a cell comprising administering to the cell or expresssing in the cell an antisense oligonucleotide comprising a nucleic acid sequence comple=mentary to or capable of hybridizing under stringent conditions toa nucleic acid of the i nvention. In one aspect, the antisense oligonucleotide is between about 10 to 50, about 220 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 bases in length.
The invention provides methods of inhibiting the translation. of a protease message in a cell comprising administering to the cell or expressing in the cell an antisen_se oligonucleotide comprising a nucleic acid sequence complementary to or capables of hybridizing under stringent conditions to a nucleic acid of the in-vention. The invention provides double-stranded inhibitory RNA (RNAI) molecules comaprising a subseqmuence of a sequence of the invention. In one aspect, the RNAi is about 15, 16, 17, 18,19, 20,21, 22, 23, 24, 25 or more duplex nucleotides in length. The invention providess methods of inhibiting the expression of a protease in a cell compr sing admini sstering to the cell or expressing in the cell a double-stranded inhibitory RNA (iRNAD), wherein the RNA comprises a subsequence of a sequence of the imvention,
/ Po @® WO 2004/033668 PCT/US2003/032819
The invention provides an isolated or recombinant polypeptide comparising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 562%, 57%, 58%, 5924, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 13%, 71424, 75%, 16%, 11%, 18%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 868%, 87%, 88%, 892%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (10024) sequence identity to an exemplary polypeptide or peptide of the invention over aregion of at least about 25, 50, 75, 100, 125, 150, 175, 200, 223, 25 0, 275,300, 325, 350 or more residues, or over the full length of the polypeptide, and the sequence identities are determined by analysis with a sequence comparison algorithem or by a visual inspection. Exemplary polypeptide or peptide sequences of the inventiora include SEQ IID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:1+0;
SEQ ID NO:122; 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; SEQLD
NO:32; SEQ IID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID
NO:42; SEQ IID 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 IID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID
NO:72; SEQ IID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID
NO:82; SEQ IID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID
NO:92; SEQ IID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID
NO:102; SEQ LD NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQID
NO:112; SEQ LDNO:114; SEQ ID NO:116; SEQ ID NO:1 18; SEQ ID NO:120; SEQ ID
NO:122; SEQ I DNO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ ID
NO:132; SEQ I DNO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQQ ID
NO:142; SEQ IDNO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID
NO:165; SEQ I'DNO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQQ ID
NO:200; SEQ I'D NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQQ ID
NO:230; SEQ IID NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or- a protease encode=d by SEQ ID NO: 145, and subsequences thereof and variants thereof
Exemplary polypeptides also include fragments of at least about 10, 15, 20, 25, 30, 3s, 40,45, 50,75, 1 00, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more residues in ~~ length, or over the full length of an enzyme. Exemplary polypeptide or peptide seque=nces of the invention include sequence encoded by a nucleic acid of the invention. Exempelary polypeptide or peeptide sequences of the invention include polypeptides or peptides
WE 2004/033668 PCT/US2003/032819 ® specifically bound by an antibody of the invention. In one aspect, a po lypeptide of the iravention can have at least one protease activity.
Another aspect of the invention provides an isolated or recombinant polypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 175, 80, 85,90, 95 or 100 or more consecutive bases of a polypeptide or peptide sequence off the invention, sequences substantially identical thereto, and the sequences complementary thereto. The peptide can be, e.g., an immunogenic fragzment, emotif (e=.g., a binding site), a signal sequence (e.g., as in Table 4), a prepro sequence or an . active site.
In one aspect, protease activity comprises catalyzing hydrolysis of peptide bonds. The protease activity can comprise an endoprotease activity ancl/or an exoprotease ac=tivity. The protease activity can comprise a carboxypeptidase activitsy, an arminopeptidase activity, a serine protease activity, a metalloprotease activity, a cysteine protease activity and/or an aspartic protease activity. In one aspect, pro-tease activity can comprise activity the same or similar to a chymotrypsin, a trypsin, an elastase, a kallikrein armd/or a subtilisin activity. The protease activity can comprise a peptidase activity, such as adipeptidylpeptidase or a carboxypeptidase activity.
In one aspect, the protease activity is thermostable. The polypeptide can re®ain a protease activity under conditions comprising a temperature ram ge of between ab out 1°C to about 5°C, between about 5°C to about 15°C, between aboxat 15°C to about °C, between about 25°C to about 37°C, between about 37°C to about 95°C, between ab-out 55°C to about 85°C, between about 70°C to about 75°C, or betwee=n about 90°C to abeout 95°C, or more. In another aspect, the protease activity can be thexmotolerant. The po lypeptide can retain a protease activity after exposure to a temperatur<e in the range 25 froom greater than 37°C to about 95°C, or in the range from greater than 55°C to about 85=°C. In one aspect, the polypeptide can retain a protease activity after exposure to a termperature in the range from greater than 90°C to about 95°C at pH45.
In one aspect, the isolated or recombinant polypeptide can comprise the poRypeptide of the invention that lacks a signal sequence. In one aspect, the isolated or recombinant polypeptide can comprise the polypeptide of the invention <omprising a heterologous signal sequence, such as a heterologous protease or non-protease signal sequence. Co
In one aspect, the invention provides a signal sequence comprising a pepotide comprising/ consisting of a sequence as set forth in residues 1 to 12,1t0 13,1 to
/ a
C WO 2004/033668 PCT/US2003/032819 4.11015 11016,11017, 110 18,1019, 11020, 1 to 31, 11022, 1 t023,1t024, 1 to 25,1026, 11927, 1t028, 1 to 28,1t030,1t031, 11032, 1 t033,1t034,1t035,1to 36,1t037,1t038,1t039, 1 to 40,1t041,1t042,1to0 43, 1 to 44 (or a longer peptide) : of a polypeptide of the invention. In one aspect, the invention provides a signal sequence comprising a peptide comprisingg/ consisting of a sequence as set forth Table 4.
The invention provides isolated or recombinant peptides comprising an amino acid sequence having at leeast 95%, 96%, 97%, 98%, 99%, or more sequence identity to residues 1 to 37 of SEQ ID NO:2, at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to residues 1 te 36 of SEQ ID NO:4, at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to residues 1 to 32 of SEQ ID NO:6, at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to residues 1 to 28 of SEQ ID NO:10, at least 95%, 96%, 97%, 98%, 99%, or rnore s¢quence identity to residues 1 to 33 of SEQ ID
NO:14, and least 95%, 96%, 97%, 98%, 99%, or more sequence identity to the other signal sequences as set forth in tFae SEQ ID listing, wherein the sequence identities are determined by analysis with a Seequence comparison algorithm or by visual inspection.
These peptides can act as signal sequences on its endogenous protease, on another protease, or a heterologous protein (a non- protease enzyme or other protein).
In one aspect, the invention provides chimeric proteins comprising a first domain comprising a signal sequence of the invention (e.g., see Table 4) 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 protease.
The invention prowsides chimeric polypeptides comprising at least a first domain comprising signal peptide (SP), a prepro sequence and/or a catalytic domain (CD) of the invention and at least a seceond domain comprising a heterologous polypeptide or oh peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP), prepro Secjuence and/ or catalytic domain (CD). In one aspect, the heterologous polypeptide or peptide is not a protease. The heterologous polypeptide or peptide can be amino terminal to, carboxy terminal to or on both ends of the signal peptide (SP), prepro sequence andl/or catalytic domain (CD).
The invention prov-ides isolated or recombinant nucleic acids encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises at least a first domain : comprising signal peptide (SP), a pprepro domain and/or a catalytic domain (CD) of the invention and at least a second dormnain comprising a heterologous polypeptide or peptide,
wherein the heterologous polypeptide or peptide is net naturally associated with the signal ; peptide (SP), prepro domain and/ or catalytic domaira (CD).
In one aspect, the protease activity commprises a specific activity at about 37°C in the range from about 1 to about 1200 units per milligram of protein, or, about 100 to about 1000 units per milligram of protein. In another aspect, the protease activity «comprises a specific activity from about 100 to abou 1000 units per milligram of protein, or, from about 500 to about 750 units per milligram of protein. Alternatively, the protease activity comprises a specific activity at 37°C in the range from about 1 to about 750 units per milligram of protein, or, from about 500 to about 1200 units per milligram of protein. In one aspect, the protease activity comprises a specific activity at 37°C in the range from about 1 to about 500 units per milligram ef protein, or, from about 750 to about 1000 units per milligram of protein. In anothex- aspect, the protease activity
Co comprises a specific activity at 37°C in the range frorm about 1 to about 250 units per milligram of protein. Alternatively, the protease activity comprises a specific activity at 37°C in the range from about 1 to about 100 units per milligram of protein. In another aspect, the thermotolerance comprises retention of at least half of the specific activity of the protease at 37°C after being heated to the elevatecd temperature. Alternatively, the thermotolerance can comprise retention of specific activity at 37°C in the range from about 1 to about 1200 units per milligram of protein, or, from about 500 to about 1000 units per milligram of protein, after being heated to the elevated temperature. In another aspect, the thermotolerance can comprise retention of specific activity at 37°C in the range from about 1 to about 500 units per milligram Of protein after being heated to the elevated temperature. :
The invention provides the isolated or recombinant polypeptide of the invention, wherein the polypeptide comprises at least one glycosylation site. In one aspect, glycosylation can be an N-linked glycosylatio nn. In one aspect, the polypeptide can be glycosylated after being expressed in a P. pastoris or a S. pombe.
In one aspect, the polypeptide can retain a protease activity under conditions comprising about pH 6.5, pH 6, pH 5.5, pF 5, pH 4.5 or pH 4. In another aspect, the polypeptide can retain a protease activity vander conditions comprising about pH 7,pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pI 10.5 or pH 11. In one aspect, the polypeptide can retain a protease activity after exposumre to conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In anoth er aspect, the polypeptide can retain a a protease activity after exposure to conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, PH 10.5or pH 11.
The invention provides protein preparations comprising a polypeptide of the invention, wherein the protein prepraration comprises a liquid, a solid or a gel.
The invention provides ‘heterodimers comprising a polypeptide of the invention and a second protein or domain. The second member of the heterodimer can be: a different protease, a different enzymes or another protein. In one aspect, the Second : domain can be a polypeptide and the heterodimer can be a fusion protein. In one aspect, the second domain can be an epitope or atag. In one aspect, the invention provides homodimers comprising a polypeptide ©f the invention.
The invention provides f- mmobilized polypeptides having a protease 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 a the invention and a second domain. In one aspect, the polypeptide can be immobilized on acell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array ora capillary tube.
The invention provides aTrays comprising an immobilized nucleic acid of the invention. The invention provides a=mrays comprising an antibody of the invention.
The invention provides isolated 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 fo od supplements for an animal comprising a polypeptide of the invention, e.g., a polypeptide encoded by the nucleic acid of the invention. In one aspect, the polypeptide in the food supplement can be glycosylated.
The invention provides edible enzyme de livery matrices comprising a polypeptide of the invention, e.g., a polypeptide encoded by the nucleic acid of the invention. In one aspect, the delivery matrix comprises a pellet. Im one aspect, the polypeptide can be glycosylated. In one aspect, the protease activity is thermotolerant. In another aspect, the protease activity is thermostable.
The invention provides method of isolating or identifying a polypeptide having a protease 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 a protease activity.
The invention provides methods of making an anti-protease antibody comprising administering to a non-human anixnal 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 am anti-protease antibody. The invention provides methods of making an anti-protease #mmune 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 nuclei ¢ acid of the invention operably linked to a promoter; and (b) expressing the nucleic acid ©f step (a) under conditions that allow } expression of the polypeptide, thereby producing a recombinant polypeptide. In one aspect, 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 method. s for identifying a polypeptide having a protease 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 protease 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 thae reaction product detects a polypeptide having a protease activity.
The invention provides method s for identifying a protease substrate comprising the following steps: (a) providing & polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the iravention; (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 a protease substrate.
rd . {
The invention provides methods of determining whether a test compourrmd 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) providingz 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 provide=s methods for identifying a modulator of a protease activity comprising the following stepps: (a) providing a polypeptide of the invention or =a 10. polypeptide encoded by a nucleic acici 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 protease, wherein a change in the protease activity measurecd in the presence of the test compound compared to the activity in the absence of the test compound provides a determination tBaat the test compound modulates the protease activity. In one aspect, the protease activity can be measured by providing a protease substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an imcrease in the amount of the substrate or a decrease in the amount of a reaction product. Ma 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 protease activity. An increase in the amount of the substrates 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 protease activity.
The invention provides <computer systems comprising a processor and a data storage device wherein said data s®orage 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). In one aspect, the computer system can further comprise a sequence comparison algorithm and a d ata storage device having at least one reference sequence stored thereon. In another aspect, the sequence comparison algorithm comprises a computer program that indi cates polymorphisms. In one aspect, the computer system can further comprise awn 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 identi fies 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 co mparing 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 mucleic 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 clifferences between the first sequence and the second sequence can further comprise the step of identifying polymorphisms. In one aspect, the method can further comprise an iclentifier that identifies one or more features in a sequence. In another aspect, 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 a protease activity, from an environmental sample comprising the steps of: (a) providing an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptides having a protease 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 hybridlization to the amplification primer pair; and, (¢) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the environzmental sample, thereby isolating or recovering a nucleic acid encoding a polypeptide having a protease activity from an environmental sample. One or each member of t he amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of a sequence of the invention. In one aspect, the amplification primer sequence pair is an amplification pair of the invention.
The invention provides methods feor isolating or recovering a nucleic acid encoding a polypeptide having a protease 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 environmeratal sample such that nucleic acid in the sample is accessible for hybridization to a polymacleotide 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 mmcleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide having a protease activity from an environmental sample. The environmental sample can comprise a waater sample, a liquid sample, a soil sample, an air sample or a biological sample. In one asspect, the biological sample can be derived from a bacterial cell, a protozoan cell, an insec=t 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 a protease activity compprising the steps of: (a) providing a template nucleic acid comprising a nucleic acid of the Envention; and (b) modifying, deleting or adding one or more nucleotides in the tempdate sequence, or a combination thereof, to generate a variant of the template nucleic acid. In one aspect, the method can further comprise expressing the variant nucleic acid to generate a variant protease 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, casssette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagene=sis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof. In another aspect, the mmodifications, additions or deletions are introduced by a method comprising recom bination, recursive sequence recombination, phosphothioate-modified DNA mutagemesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatech repair mutagenesis, repair- deficient host strain mutagenesis, chemical mutagenesis , radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeri_c nucleic acid multimer creation and a combination thereof,
In one aspect, the method can be iterativesly repeated until a protease 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. In one aspect, the variant protease polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature. In another aspect, the variant protease polypeptide has increased glycosylation as compared to the protease encoded by a €emplate nucleic acid.
Alternatively, the variant protease polypeptide has a prot-ease activity under a high temperature, wherein the protea_se encoded by the template nucleic acid is not active under the high temperature. In ene aspect, the method can be iteratively repeated until a protease coding sequence havin. g an altered codon usage from that of the template nucleic + acid is produced. In another aspect, the method can be iteratively repeated until a protease gene having higher or Mower level of message expression or stability from that of the template nucleic acid is procluced.
The invention preovides methods for modifying codons in a nuclSic acid encoding a polypeptide having za protease activity to increase its expression in a host cell, the method comprising the follawwing steps: (a) providing a nucleic acid of the invention encoding a polypeptide having z protease 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 €he 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 prefezrred codon is a codon under-represented in coding 16 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 za protease 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 a protease.
The invention provides methods for modifying codons in a nucleic acid encoding a polypeptide having =a protease activity to increase its expression in a host cell, the method comprising the follwing steps: (a) providing a nucleic acid of the invention encoding a protease polypeptides; 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 cocling sequences in genes in the host cell and a non- preferred or less preferred codom 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 aa protease 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 acied 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-regpresented in coding sequences in genes in a host cell and a non-preferred or less prefe=1red 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. In one aspec=t, the host cell can be a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell ora mammalian cell.
The invention provides methods for producing a liborary of nucleic acids encoding a plurality of modified protease active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived frorm a first nucleic acid comprising a sequence encoding a first active site or a first substramte binding site the method comprising the following steps: (a) providing a first nucle=ic acid encoding a first ’ active site or first substrate binding site, wherein the first nucleic a_cid sequence comprises a sequence that hybridizes under stringent conditions to a nucleic zcid of the invention, and the nucleic acid encodes a protease active site or a protease sulbstrate binding site; (b) providing a set of mutagenic oligonucleotides that encode naturallye/-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; a-nd, (c) using the set of mutagenic oligonucleotides to generate a set of active site-encodingg or substrate binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a librar-y of nucleic acids encoding a plurality of modified protease active sites or substrate banding sites. In one aspect, the method comprises mutagenizing the first nucleic acid of step (a) by a method . comprising an optimized directed evolution system, gene site-satur=ation mutagenesis (GSSM), synthetic ligation reassembly (SLR), error-prone PCR, sheiffling, 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 saturated : mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a cormbination thereof. In another aspect, the method comprises mutagenizing the first nucleic acid of step (a) or variants 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 invention provides methods for making a small molecule comprising : the following steps: (a) providing a plurality of biosynthetic enzymes capable of . synthesizing or mod Sfying a small molecule, wherein one of the enzymes comprises a protease enzyme encoded by a nucleic acid of the invention; (b) providing a substrate for at least one of the emmzymes of step (a); and (c) reacting the substrate of step (b) with the enzymes under cond itions that facilitate a plurality of biocatalytic reactions to generate a : 10 small molecule by a series of biocatalytic reactions. The invention provides methods for . modifying a small molecule comprising the following steps: (a) providing a protease enzyme, wherein thes 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 protease enzyme, thereby modifying a small molecule by a protease enzymatic reaction. In one aspect, the method ¢ an comprise a plurality of small molecule substrates for the enzyme of step (a), thereby g=enerating a library of modified small molecules produced by at least one enzymatic reactEon catalyzed by the protease enzyme. In one aspect, 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. In another aspect, the method can further comprise the step of testing the library to determine if a particular modified small molecule which exh Z bits 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 re=actions 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 a bsence of the particular modified small molecule with a desired activity, and identify~ing at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.
The imvention provides methods for determining a functional fragment of a protease enzyme coraprising the steps of: (a) providing a protease 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
/ i ( WYO 2004/033668 PCT/US2003/032819 residues from the sequence of step (a) and testing the remaining subsequence fora protease activity, thereby determining a functional fragment of a pro tease enzyme. In one aspect, the protease activity is measured by providing a protease substrate and detecting a «decrease in the amount of the substrate or an increase in the amount &f a reaction product,
The invention provides methods for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the mmethod comprising &be following steps: (a) making a modified cell by modifying the gen etic composition ofa
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; (Cc) measuring at least one metabolic parameter of the cell by monitorang the cell culture
Of step (b) in real time; and, (d) analyzing the data of step (c) to determmine if the measured
Parameter differs from a comparable measurement jn an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time neetabolic flux analysis. In one aspect, the genetic composition of thes 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. In one aspect, the amethod can further ceomprise selecting a cell comprising a newly engineered phenotype. Xn another aspect, thae method can comprise culturing the selected cell, thereby generatin_g a new cell strain comprising a newly engineered phenotype.
The invention provides methods of increasing thermotowlerance or th ermostability of a protease polypeptide, the method comprising glyceosylating a protease 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 in-vention, thereby increasing the thermotolerance or thermostability of the protease poslypeptide. In one aspect, the protease specific activity can be thermostable or thermotolerant at a temperature in the range from greater than about 377°C to about 95°C,
The invention provides methods for overexpressing a re«combinant protease polypeptide in a cell comprising expressing a vector comprisirag a nucleic acid cornprising a nucleic acid of the invention or a nucleic acid sequence off the invention, wimerein the sequence identities are determined by analysis with a sequence comparison alg-orithm or by visual inspection, wherein overexpression is effected bw use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.
The invention provides methods of making a transgenic gplant comprising the following steps: (a) introducing a heterologous nucleic acid sequenc-e into the cell,
wherein the heterologous nucleic sequence comprises a nucleic acid sequence of the invention, thereb>y producing a transformed plant cell; and (b) producing a transgenic plant from the transformed cell. In one aspect, the step (a) can further comprise introducing the kneterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts. In another aspect, the step (a) can further comprise introducing the heterologous nucleic acid sequence directly to plant tissue by DNA particle bombardment. Alternatively, the step (a) can further comprise introducing the™" heterologous nucleic acid sequence into the plant cell DNA using an Agrobacterium tumefaciens host. In one aspect, the plant cell can be a potato, corn, rice, wheat, tobacco, or barley cell. ‘
Thhe invention provides methods of expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps: (a) transforming the plant cel 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 16 plant under cond itions wherein the heterologous nucleic acids sequence is expressed in the plant cell. Tlhe 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 sequence of the invention; (b) growing tine plant under cond itions wherein the heterologous nucleic acids sequence is expressed in the plant cell.
Thhe invention provides methods for hydrolyzing, breaking up or disnz pting a protein-compri sing composition comprising the following steps: (a) providing a polypeptide of tire invention having a protease activity, or a polypeptide encoded by = nucleic acid of thae invention; (b) providing a composition comprising a protein; and (c) contacting the poelypeptide of step (a) with the composition of step (b) under conditions wherein the prote=ase hydrolyzes, breaks up or disrupts the protein-comprising composition. In one aspect, the composition comprises a plant cell, a bacterial cell, aa. yeast cell, an insexct cell, or an animal cell. Thus, the composition can comprise any gplant or plant part, any" protein-containing food or feed, a waste product and the like. The invention provides methods for liquefying or removing a protein from a composition comprising the following steps: (a) providing a polypeptide of the invention having a protease activity, or a polypeptide encoded by a nucleic acid of the invention; (b) providing a composition comprising a protein; and (c) contacting the polypeptide of step
- @® WO 2004/03366-8 PCT/USZ2003/032819 to. (a) with the composition of step (b) under conditions wherein the protease remmoves or ’ : liquefies the gprotein. :
The invention provides detergent compositions comprising a polypeptide of the inventi on, or a polypeptide encoded by a nucleic acid of the invention, wherein the } 5 polypeptide masa protease activity. The protease can be a nonsurface-active protease or a surface-activez protease. The protease can be formulated in a non-aqueous liquid composition, -a cast solid, a granular form, a particulate form, a compressed tablet, a gel form, a paste «ra slurry form. The invention provides methods for washing aan object comprising th e following steps: (a) providing a composition comprising a pol-ypeptide of the invention having a protease activity, or a polypeptide encoded by a nucleiec acid of the invention; (b) providing an object; and (¢) contacting the polypeptide of step (Ca) and the object of step (b) under conditions wherein the composition can wash the obje=ct.
The invention provides textiles or fabrics, including, e.g., threa-ds, comprising a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention. In one aspect, the textiles or fabrics comprise cellulose-contain-ing fibers.
The invention provides methods for removing protein stains from a compositicon comprising thes following steps: (a) providing a composition comprising a polypeptide of the invention lmaving a protease activity, or a polypeptide encoded by a nucleic= acid of the invention; (b) providing a composition having a protein stain; and (c) contacting the polypeptide of step (a) and the composition of step (b) under conditions wherein the protease can re=move the stain. The invention provides methods for improving the finish of a fabric com prising the following steps: (a) providing a composition compri sing a polypeptide of “the invention having a protease activity, or a polypeptide encodeed by a nucleic acid of the invention; (b) providing a fabric; and (c) contacting the polypeptide of step (a) and the fabric of step (b) under conditions wherein the polypeptide can treat the fabric thereby improving the finish of the fabric. In one aspect, the fabric is a vovool or a silk.
The invention provides feeds or foods comprising a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention. The inv—ention provides methods for hydrolyzing proteins in a feed or a food prior to consumpation by an animal compris ng the following steps: (a) obtaining a feed material comprising a protease of the imvention, or a protease encoded by a nucleic acid of the invention; and ®) adding the polypeptide of step (a) to the feed or food material in an amount sufficient for a sufficient time period to cause hydrolysis of the protein and formation of a tre=ated food
® or feed, thereby hydrolyzing the proteins in the food or the feed prior to consumption by the animal. In one aspect, the invention provides methods for hydrolyzi ng proteins in a feed ora. food after consumption by an animal comprising the following: steps: (a) : obtaining a feed material comprising a protease of the invention, or a protease encoded by anucleic= acid of the invention; (b) adding the polypeptide of step (ato the feed or food material; and (c) administering the feed or food material to the animal, wherein after - consumption, the protease causes hydrolysis of the proteins in the feed or food the digestive= tract of the animal. The food or the feed can be, e.g., com. + The invention provides methods for improving texture ard flavor of a dairy prosduct comprising the following steps: (a) providing a polypepticlle of the invention havinga protease activity, or a protease encoded by a nucleic acid of the invention; (b) providings a dairy product; and (c) contacting the polypeptide of step (a) and the dairy product Of step (b) under conditions wherein the protease can improve the texture or flavor of” the dairy product. In one aspect, the dairy product comprises za cheese or a yogurt. "The invention provides dairy products comprising a protease of the invention, or is encode=d by a nucleic acid of the invention. The invention provides maethods for tenderizi ng a meat or a fish comprising the following steps: (a) providing a polypeptide of the invermtion having a protease activity, or a protease encoded by a nucleic acid of the inventiorn; (b) providing a composition comprising meat or fish; and (<) contacting the polypeptide of step (a) and the composition of step (b) under conditions wherein the polypeptide can tenderize the meat or the fish. The invention provides rmethods for producin_g a gluten-free product comprising the following steps: (a) providing a polypeptide of the invention having a protease activity, or a protease encoded by a nucleic acid of tiae invention; (b) providing a product comprising gluten; and (c) contacting the polypeptIde of step (a) and the product of step (b) under conditions wherein the polypept3de can hydrolyze gluten thereby producing the gluten-free prociuct. In one aspect, tae gluten-free product is a cereal, a bread or a beer. The invention provides gluten-free food compositions comprising a polypeptide of the invention, or a protease encoded "by a nucleic acid of the invention, wherein the polypeptide conprises a protease activity.
The invention provides methods for improving the extraction of oil from an oil-ric-h plant material comprising the following steps: (a) providing &a polypeptide of the invermtion having a protease activity, or a protease encoded by a nuck eic acid of the inventiora; (b) providing an oil-rich plant material; and (c) contacting thes polypeptide of
/ ! step (a) and the oil-rich plant material. In one aspect, the oil-rich plant material comprises an Oil-rich seed. The oil can be a soybean oil, an olive oil, a rapeseed (c-anola) oil or a sunflower oil. : :
The invention provides methods for preparing a fruit or vegetable jirice, syrup, puree ox extract comprising the following steps: (a) providing a polypeptide of the invention havimg a protease activity, or a protease encoded by a nucleic acid of the invention; (b) goroviding a composition or a liquid comprising a fruit or vegetable material; and («) contacting the polypeptide of step (a) and the composition, thereby : preparing the fruit or vegetable juice, syrup, puree or extract.
The invention provides papers or paper products or paper pulp comprising a protease of thae invention, or a polypeptide encoded by a nucleic acid of the inven&ion.
The invention provides methods for treating a paper or a paper or wood pulp comprising the following steps: (a) providing a polypeptide of the invention having a protease : activity, or a protease encoded by a nucleic acid of the invention; (b) providing a composition comprising a paper or a paper or wood pulp; and (c) contacting the polypeptide of step (a) and the composition of step (b) under conditions wherein thes protease can treat the paper or paper or wood pulp. “The invention provides pharmaceutical compositions comprising a polypeptide of he invention, or a polypeptide encoded by a nucleic acid of the invemtion.
In one aspect, the pharmaceutical composition acts as a digestive aid or as a topical skin care. The invention provides methods of treating an imbalance of desquamation comprising topical application of a pharmaceutical composition of the invention. In one aspect, the treatment is prophylactic. The invention provides oral care products comprising a polypeptide of the invention having a protease activity, or a protease encoded by a nucleic acid of the invention. The oral care product can comprise a toothpaste, a demtal cream, a gel or a tooth powder, an odontic, a mouth wash, a pre~ or post brushing rimse formulation, a chewing gum, a lozenge or a candy. The inventio n provides contac# lens cleaning compositions comprising a polypeptide of the invention having a protease activity, or a protease encoded by a nucleic acid of the invention.
The invention provides methods for treating solid or liquid animal wan ste products compri sing the following steps: (a) providing a polypeptide of the inventio n having a proteas e activity, or a protease encoded by a nucleic acid of the invention; (b) providing a solid or a liquid animal waste; and (c) contacting the polypeptide of step (a) and the solid or Riquid waste of step (b) under conditions wherein the protease can tre=at the waste. The invention provides processed waste products comprising a polypeptide of the invention having a protease activity, or a protease encoded by a nucleic acid of the inv-ention.
The invention provides hairball prevention and/or remeclies comprising a polypeptide of the invention having a protease activity, or a protease encoded by a nucleic acid of the invention. The invention provides blood or organic spot removers comprising apolypeptide of the invention having a protease activity, or a protease encoded by a nucleic acid of the invention.
The details of one or more embodiments of the inventiom are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. -
All publications, patents, patent applications, GenBank sequences and
AT"CC deposits, cited herein are hereby expressly incorporated by reference for all purposes.
DESCRIPTION OF DRAWINGS
The patent or application file contains at least one drawi ng executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Figure 1 is a block diagram of a computer system.
Figure 2 is a flow diagram illustrating one aspect of a process for cormparing a new nucleotide or protein sequence with a database of seq uences in order to determine the homology levels between the new sequence and the sequiences in the database.
Figure 3 is a flow diagram illustrating one aspect of a process in a cornputer for determining whether two sequences are homologous.
Figure 4 is a flow diagram illustrating one aspect of an i dentifier process 300 for detecting the presence of a feature in a sequence.
Figure 5 is a an illustration of results of testing SEQ ID INO:144 (encoded by SEQ ID NO:143) in a gelatin in fluorescent liquid end point assay, as described in detail in Example 1, below.
( : . @® WO 2004/033668 PCT/US2003/032819
Figure 6 is an illustration of a standard curve of (PNA) (para-nitroanalide)®, generated to allow conversion of pBNA absorbance (A405nm) to moles of pNA, as described in detail in Example 1, beelow.
Figure 7 is an illustration of a standard curve of subtilisin A protease, as described in detail in Example 1, bezslow.
Figure 8 is a an illustration of results if a protease activity using the small peptide substrate p-nitroanalide link<ed Alanine-Alanine-Proline-Phenylalanine, as described in detail in Example 1, bealow.
Like reference symbeols in the various drawings indicate like elements.
DET-AILED DESCRIPTION
The invention provicles polypeptides having a protease activity, polynucleotides encoding the polyp-eptides, and methods for making and using these polynucleotides and polypeptides. Mn one aspect, the proteases of the invention are used to catalyze the hydrolysis of peptide= bonds. The proteases of the invention can be used to make and/or process foods or feeds, textiles, detergents and the like. The proteases of the invention can be used in pharmaceu-tical compositions and dietary aids. ~ The protease preparations of the invention (including those for treating or processing feeds or foods, treating fabers and textiles, waste treatments, plant treatments, and the like) can further comprise ore or more enzymes, for example, pectate lyases, cellulases (endo-beta-1,4-glucanasess), beta-glucanases (endo-beta-1,3(4)-glucanases), lipases, cutinases, peroxidases, laccamses, amylases, glucoamylases, pectinases, reductases, oxidases, phenoloxidases, ligninasess, pullulanases, arabinanases, hemicellulases, mannanases, xyloglucanases, xylana ses, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, pectin methylesterases, cellobiohydreolases, transglutaminases; or mixtures thereof.
Definitions
The term “protease” imcludes all polypeptides having a protease activity, including a peptidase and/or a proteirase activity. A protease activity of the invention can comprise catalysis of the hydroly~sis of peptide bonds. The proteases of the invention can catalyze peptide hydrolysis reactions in both directions. The direction of the reaction can be determined, €.g., by manipulating substrate and/or product concentrations, temperature, selection of protease anc] the like. The protease activity can comprise an endoprotease activity and/or an exopr-otease activity. The protease activity.can comprise a protease activity, e.g., a carboxypeptidase activity, a dipeptidylpeptidase or an aminopeptidase activity, a serine protease activity, a metalloproteinase activity, a cysteine protease activity and/or an aspartic protease activity. In one aspect, protease activity can comprise activity the same or similar to a chymotrypsin, a trypsin, an elastase, a kallikrein . and/or a subtilisin activity.
In describing a polypeptide of the iravention having a protease activity, e.g., an exemplary polypeptide having a sequence as se® forth in SEQ ID NO:2; SEQ ID NO:4; oo
SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NQ:14; SEQ ID
NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID ENO:22; SEQ ID NO:24; SEQ ID
NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID INO:32; SEQ ID NO:34; SEQID
NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID ENO:42; SEQ ID NO:44; SEQID
NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID INO:52; SEQ ID NO:54; SEQID
NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID INO:62; SEQ ID NO:64; SEQID
NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID INO:72; SEQ ID NO:74; SEQID :
NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID INO:82; SEQ ID NO:84; SEQID
NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID INO:92; SEQ ID NO:94; SEQ ID
NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID
NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:1 12; SEQ ID NO:114; SEQ ID
NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ ID
NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID
NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO: 144; SEQ ID
NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID
NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ JID NO:200; SEQ ID NO:205; SEQ ID
NO:211; SEQ ID NO:219; SEQ ID N0:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID
NO:242; SEQ ID NO:249; SEQ ID NO:255; a polypeptide encoded by SEQ ID NO:145, it is meant that the polypeptide has a protease activ-ity with and/or without a signal sequence, or, with and/or without a prepro sequenc € (e.g., a "prepro" domain), if the polypeptide has a signal sequence and/or a prepro Ssequence (e.g., a "prepro" domain).
Thus, the invention includes polypeptides (having = protease activity) in inactive form, €.g., as a proprotein before “maturation” or processing of its prepro sequence (e.g, by a proprotein-processing enzyme, such as a proproteira convertase) to generate an “active” oo mature protein, or, before “activation” by a post-tramnslational processing event, e.g., an endo- or exo-peptidase or proteinase action, a phos “phorylation event, an amidation, a glycosylation or a sulfation, a dimerization event, and the like, in addition to including all
- (
C WO 2004/033668 PCT/US2003/032819 active forms and active subsequences (e.g., catalytic domains or active sites) of the
Protease.
A polypeptide can be routinely assayed for protease activity (e.g., tested to see if the protein is within the scope Of the invention) by any method, €.g., protease activity can be assayed by the hydrolwsis of casein in Zymograms, the release of fluorescence from gelatin, or the release of p-nitroanalide from various small peptide ) substrates (these and other exemplary” protease assays are set forth in the Examples, below).
The term “antibody” dincludes a peptide or polypeptide derived from, modeled after or substantially encode by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, sce, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993);
Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem.
Biophys. Methods 25:85-97. The term 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 CH! 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 CHI 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 3<11:544-546), which consists of a VH domain; and (vi) an isolated complementarity deterrmining region (CDR). Single chain antibodies are also included by reference in the term '* antibody."
The terms “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.
As used herein, the termss “computer,” “computer program” and “processor” are used in their broadest general contexts and incorporate all such devices, as described in 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.
The term “expression cassette” ass used herein refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as a protease of the inveration) 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. Thus, expression cassettes also include plasmids;- expression vectors, recombinant viruses, any foxm of recombinant “naked DNA? vector, and the like. : “Operably linked" as used hereim refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory sequence to a transcribed sequence.
For example, a promoter is operably linked toa coding sequence, such as a nucleic acid of the invention, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some _ transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
A "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that 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, aviral lipid envelope, etc.). Vectors include, buat 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. 5,217,879), and include both the expressiorn and non-expression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome. }
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As used herein, the term “promoter” imncludes all sequences capable of driving transcription of a coding sequence in a cell, e=.g., a plant cell. Thus, promoters used in the constructs of the invention include cis-acting transcriptional control elements : and regulatory sequences that are involved in regulating or modulating the timing and/or : $5 rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription - terminator, an origin of replication, a chromosomal irategration sequence, 5' and 3’ untranslated regions, or an intronic sequence, which zre 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. “Constitutive” 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 developmen_tal conditions. Examples of environmental conditions that may affect transcriptior by inducible promoters include anaerobic conditions, elevated temperature, drought, Or the presence of light. “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 expresssed. Such factors are known to exist in mammals and plants so as to allow for specific tissues to develop.
The term "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 t=echniques, including angiosperms (monocotyledonous and dicotyledonous plants), as we 11 as gymnosperms. It includes plants of a variety of ploidy levels, including polyploic, diploid, haploid and hemizygous states. As used herein, 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 - 30 various recombinant constructs (e.g., expression cassettes) of the invention. “Plasmids” can be commercially available, publicly available on an unrestricted basis, or can be constructed from available= plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan. :
®
The term “gene” itacludes a nucleic acid sequence comprising a segment of
DNA involved in producing a traraiscription product (e.g., a message), which in turn is translated to produce a polypeptidlle chain, or regulates gene transcription, reproduction or stability. Genes can include regicwns preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between indiv=idual coding segments (exons).
The phrases “nucle=ic acid” or “nucleic acid sequence” includes— oligonucleotide, nucleotide, polymaucleotide, or to a fragment of any of these, to DNA or
RNA (e.g., mRNA, rRNA, tRNA, iRNA) 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 many DNA-like or RNA-like material, natural or synthetic in origin, including, e.g. _, iRNA, ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g., iRNPs). The term ermcompasses nucleic acids, i.e., oligonucleotides, .- containing known analogues of namtural 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; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156. “Amino acid” or “z=mmino acid sequence” 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. The terms “polypeptide” and “protein” include amino acids joined to eacka other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may containe. modified amino acids other than the 20 gene-encoded amino acids. The term “polypepti~de” 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 term “isolated’= includes a material removed from its original environment, e.g., the natural environment if it is naturally occurring. For example, 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 polynucleo-tides 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. As used herein, an issolated 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 libmrary can be conventionally : purified to electrophoretic homogeneity. In alternative aspects, the invention provides nucleic acids which have been purified from genomic IDNA or from other sequences in a § library or other environment by at least one, two, three, four, five or more orders of magnitude.
As used herein, the term “recombinant” can include nucleic acids adjacent to a “backbone” nucleic acid to which it is not adjacent Fn its natural environment. In one aspect, nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid “backbone molecules.” “Backbone molecules” according to : the invention include nucleic acids such as expression veectors, self-replicating nucleic } acids, viruses, integrating nucleic acids, and other vectomrs or nucleic acids used to .. maintain or manipulate a nucleic acid insert of interest. In one aspect, the enriched nucleic acids represent 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% 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 t-he desired polypeptide or protein. “Synthetic” polypeptides or protein are those prepared by chemical synthesis, as : described in further detail, below. .
A promoter sequence can be “operably lirked to” a coding sequence when
RNA polymerase which initiates transcription at the prormoter will transcribe the coding sequence into mRNA, as discussed further, below. “Oligonucleotide” includes either a single= stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' wphosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
The phrase “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 nucl_eotide or amino acid residue (sequence) identity, when compared and aligned for maxcimum correspondence, as measured using one any known sequence comparison algorithem, as discussed in detail below, or by visual inspection. In alternative aspects, the inventi on provides nucleic acid and polypeptide sequences having substantial identity to an exemmplary sequence of the invention, e.g., 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: M7; SEQ ID NO:19; SEQ ID NO:21; SEQ ID N6:23; SEQ
ID NO:25; SEQ ID NO:27; SEEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID
NO:35; SEQ ID NO:37; SEQ 1D 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; SEQID
NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID
NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID
NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQID
NO:85; SEQ ID NO:87; SEQ 1D NO:89; SEQ ID NO:91; 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; SE«Q ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID
NO:115; SEQ ID NO:117; SE«Q ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID
NO:125; SEQ ID NO:127; SE«Q ID NO: 129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID
NO:135; SEQ ID NO:137; SE«Q ID NO: 139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID
NO:145; SEQ ID NO:146; SE«Q ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID
NO:171; SEQ ID NO:179; SE«Q ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID .
NO:204; SEQ ID NO:210; SE«Q ID NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID d
NO:234; SEQ ID NO:241; SE«Q ID NO:248 and/or SEQ ID NO:254 (nucleic acids); SEQ
ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12;
SEQID NO:14; SEQ IDNO:1 6; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ
ID NO:24; SEQ ID NO:26; SEEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID
NO:34; SEQ ID NO:36; SEQ HD NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID
NO:44; SEQ ID NO:46; SEQ HD NO:48; SEQ ID NO:50; SEQ ID NO:52; SEQ ID
NO:54; SEQ ID NO:56; SEQ XD NO:58; SEQ ID NO:60; SEQ IDNO:62; SEQ ID
NO:64; SEQ ID NO:66; SEQ ED NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID ’ NO:74; SEQ ID NO:76; SEQ ED NO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID . 7 NO:84; SEQ ID NO:86; SEQ I'D NO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID
NO:94; SEQ ID NO:96; SEQ ILD NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID
NO:104; SEQ ID NO:106; SEQQ ID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQ ID
( ro
NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:1208; SEQ ID NO:122; SEQID
NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130=; SEQ ID NO:132; SEQ ID
NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140 sSEQID NO:142; SEQ ID
NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159 s SEQID NO:165; SEQ ID % NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID NO:194 = SEQ ID NO:200; SEQ ID
NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223= SEQ ID NO:230; SEQ ID
NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:2555, or the polypeptide encoded by SEQ ID NO: 145, over a region of at least about 13, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 80 ©, 850, 900, 950, 1000 or more residues, or a region ranging from between about 50 resiciues 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.
A “substantially identical” amino acid sequence= also can include a sequence that differs from a reference sequence by one or more= conservative or non- conservative amino acid substitutions, deletions, or insertions, mparticularly when such a substitution occurs at a site that is not the active site of the molescule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for anothe=r 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 aci d or glutamine for asparagine). One or more amino acids can be deleted, for exam _ple, from a protease, : resulting in modification of the structure of the polypeptide, wit hout significantly altering its biological activity. For example, amino- or carboxy-terminal amino acids that are not required for protease activity can be removed. “Hybridization” includes 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 czan 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 p-rehybridization 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 hybridi_zation temperature, altering the time of hybridization, as described in detail, below. Mn alternative aspects,
nucleic acids of the inveention are defined by their ability to hybridize under various : stringency conditions (e.g., high, medium, and low), as set forth herein. : “Varian€” includes polynucleotides or polypeptides of the invention : modified at one or mores base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of a protease of the invention (which can be assayed by, e.g... the hydrolysis of casein in zymograms, the release of fluorescence from gelatin, or the release of p-nitroanalide from various small peptide substrates). Variants can be produced by any number of means included methods such ass, for cxample, error-prorme PCR, shuffling, oligonuclcotide-directed mutagenesis, assembly
PCR, sexual PCR mutaegenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSS and any combination thereof. Techniques for producing variant protease having activites at a pH or temperature, for example, that is different from a wild— type protease, are included herein.
The terrmn “saturation mutagenesis” or “GSSM” includes a method that uses degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as described in detail, below.
The term “optimized directed evolution system” or “optimized directed evolution” includes a rmmethod for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below.
The terrm “synthetic ligation reassembly” or “SLR” includes a method of ligating oligonucleotide fragments in a non-stochastic fashion, and explained in detail, below.
Generating and Manip=ulating Nucleic Acids
The inveention provides nucleic acids (e.g., SEQ ID NO:1; SEQ ID NO:3;
SEQ ID NO:5; SEQ IID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID
NO:15; SEQ ID NO:1°Z; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID
NO:25; SEQ ID NO:2°/; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID
NO:35; SEQ ID NO:3°7; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID
NO:45; SEQ ID NO:47Z; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID
NO:55; SEQ ID NO:5°7; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID
NO:65; SEQ ID NO:6"7; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID
NO:75; SEQ ID NO:7°7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID
} p 0
NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91 ; SEQ ID MO0:93; 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:11 1; SEQ ID NO:113; SEQ ID
NO:115; SEQ ID NO:117; SEQ ID NO:1 19; SEQ ID NO:121; SEQ ID NO:123; SEQ ID
NO:125; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID
NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID
NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:154; SEQ ID
NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID
NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID N0O:222; SEQ ID NO:229; SEQ ID
NO:234; SEQ ID NO:241; SEQ ID NO:248 and/or SEQ ID NO:254 z nucleic acids encoding polypeptides as set forth in 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 N«©O:26; SEQ ID
NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID N©:36; SEQ ID
NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID N€:46; SEQ ID
NO:48; SEQ ID NO:50; SEQ ID NO:52; SEQ ID NO:54; SEQ ID N©:56; SEQ ID
INO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID
INO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NQO:76; SEQ ID
INO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID
INO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ ID NQO:96; SEQ ID
IN0:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID
IN0:108; SEQ ID NO:110; SEQ ID NO:1 12; SEQ ID NO:114; SEQ IID NO:116; SEQ ID
NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ IID NO: 126; SEQ ID
NO0:128; SEQ ID NO:130; SEQ ID NO: 132; SEQ ID NO: 134; SEQ IID NO:136; SEQ ID
NO:138; SEQ ID NO:140; SEQ ID NO: 142; SEQ ID NO:144; SEQ IID NO: 147; SEQ ID
NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ IID NO:180; SEQ ID
NO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ IID NO:21 1; SEQ ID
NI0:219; SEQ ID N0:223; SEQ ID NO:230; SEQ ID NO:235; SEQ IID NO:242; SEQ ID
N0:249 or SEQ ID NO:255, or the polypeptide encoded by SEQ ID NF 0:145, including expression cassettes such as expression vectors, encoding the polypeptides of the imwvention. The invention also includes methods for discovering new pxotease sequences ussing the nucleic acids of the invention. The invention also includes methods for inhibiting the expression of protease genes, transcripts and polypeptides using the nucleic acids of the invention. Also provided are methods for modifying the nwicleic acids of the invention by, e.g ., synthetic ligation reassembly, optimized directed evolution systems and/or saturation. mutagenesis.
The nucleic acids of the invention can be made, isolated and/or manipulated by, .g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. In practicing the methods of the invention, 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 ox device known in the art, which are well described in the scientific and patent literature.
General dechniques
The nucleic acids used to practice this invention, whether RNA, iRNA., antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a v-ariety of sources, genetically engineered, amplified, and/or expresse</ generated recombinantly. Recombinant polypeptides (e.g., proteases) generated from N 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, ye ast, insect or plant cex1l expression systems.
A Tternatively, 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. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free
Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Nara g (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981)
Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066.
Techniques for the manipulation of nucleic acids, such as, e.g., subclo ning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, 26 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.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. Joh
Wiley & Sons, Irac., New York (1997); LABORATORY TECHNIQUES IN :
BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH
NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed...
Elsevier, N.Y. (1 993).
o WO 2004/033668 : PCT/U S2003/032819
Amother useful means of obtaining and manipulating mcleic acids used to practice the methmods of the invention is to clone from genomic samples, an d, if desired, screen and re-clo-ne inserts isolated or amplified from, €.g., genomic cloness or cDNA clones. Sources eof nucleic acid used in the methods of the invention inclucle genomic or § cDNA libraries c-ontained in, e.g., mammalian artificial chromosomes (MA_Cs), see, c.g.,
U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see. e.g.,
Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (Y_ACY; bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon (1998)
Genomics 50:3063-316; P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids. . In one aspect, a nucleic acid encoding a polypeptide of the imnventionis : assembled in app-ropriate phase with a leader sequence capable of directing secretion of oo 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 poelypeptide, such as N-terminaal identification peptides which impart desired characteristics, such as increased stabilitwy or simplified purification. Peptides and polypeptides of —the invention can also be syntheesized 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 recombimnantly synthesized peptide, to identify and isolate antibodi_es and antibody-expressfing B cells, and the like. Detection and purification facilit-ating domains include, e.g., metaal chelating peptides such as polyhistidine tracts and histicline- tryptophan modules that allow purification on immobilized metals, protein _A domains : that allow purification on immobilized immunoglobulin, and the domain utilized in the
FLAGS extensior/affinity purification system (Immunex Corp, Seattle WA). The inclusion of a clezvable linker sequences such as Factor Xa or enterokinase (Invitrogen,
San Diego CA) between a purification domain and the motif-comprising pe ptide or polypeptide to facilitate purification. For example, an expression vector car include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and am enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Doobeli (1998) Protein Expr. Purif. 12:404-414). The histidime residues . facilitate detectior1 and purification while the enterokinase cleavage site provides a means for purifying the espitope from the remainder of the fusion protein. Technoleogy pertaining to vectors «encoding fusion proteins and application of fusion proteirns are well described in the scieratific and patent literature, see e.g., Kroll (1993) DNA Cezll. Biol, 12:441-53.
Transcriptional and translational control sequences
The invention provides nucleic acid (e.g., DNA) sequaences of the invention Operatively linked to expression (e.g., transcriptional or treanslational) control sequence(ss), e.g., promoters or enhancers, to direct or modulate RNA. synthesis/ expression The expression control sequence can be in an expressiomn vector. “Exemplary bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL a-nd trp. Exemplary eukaryotic promoters include CMV immediate early, HSV thymidin_e kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I.
Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or €xp promoters, the lacl promoter, the lacZ promoter, the T=3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PI. promoter, promoters from opero- ns encoding glycolytic enzymes such as 3-phosphoglycer—ate kinase (PGK), and the acid phosphatase promoter. Eukaryotic promoters include tte CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock proxmoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metallo ®hionein-I promoter.
Other prommoters known to control expression of genes in prokaryotic or eukaryotic cells or their virtases may also be used.
Tis-sue-Specific Plant Promoters
The invention provides expression cassettes that can oe expressed in a tissue-speck fic manner, e.g., that can express a protease of the invent#on in a tissue- specific mamner. The invention also provides plants or seeds that exgoress a protease of the invention in a tissue-specific manner. The tissue-specificity can We seed specific, stem specific, leaf specific, root specific, fruit specific and the like.
In one aspect, a constitutive promoter such as the CaN-IV 358 promoter can be used for expression in specific parts of the plant or seed or througEout the plant. For example, fo r overexpression, 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. Such. promoters are referred to herein as "constitutive" promot=ers and are active under most -environmental conditions and states of development or ce=Il differentiation.
Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of
Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skil 1. Such genes include, e.g., ACT!1 from Arabidopsis (Fuang (1996) Plant Mol. Biol. 33:1 25-1 39); Cat3 from Arabidopsis (GenBank No. U43147,
Zhong (1996) Mol. Gen. Geret. 251 1196-203); the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe (1994) P Zan
Physiol. 104:1167-1176); GP¢l from maize (GenBank No. X15596; Martinez (1989) J.
Mol. Biol 208:551-565); the Gpc2 from maize (GenBank No. U45855, Manjunath (19997)
Plant Mol. Biol. 33:97-112); plant promoters described in U.S. Patent Nos. 4,962,028; 3,633,440,
The invention_ uses tissue-specific or constitutive promoters derived from viruses which can include, e. =, the tobamovirus subgenomic promoter (Kumagai (1995)
Proc. Natl. Acad. Sci. USA 9°2:1679-1683; 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 (1 996) Plant Mol. Biol. 31:1129-1 139).
Alternatively, the plant promoter may direct expression of protease- 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. Examples of environmental conditions that may affect tramscription include anaerobic conditions, elevated temperature, the presence of 14ght, or sprayed with chemicals/hormones. For example , the invention incorporates the drawught-inducible promoter of maize (Busk (1997) supra); she cold, drought, and high salt in_ducible promoter from potato (Kirch (1997) Plant Mol.
Biol. 33:897 909).
Tissue-specific promoters can promote transcription only within a certain time frame of developmental sstage within that tissue. See, e.g., Blazquez (1998) Plant.
Cell 10:791-800, characterizing the Arabidopsis LEAFY gene promoter. See also Caron (1997) Plant J 12:367-77, describing the transcription factor SPL3, which recognizes =a conserved sequence motif in thie promoter region of the 4. thaliana floral meristem identity gene AP1; and Mande:1 (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter elF4. Tissue specific promoters which are active : throughout the life cycle of a pearticular tissue can be used. In one aspect, the nucleic acids of the invention are operably linked to a promoter active primarily only in cotton
Wa 2004/033668 PCT/US2003/032819 ® fi ber cells. In one aspect, the nucleic acids of the invention are operably linked to a promoter active primarily during the stages of cotton fibe=r cell elongation, e.g., as described by Rinehart (1996) supra. The nucleic acids czan be operably linked to the
FbI2A gene promoter to be preferentially expressed in cawtton fiber cells (Ibid) . See also,
John (1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; Y ohn, et al., U.S. Patent Nos. 5,608,148 and 5,602,321, describing cotton fiber-specificc 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 r-oot-specific promoters include tkxe promoter from the alcohol dehydrogenase gene (Del disle (1990) Int. Rev. Cytol. 1223:39-60). Other promoters that can be used to express the nucleic acids of the iravention 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. 11:1285 1295, describing a leaf-specific p=omoter in maize); the ORF13 promoter from 4grobactezrium rhizogenes (which exhibits high activity in roots, sec, e.g., Hansen (1997) supra); a maize pollen specific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161 168); a tomato promoter active dwiring fruit ripening, senescence and abscission of leaves and, to a lesser extent, of fl owers can be used (see, e.g., Blume (1997) Plant J. 12:7 31 746); a pistil-specific promoter from the potato SK2 gene (see, e.g., Ficker (19°97) Plant Mol. 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 BEL1 gene (see, e.g., Reiser (1995) Cell 83:735—742, GenBank No. U39944); arxd/or, the promoter in Klee, U.S. Patent No. 5,589,583, «describing a plant promoter : region is capable of conferring high levels of transcriptiom in meristematic tissue and/or . rapidly dividing cells.
Alternatively, plant promoters which are imducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids of the invention. For excample, the invention can use the auxin-response elements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) PAant Physiol. 115:397-407); the aLaxin-responsive Arabidopsis GST6 promoter (also respownsive 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 plamt biotin response element (Streit
C WO 2004/033668 PCT/US2003/032819 (1997) Mol. Plant Microbe Interact. 10=933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (19296) Science 274:1900-1 902).
The nucleic acids of the -invention can also be operably linked to plant promoters which are inducible upon EXpo0sure to chemicals reagents which can be applied * § to the plant, such as herbicides or antibi otics. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997)
Plant Cell Physiol. 38:568-577); applicamtion of different herbicide safeners indtices distinct gene expression patterns, includ. ing expression in the root, hydathodes, and the shoot apical meristem. Coding sequences can be under the control of, e.g, a : tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) argi nine decarboxylase gene (Masgrau (1997) Plant
J. 11:465-473); or, a salicylic acid-respo-nsive element (Stange (1997) Plant J, 11:1315-1324). Using 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 fEnvention can be induced at a particular stage of development of the plant. Thus, the inve=ntion 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.
One of skill will recognize= that a tissue-specific plant promoter may drive expression of operably linked sequences din tissues other than the target tissue. Thus, a tissue-specific promoter is one that drivess 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 in-vention can also be operably linked to plant promoters which are inducible upon expo sure 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 protease-producing nucleic acids of the invention will allow the groweer to select plants with the optimal protease expression and/or activity. The developm_ent of plant parts can thus controlled. In this way the invention provides the means to facilitate the harvesting of plants and plant parts.
For example, in various embodiments, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, is used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbi cide safeners induces distinct gene expression patterns, including expression in the root, kaydathodes, and the shoot apical meristem.
Coding sequences of the invention are also urader 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. 11:1315-1324). : :
In some aspects, proper polypeptide expression may require polyadenylation region at the 3'-end of the co«ding region. The polyadenylation region can be derived from the natural gene, from a wariety of other plant (or animal orother) genes, or from genes in the Agrobacterial T-IDONA.
Expression vectors and cloning vehicl es
The invention provides expres=sion vectors and cloning vehicles comprising nucleic acids of the invention, e.g ., sequences encoding the proteases of the : invention. Expression vectors and cloning ve=hicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phageemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, ademovirus, foul pox virus, pseudorabies and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast). 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), pB luescript plasmids, pNH vectors, (lambda-
ZAP vectors (Stratagene); ptrc99a, pKX223-3, pDR540, pRIT2T (Pharmacia); ~ Eukaryotic: pXT1, pSG5 (Stratagene), pSVK_3, pBPV, pMSG, pSVLSV40 (Pharmacia).
However, any other plasmid or other vector may be used so long as they are replicable and viable in the host. Low copy number or Enigh copy number vectors may be employed with the present invention.
The expression vector can conmprise a promoter, a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying express ion. Mammalian expression vectors can comprise an origin of replication, any necessaary ribosome binding sites, a polyadenylation site, splice donor and accepteor sites, transcriptional termination sequences, and 5' flanking non-transcribed se quences. In some aspects, DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
: i } @® WO 2004/033668 PCT/US2003/032819
In one aspect, the expression vectors contaimm one or more selectable marker genes to "permit selection of host cells containing the ~vector. Such selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for Co . eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter megions can be selected from any desired gene using chloramphenicol transferase (CAT) ve ctors or other vectors with selectable markers. - '
Vectors for expressing the podypeptide or fragment thereof in eukaryotic cells can 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 nucleic acid sequence can bse inserted into a vector by a variety of procedures. In general, the sequence is ligated to the desired position in the vector : following digestion of the insert and the vecteor with appropriate restriction . endonucleases. Alternatively, blunt ends in both the insert and the vector may be ligated. :
A variety of cloning techniques are known ime the art, e.g., as described in Ausubel and
Sambrook. Such procedures and others are ®eemed to be within the scope of those skilled . ' 20. in the art.
The vector can 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, phagse DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and prhage 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 can be used include the commercially available plasmids comprising genetic elemerats of the well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden),
GEMI (Promega Biotec, Madison, WI, USA pQE?70, pQEG60, pQE-9 (Qiagen), pD10, psiX174 pBluescript I KS, pNH8A, pNH16a_, pNHI 34, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRITS (Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG443, pXT1, pSG (Stratagene) pSVK3, pBPV,
: PMSG, and pSVL (Pharmacia). However, any other vector may be used as long as it is : replicable and viable in the host cell.
The nucleic acids of the imvention 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. £., cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus by transcription of an episomal mini-chromosome containimg supercoiled DNA, see, e.g., Covey-£1990) i
Proc. Natl. Acad. Sci. USA 87:1633-163 7. Alternatively, 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 thes invention can comprise a marker gene that confers a selectable phenotype on a plan cell or a seed. For example, the marker may encode biocide resistance, particularly aratibiotic 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 Agrobacterium 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. 37:471-476), cauliflower mosaic virus (see, e.g., Cecchini (1997) Mol. Plant Microbe
Interact. 10:1094-1101), maize Ac/Ds transposable element (see, e.g., Rubin (1997) Mol.
Cell. Biol. 17:6294-6302; Kunze (1996) «Curr. Top. Microbiol. Immunol. 204:161-194), and the maize suppressor-mutator (Spm) transposable element (see, e.g., Schlappi (1996)
Plant Mol. Biol. 32:717-725); and derivatives thereof.
In one aspect, 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. Furthermore, for : integrating expression vectors, the expression vector can contain at least one sequence homologous to the host cell genome. It ccan contain two homologous sequences which flank the expression construct. The integrrating vector can be directed to a specific locus in the host cell by selecting the appropriate homolog ous sequence for inclusion in the vector. Constructs for integrating vectors are well kanown in the art.
Expression vectors of the invention mmay also include a selectable marker gene to allow for the selection of bacterial strains thamt have been transformed, e.g., genes § which render the bacteria resistant to drugs such as a-mpicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracyclines. Selectable markers can also include biosynthetic genes, such as those in the histicline, tryptophan and leucifz : biosynthetic pathways.
Host cells and transformed cells
The invention also provides a transformmed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding =a protease 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 cellss, or plant cells. Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cells include Drosophila S2 and Spodoptera S/%. Exemplary animal cells include ,-
CHO, COS or Bowes melanoma or any mouse or hunman 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 ares well known and described in the technical and scientific literature. See, e.g., Weising (C1988) Ann. Rev. Genet. 22:421- 477; U.S. Patent No. 5,750,870.
The vector can be introduced into the haost cells using any of a variety of techniques, including transformation, transfection, tramsduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipyofection, or electroporation (Davis,
L., Dibner, M., Battey, 1., Basic Methods in Molecular Biology, (1986)). =0 In one aspect, the nucleic acids or vectors of the invention are introduced into the cells for screening, thus, the nucleic acids ente=r 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 CaPOy precipitation, liposome fusion, lipofection (e.g., LIPOFESCTIN™), 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. —=" .
Where appropriates, the engineered host cells can be cultured in conventional nutrient media mod ified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induce-d 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 polypeptice or fragment thereof,
Cells can be harvessted by centrifugation, disrupted by physical or chemical means, and the resulting crude ex=tract is retained for further purification. Microbial cells employed for expression of prote=ins can be disrupted by any convenient method, including freeze-thaw cycling, somnication, mechanical disruption, or use of cell lysing agents. Such methods are well kenown 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 chroma tography, 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) c-an be employed for final purification steps.
Various mammalizan cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasss and other cell lines capable of expressing proteins from a compatible vector, such ass the C127, 3T3, CHO, HeLa and BHK cell lines.
The constructs in bnost cells can be used in a conventional manner to } produce the gene product encodecd by the recombinant sequence. Depending upon the ‘ host employed in a recombinant poroduction procedure, the polypeptides produced by host cells containing the vector may bes glycosylated or may be non-glycosylated.
® WO 2004/033668 PCT/US2003/032819
Polypeptides of the invention may or may not also include am 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 DMA construct may be linearized prior to conducting an in vitro transcription reactiosn. 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 fra_gment thereof,
The expression vectors can contain one or mone 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. “.
Amplification of Nucleic Acids
In practicing the invention, nucleic acids of the invention and nucleic acids . encoding the proteases of the invention, or modified nucleic acids of the invention, can be reproduced by amplification. Amplification can also be used to clone or modify the nucleic acids of the invention. Thus, the invention provides amplification primer sequence pairs for amplifying nucleic acids of the invention. One of skill in the art can design amplification primer sequence pairs for any part of or €he full length of these sequences.
In one aspect, the invention provides a nucleic acid amplified by a primer pair of the invention, e.g., a primer pair 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 or 30 or more residues of a nucleic acid of the invention, and about the first (the 5%) 15, 165, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of the complementary strand (e.g., of SEQ ID NO:1; SEQ ID NO:3:
SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:1 1Z SEQID 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 N0O: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 N0:49; SEQ ID NO:51; 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; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; 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; SEQ ID NO:89; SEQ ID NO:91; 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:11 “7; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID
NO:125; SEQ ID NO:12 77; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID
NO:135; SEQ ID NO:13 7; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143: SEQ ID
NO:145; SEQ ID NO:14 6; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID
NO:171; SEQ ID NO:17 9; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID
NO:204; SEQ ID NO:21 0; SEQ ID NO:218; SEQ ID N0:222; SEQ ID NO:229; SEQ ID
NO:234; SEQ ID NO:24 1; SEQ ID NO:248 and/or SEQ ID NO:254).
The inveration provides an amplification primer sequence pair for amplifying a nucleic acic encoding a polypeptide having a protease 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, or about 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 consecutive bases of the sequence. The invention provides amplification primer paizrs, 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, or 25 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, or 25 residues of the complementary strand of the first member. The invention provides proteases gener-ated by amplification, e.g., polymerase chain reaction (PCR), using an amplification parimer pair of the invention. The invention provides methods of making a protease by amplification, e.g., polymerase chain reaction (PCR), using an amplification primer paix of the invention. In one aspect, the amplification primer pair amplifies a nucleic acid from a library, e.g., a gene library, such as an environmental library,
Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such ass the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an arr-ay ora blot), detect the nucleic acid, or quantify the amount of a. specific nucleic acid in a sample. In one aspect of the invention, message isolated from a cell or a cDNA library ame amplified.
, ; o WO 2004/033668 PCT/US2003/032819
The skilled artisan can select and design suitable osligonucleotide amplification primers. Amplification methods are also well knovevn in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO : oo METHODSS AND APPLICATIONS, ed. Innis, Academic Press, IN.Y. (1990) and PCR § STRATEGEIES (1995), ed. Innis, Academic Press, Inc., N.Y, liga se chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science= 241:1077; Barringer (1990) Genme 89:117); transcription amplification (see, e.g., Kwoh_ (1989) Prog Natl.
Acad. Sci. WJISA 86:1173); and, self-sustained sequence replicatior (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1 874); Q Beta replicase amplification (see, e.g.
Smith (19977) J. Clin. Microbiol. 35: 1477-1491), automated Q-betaa replicase amplification assay (see, e.g., Burg (1 996) Mol. Cell. Probes 10:2257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississ auga, Ontario); see also
Berger (1987) Methods Enzymol. 152:307-3 16; Sambrook; Ausut=el; U.S. Patent Nos. 4,683,195 amd 4,683,202; Sooknanan (1995) Biotechnology 13:56 3-564.
Determiningy the degree of sequence identity
The invention provides nucleic acids comprising sequences having at least about 50%, _51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 6%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 11%, 72%, 13%, 74%, 75%, 76%, 77%, 718%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90", 91%, 92%, 93%, 94%, 95%, S6%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention (e.g, SEQ ID NO:1; SEQ JID NO:3; SEQ ID
NO:5; SEQ HD NO:7; SEQ ID NO:9; SEQ ID NO:1 1; SEQ ID NO=13; SEQ ID NO:15;
SEQ ID NO=17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SSEQ ID NO:25; SEQ
ID NO:27; S_EQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ MD NO:35; SEQ ID
NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID INO:45; SEQ ID
NO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID INO:55; SEQ ID
NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID ™N0:65; SEQ ID
NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID MNO0:75; SEQ ID
NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID ™N0:85; SEQ ID
NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID M0:95; SEQ ID
NO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ IID NO:105; SEQ ID
NO:107; SEQQ ID NO:109; SEQ ID NO:111; SEQID NO:113; SEQ ID NO:115; SEQ ID
NO:117; SEQ IDNO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID
NO:127; SEQ ID NJ0:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NJO:135; SEQ ID
NO:137; SEQ ID N0:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NJ0:145; SEQ ID
NO:146; SEQ ID NJO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID NJO:171; SEQ ID
NO:179; SEQ ID NJ0:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NJ0:204; SEQ ID § NO:210; SEQ ID NJ0:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NJO:234; SEQ ID
NO:241; SEQ ID NJ0O:248 and/or SEQ ID NO:254, and nucleic acids encending SEQ ID .
NO:2, SEQ ID NO =4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SE€Q ID-NO:12,
SEQ ID NO:14, SEEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ [¥D NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID N0O:28, SEQ ID NO:30, SEQ ID NO»:32, SEQID
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 ID NO:72=, SEQ ID
NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82=, SEQ ID
NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92=, SEQ ID
NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:1 02, SEQ ID
NO:104, SEQ ID N¥O:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NC O:112, SEQ ID
NO:114, SEQ ID NIO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NT 0:122, SEQ ID
NO:124, SEQ ID N¥O:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID N"0:132; SEQ ID
NO:134; SEQ ID NXO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NJO:142; SEQ ID
NO:144 and/or SEQQ ID NO:147) over a region of at least about 50, 75, 10, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, : 1100, 1150, 1200, ® 250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues. The : invention provides ~polypeptides comprising sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 642%, 65%, 66%, 67%, 68%, 69%, 10%, 71%, 12%, 73%, 74%, 15%, 16%, 17%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 942%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary polypeptide of the imvention. The extent of sequence identity (homology may be determined using ary computer program and associated parameters, inchmding those described herein, sumch as BLAST 2.2.2. or FASTA version 3.0t78, with tFae default parameters.
Hommologous sequences also include RNA sequences in which uridines replace the thymine s in the nucleic acid sequences. The homologous sequiences may be
. ‘oe
C WO 2004/033665 . PCH/US2003/032819 obtained using any of the procedures described herein or may result from the correction of a sequencimg error. It will be appreciated that the nucleic acid sequences as set forth herein can be mepresented in the traditional single character format (see, e.g., Stryer,
Lubert. Biochesmistry, 3rd Bd., W. H Freeman & Co., New York) or in amy other format whichrecords the identity of the nucleotides in a sequence.
Various sequence comparison programs identified herein zare used in this aspect of the imvention. 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 lim ted to,
TBLASTN, BL.ASTP, FASTA, TFASTA, and CLUSTALW (Pearson an Lipman, Proc.
Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Bio. 215(3):403- 410, 1990; Thowmpson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al.
Methods Enzyrmol. 266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410, - 1990; Altschul et al., Nature Genetics 3:266-272, 1993).
Homology or identity can be measured using sequence ana lysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, \V1 53705).
Such software matches similar sequences by assigning degrees of homolo £y to various deletions, substitutions and other modifications. The terms “homology” and “identity” in the context of tvvo or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentag:e 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 measumred using any number of sequesnce comparison algorithms or by manual alignment and visual inspection. For sequence comparison, one sequence can act as a reference sequence, e.g, a sequence of thes invention, to which test Sequences are compared. When using a sequence compaxrison algorithm, test and reference Sequences are entered ixto a computer, subsequence coowrdinates are designated, if necessary, and sequence algoritdhm program parameters are designated. Default program parameters can be used, or alteemative parameters can b-e designated. The sequence comparison algorithm then calculates the percent sequence- identities for the test sequences relative to the reference sesquence, based on the program p arameters.
A “comparison window”, as used herein, includes reference to a segment : of any one of the numbers of contiguous residues. For example, in alternative aspects of the inventiom, contiguous residues ranging anywhere from 20 to the fur 1l length of an exemplary prpolypeptide 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. If the reference sequence has the requisite sequence identity to an exemplary ppolypeptide or nucleic acid sequence of the invention, e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, “70%, 11%, 72%, 13%, 14%, 75%, 16%, 17%, 18%, 79%, B0%, 81%%, 82%, 83%, 84%, B5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, O5%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence of the invention, that sequence is within the scope of the invention. In alternative embodiments, subseq uences ranging from about 220 to 600, about 50 to 200, and about 100 to 150 are comps ared to a reference sequence of~ the same number of contiguous positions after the two sequences are optimally al igned. 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. Mol. Biol. 48:443, 1970, by the search for similarity method of person & Lipman, Proc. Nat'l. Aca-d. Sci. USA } 85:2444, 19 88, by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.
Other algorithms for determining homology or identity include, for excample, in addition to a BLAST" program (Basic Local Alignment Search Tool at the Natieonal Center for
Biological Imformation), ALIGN, AMAS (Analysis of Multiply Align ed Sequences),
AMPS (Pro&ein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical
Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequaence Comparative
Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points,
BMB, CLUSSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS,
Smith-Wate rman algorithm, DARWIN, Las Vegas algorithm, FNAT Forced Nucleotide
Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSSAP (Fristensky
Sequence Amalysis Package), GAP (Global Alignment Program), GEINAL, GIBBS,
GenQuest, I SSC (Sensitive Sequence Comparison), LALIGN (Local SSequence
Alignment), LCP (Local Content Program), MACAW (Multiple Aligrament Construction & Analysis "Workbench), MAP (Multiple Alignment Program), MBLIKP, MBLKN, PIMA (Pattern-Ind uced Multi-sequence Alignment), SAGA (Sequence Aligrament by Genetic
® WO 2004/033668 PCT/US2003/0328 19
Algorithm) and WHAT-IF. Stach alignment programs can also be used to screen genome databases to identify polynucleeotide sequences having substantially identical sequenc=es.
A number of genome database=s are available, for example, a substantial portion of thes human genome is available as part of the Human Genome Sequencing Project (Gibbss, 1995). Several genomes have been sequenced, e.g., M. genitalium (Fraser et al., 1995), : M. jannaschii (Bult et al., 199€6), H. influenzae (Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast (S. cerevisiae) (Mewes et al., 1997), and D. elanogzaster (Adams et al., 2000). Significant progress has also been made in sequencing the genOmes : of model organism, such as meouse, C. elegans, and Arabadopsis sp. Databases conta3ining genomic information annotated with some functional information are maintained by different organization, and are= accessible via the internet.
BLAST, BLASST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are descrigbed, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389- 3402; Altschul (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available ®hrough the National Center for Biotechnology Informa tion.
This algorithm involves first iclentifying high scoring sequence pairs (HSPs) by identifying short words of lenggth W in the query sequence, which either match or sati_sfy some positive-valued thresholed score T when aligned with a word of the same length ina database sequence. T is referred to as the neighborhood word score threshold (Altschmul : (1990) supra). These initial nezighborhood word hits act as seeds for initiating searches to find longer HSPs containing tkaem. The word hits are extended in both directions alorg each sequence for as far as the= cumulative alignment score can be increased. Cumulative scores are calculated using, fomr nucleotide sequences, the parameters M (reward scores for a pair of matching residues; al~ways >0). For amino acid sequences, a scoring matrix is 26 used to calculate the cumulative score. Extension of the word hits in each direction a-re 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 rmegative-scoring residue alignments; or the end of eithemr sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequerces) uses as defaults a wordlength (CW) of 11, an expectation (E) of 10, M=5, N=-4 and a = comparison of both strands. F<or amino acid sequences, the BLASTP program uses ass defaults a wordlength of 3, ancl expectations (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)
W/O 2004/033668 PCT/US2003/032819 ® alignments (B) of 50, expectation (E) of 10, M=5, N=-<%, and a comparison of both strands. The BLAST algorithm also performs a statisticzal analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (15393) Proc. Natl. Acad. Sci. USA ©0:5873). One measure of similarity provided by BLASST algorithm is the smallest sum gprobability (P(N)), which provides an indication of the yorobability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a mucleic acid is considered similar to a references sequenmce if the smallest sum Probability
An a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, nore preferably less than about 0.01, and most preferab ly less than about 0.001. In one aspect, protein and nucleic acid sequence homologies ar-e evaluated using the Basic Local
Alignment Search Tool ("BLAST"). For example, five specific BLAST programs can be used to perform the following task: (1) BLASTP and B LAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide qQuery sequence against a nucleotide sequence database; (3) BLASTX compares the six- fame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN conmpares 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 " zagainst the six-frame translations of a nucleotide sequermce database. The BLAST porograms 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 obtain_ed from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by rneans of a scoring matrix, many of which are known ir. the art. Preferably, the scoring matrix used is the BLOSUMG62 matrix (Gonnet et al., Science 256:1443-1445, 1992;
Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, the PAM or PAM250 rmatrices may also be used (see, e.g., Schwartz and Daylnoff, eds., 1978, Matrices for ‘
Detecting Distance Relationships: Atlas of Protein Seqmuence and Structure, Washington:
Iational Biomedical Research Foundation). -
In one aspect of the invention, to determ&ne if a nucleic acid has the requisite sequence identity to be within the scope of the invention, the NCBI BLAST 2.2.2 programs is used, default options to blastp. There are about 38 setting options in the
E3LAST 2.2.2 program. In this exemplary aspect of the invention, all default values are tased except for the default filtering setting (i.e., all parammeters 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 i-n Karlin-Altschul violations due to short length of sequence.
The default values used in this exemplary aspect of the invention include: "Filter for low complexity: OBEN : : Word Size: 3
Matrix: Blosum62 =
Gap Costs: Existence:11
Extension; 1"
Other default settings «an be: filter for low complexity OFF, word size of 3 for protein, BLOSUM62 matrix, gap existence penalty of -11 and a gap extension penalty of -1. An exemplary NCBI BLAST 2.2.2 program setting has the "-W™ option default to 0. This means that, if not set, the word size defaults to 3 for proteins and 11 for nucleotides.
Computer systems and computer program products
To determine and iden tify sequence identities, structural homologies, motifs and the like in silico, 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 thes invention. As used herein, the words “recorded” and “stored” refer to a process for stomring 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 ir1vention.
Another aspect of the invention is a computer readable medium having recorded thereon at least one nucleic acid and/or polypeptide sequence of the invention.
Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, 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 kenown to those skilled in the art.
Aspects of the invention include systems (e.g., internet based systems), particularly computer systems, which store and 1manipulate the sequences and sequence : information described herein. One example of za computer system 100 is illustrated in ~ block diagram form in Figure 1. As used hereim, “a computer system” refers to the hardware components, software components, amd 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 Corporatiore, or similar processor from Sun,
Motorola, Compaq, AMD or International Busiriess 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, arad one or more data retrieving devices for oo retrieving the data stored on the data storage cormponents. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.
In one aspect, 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 reco rded 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 dri ve, a magnetic tape drive, or a modem . capable of connection to a remote data storage system (e.g., via the internet) etc. In some embodiments, the internal data storage device 1 10 is a removable computer readable medium such as a floppy disk, a compact disk, & magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate sofiware 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 whitch 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 fox accessing and processing the nucleotide or amino acid sequences of the invention can reside in main memory 115 during execution. In some aspects, the computer systerma 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 = 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 Comupare a nucleotide sequence with other nucleotide sequences and/or compounds stored within a data storage means, For example, the sequence comparison algorithnm 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 homo _logies or structural motifs. —-
The parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. In some aspects, the parameters may be the default parameters used by the al gorithms in the absence of instructions from the user. Figure 2 is a flow diagram illustrating one aspect of a process 200 for comparing a new nucleotide or protein seque=nce with a database of sequences in order to determine the homology levels between the rmew 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 sumch 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. As discussed above, the memory could “be any type of memory, including RAM or an internal storage device. The process 200 thhen 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 amt 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 be-tween the new sequence and the first sequence in the database. Well-known metho-ds are known to those of skill in the art for comparing two nucleotide or protein Sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during - comparison are normally entered by the user of the computer system. Once a compari=son of the two sequences has been performed at the state 210, a determination is mnade at a decision state 210 whether the : two sequences are the same. Of course, the temm “same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as “same” in the proce=ss 200. If a determination is made that the two sequences are the same, the processs 200 moves to a state 214 wherein the name of the sequence from the database is displayedll to the user. This state notifies the user that the sequence with the displayed name fulfil 1s the homology constraints that were entered.
Once the name of the stored sequence iss displayed to the user, 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 seq-uences do exist in the database, then theprocess 200 moves to a state 224 wherein a poimater is moved to the next sequence in the database so that it can be compared to the new se=quence. In this manner, the new sequence is aligned and compared with every sequemnce in the database. It should be noted that if a determination had been made at the decision state 212 that the sequences were not homologous, then the process 200 wouled move immediately to the decision state 218 in order to determine if any other sequencess were available in the database for comparison,
Accordingly, one aspect of the inventior is a computer system comprising a processor, a data storage device having stored thereown a nucleic acid sequence of the invention and a sequence comparer for conducting the c=omparison. The sequence comparer may indicate a homology level between the sequence=s 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 flo-w diagram illustrating one embodiment of a process 250 in a computer for determini-ng whether two sequences are homologous. The process 250 begins at a start state 252 arad 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 secquence is read and then to a state 262 wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, Gor U. If the 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 c=haracters are the same. If they are the same, then the process 250 moves to a state 268 wheerein 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 czontinues 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 27 4 to determine whether there are any more characters either sequence to read. If there are not amy more characters to read, then 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 s-equences that were the same out of the total number of sequences in the first sequence. T hus, if every character in a first 100 nucleotide sequence aligned with an every character im a second sequence, the homology level would be 100%. —
Alternatively, 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 ma-y 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. Thus, in some aspects, the computer program is a program which identifies SNPs. The method may be implemen ted by the computer systems described above and the method illustrated in F igure 3_ The method can be performed by reading a sequence of the invention and the reference s equences through the use of the computer program and identifying differences with the computer program.
In other aspects the computer based system comprises an identifier for } identifying features within a nucleic acid or polypeptides of the invention. An “identifier” refers to one or more programs which identifies certain features within a nucleic acid sequence. For example, 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 arad 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. The process 300 then moves to a_ state 306 wherein a database of sequence features is opened. Such a database would include a list of each features attributes along with the name of the feature. For example, 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 wvould be “TAATAA”. An example of such a database is produced by the University of Wisconsin Genetics
Computer Group. Alternatively, the features may be structural polypeptide motifs such as alpha helices, beta sheets, or fumctional polypeptide motifs such as enzymatic active sites, helix-turn-helix motifs or other motifs known to those skilled in the art. Once the database of features is opened at the state 306, 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 3 18-wherein the name of the found feature iss displayed to the user. The process 300 then moves to a decision state 320 wherein a destermination 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. However, if more features do exist in the database, then 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 thre 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. Thus, in one aspect, the invention provides a computer program that identifies open reading frames (ORFs).
A polypeptide oor nucleic acid sequence of the invention can be stored and manipulated in a variety of data processor programs in a variety of formats. For example, a sequence can be stored as text in a word processing file, such as Microsof WORD 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. In addition, many computer programs and databases may b« used as sequence comparison algorithms, identifiers, or sources of reference nucleotides sequences or polypeptide sequences to be compared to a nucleic acid sequence of the in vention. The programs and databases used to practice the invention include, but are not 1 imited to: MacPattern (EMBL), DiscoveryBase (Molecular
Applications Group), GeneMire (Molecular Applications Group), Look (Molecular
Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-2245, 1990), Catalyst (Molecular Simulations Inc.),
Catalyst/SHAPE (Molecular S imulations Inc.), Cerius2.DBAccess (Molecular
Simulations Inc.), HypoGen (NMolecular Simulations Inc.), Insight II, (Molecular
Simulations Inc.), Discover (MTolecular Simulations Inc.), CHARMm (Molecular
: ® WO 200- 4/033668 PCT/US2003/032819
Simulzations Inc.), Felix (Molecular Simulations Inc.), DelPhii, (Molecular Simulations
Inc.), ®QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.),
ModeRer (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein
Desigm (Molecular Simulations Inc.), WebLab (Molecular Si-mulations Inc.), WebLab
Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations
Inc.), SSeqFold (Molecular Simulations Inc.), the MDL Avail=able Chemicals Directory : databamse, the MDL Drug Data Report data base, the Comprelensive Medicinal" Chemistry databamse, Derwent’s World Drug Index database, the BioByteeMasterFile database, the
Genbamnk database, and the Genseqn database. Many other parograms and data bases would be apparent to one of skill in the art given the present disclosure.
Motifs which may be detected using the above programs include sequerces encoding leucine zippers, helix-tumn-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 stretchess, enzymatic active sites, substraate binding sites, and enzymatic cleavage sites.
Hybridization of nucleic acids
The invention provides isolated or recombinamt nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention (e.g., SEQ
ID NOO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11;
SEQ I'D NO:13; SEQ ID NO: 15; SEQ ID NO:17; SEQ ID Ne«O: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; SEEQ ID NO:41; SEQ ID
NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49; SEEQ ID NO:51; SEQ ID
NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEEQ ID NO:61; SEQ ID
NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEEQ ID NO:71; SEQ ID
NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEEQ ID NO:81; SEQ ID
NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEEQ ID NO:91; SEQ ID
NO:938 ; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEEQ 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:1L 3; SEQ ID NO:115; SEQ ID NO:117; SEQ ID NO:11+9; SEQ ID NO:121; SEQ ID
NO:123; SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO: 12:9; SEQ ID NO:131; SEQ ID
NO:1383; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID
NO:143; SEQ ID NO:1 45; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:1.558; SEQ ID
NO:164; SEQ ID NO:1-71; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:1993; SEQ ID
NO:199; SEQ ID NO:2@4; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:2222; SEQ ID :
Co NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 and/or SEQID NO:254), : ora nucleic acid that enacodes a polypeptide of the invention (e.g, SEQID NO:2, SEQ ID
NO:4, SEQ ID NO:6, S¥EEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IID NO:14,
SEQ ID NO:16, SEQ IID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NE9=24, SEQ
ID NO:26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SSEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQQ 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 N0O:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID N0O:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SSEQ ID
NO:106, SEQ ID NO:10 8, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:11 <4, SEQ ID
NO:116, SEQ ID NO:11 8, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:12=4, SEQ ID
NO:126, SEQ ID NO:12 8, SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:13=4; SEQ ID
NO:136; SEQ ID NO:13 38; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:14=4 and/or
SEQIDNO:147). The s#ringent conditions can be highly stringent conditions, medium stringent conditions and/or low stringent conditions, including the high and reduced stringency conditions desscribed herein. In one aspect, it is the stringency of thes wash conditions that set forth the conditions which determine whether a nucleic acid is within the scope of the inventiora, as discussed below.
In alternatIve embodiments, nucleic acids of the invention as defined by their ability to hybridize Lander stringent conditions can be between about five ressidues and the full length of nucMeic acid of the invention; e. g., they can be at least 5,1 0, 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. These nucleic acids can be useful as, e.g., hybridization probes, labe=ling probes, PCR oligonucleotide probes, iRNA (sing Me or double stranded), antisensze or sequences encoding antibody binding peptides (e gpitopes), motifs, active sites and the= like.
o WO 2004/033-668 : PCT/US2003/032819
In one aspect, nucleic acids of the invention are defined by their ability to hybridize umder high stringency comprises conditions of about 50% formamide at about 37°C to 425°C. In one aspect, nucleic acids of the invention are defined by their ability to hybridize umder reduced stringency comprising conditions in about 3 5% to 25% formamide atabout 30°C to 35°C. : Alternatively, nucleic acids of the invention are defined by their ability to hybridize umder high stringency comprising conditions at 42°C in 50% formamide, 5X
SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, suach as cot-1 or salmon sperm DNA (e.g., 200 n/m sheared and denatured salmon sperm DNA). In one aspect, nucl eic acids of the invention are defined by their ability to hybridize under reduced strimgency conditions comprising 35% formamide at a reduced temperature of 35°C.
Following hybridization, the filter may be washed with 6X SSC, 0.5% -.
SDS at 50°CC. These conditions are considered to be “moderate” cond itions above 25% formamide znd “low” conditions below 25% formamide. A specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of “low stringency” hybridization cornditions is when the above hybridization is conducted at 10% formamide.
The temperature range corresponding to a particular level of stringency can be furtheer narrowed by calculating the purine to pyrimidine ratio of the nucleic acid’ of interest amd adjusting the temperature accordingly. Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and lovw stringency conditions as set forth in Ausubel and Sambrook. Variations on the a bove ranges and conditions azre well known in the art. Hybridization conditions are discussed further, below.
The above procedure may be modified to identify nucl eic 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. For exzample, the hybridization temperature may be decreased im increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na* concentration of approximately
IM. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature «of hybridization. These conditions are considered to be “moderate” conditions altoove 50°C and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when the above hybridization is conducted at
55°C. A specific example of “low stringency” hybridization conditions i_s when the above hybridization is conducted at 45°C.
Alternatively, the hybridization may be carried out in buffers, such as 6X
SSC, containing formamide at a temperature of 42°C. In this case, the cconcentration 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. Fo llowing hybridization, thee filter may be washed with 6X SSC, 0.5% SDS at 50°C_ These conditions are considered to be “moderate” conditions above 25% formaxrmide and “low” conditions below 25% formamide. A specific example of “moderate” hy bridization conditions is when the above hybridization is conducted at 30% formami«e. A specific example of “low stringency” hybridization conditions is when the above Thybridization is conducted at 10246 formamide.
However, the selection of a hybridization format is not critical - it is the stringency of thes wash conditions that set forth the conditions which determine whether a nucleic acid is within the scope of the invention. Wash conditions used te 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°C or about 55° C to about 60°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°C or about 55°C to about 60°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 containimmg 0.1% SDS at room ternperatur € for 15 minutes and then washed twice by 0.1X SSC comtaining 0.1%
SDS at 680C for 15 minutes; or, equivalent conditions. See Sambrook, T ijssen and
Ausubel for a description of SSC buffer and equivalent conditions. :
These methods may be used to isolate nucleic acids of the invention.
Oligonucleotides probes and methods for using them
The invention also provides nucleic acid probes that can be used, e.g., for identifying nucleic acids encoding a polypeptide with a protease activity or fragments thereof or for ide ntifying protease genes. In one aspect, the probe compri. ses at least 10 consecutive bases of a nucleic acid of the invention. Alternatively, a proboe of the invention can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18,19, 20,21, 22,23, 24,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 610 about 30 to 70, consecutive bases of a sequence as set Forth in a nucleic 80 i
® WO 2004/033668 PCT/US2003/032819 . acid of the invention. “Whe probes identify a nucleic acid by binding and/or hybmridization.
The probes can be used in arrays of the invention, see discussion below, includi ng, e.g., capillary arrays. The pmobes of the invention can also be used to isolate other neicleic acids or polypeptides. oo :
The proboes of the invention can be used to determine whether a biological sample, such as a soil seample, contains an organism having a nucleic acid seque-nce of the : invention or an organisrm from which the nucleic acid was obtained. In such piocedures, a biological sample potentially harboring the organism from which the nucleic a-cid was isolated is obtained and nucleic acids are obtained from the sample. The nucleic acids are contacted with the probes under conditions which permit the probe to specifically hybridize to any complementary Sequences present in the sample. Where necessary, conditions which permit the probe to specifically hybridize to complementary sequences may be determined by p-lacing the probe in contact with complementary sequences from samples known to conta-in the complementary sequence, as well as control seque=nces which do not contain the complementary sequence. Hybridization conditions, stach as the salt concentration of the hybridization buffer, the formamide concentration of thes hybridization buffer, or he hybridization temperature, may be varied to identify conditions which allow €he probe to hybridize specifically to complementary nucleic acids (see discussion on specific hybridization conditions).
If the sample contains the organism from which the nucleic acid veas isolated, specific hybridi zation of the probe is then detected. Hybridization may be detected by labeling the gprobe with a detectable agent such as a radioactive isotopoe, 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 Sawuthem
Blots, Northern Blots, co lony hybridization procedures, and dot blots. Protocols for each of these procedures are p-rovided in Ausubel and Sambrook.
Alternatively, more than one probe (at least one of which is capable of specifically hybridizing te any complementary sequences which are present in thes nucleic acid sample), may be usead in an amplification reaction to determine whether the ssample contains an organism con~taining a nucleic acid sequence of the invention (e.g., an_ organism from which the nucleic acid was isolated). In one aspect, the probes cormprise oligonucleotides. In one spect, the amplification reaction may comprise a PCR reaction.
PCR protocols are descritoed in Ausubel and Sambrook (see discussion on amplifi-cation reactions). In such procedures, the nucleic acids in the sampl_e are contacted with the prob es, 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 intercalamtor such as ethidium bronnide. Alternatively, one or more of the probes may be latoeled with a radioactive isotospe and the presence of a radioactive amplification produect may be detected by automadiography after gel electrophoresis. —
Probes derived from sequences near the 3° or 55’ ends of a nucleic acid sequ-ence of the invention can also be used in chromosome walking procedures to identify clones containing additional, e.g., genomic sequences. Such mmethods allow the isolation of genes which encode additional proteins of interest from thes host organism.
In one aspect, nucleic acid sequences of the in—vention are used as probes to iden®ify and isolate related nucleic acids. In some aspects, thee 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. In ssuch procedures, a nucleic acid sample is contacted with the probe under conditions whi «ch 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 m -ethods described above.
In nucleic acid hybridization reactions, the coraditions used to achieve a partiacular level of stringency can vary, depending on the nahxre of the nucleic acids being hybridized. For example, the length, degree of complementamrity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (<.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization cond itions. An additional consideration is whether one of thes nucleic acids is immobilized, for example, on a filter. Hybridization can be carried out under conditions of lo~w stringency, moderate stringency or high stringency. A_s an example of nucleic acid hybridization, a polymer membrane containing immobilized cdenatured nucleic acids is first gprehybridized for 30 minutes at 45°C in a solution consissting of 0.9 M NaCl, 50 mM
NaH=PO4, pH 7.0, 5.0 mM Na;EDTA, 0.5% SDS, 10X Deny ardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 10” cpm (specific activity 4-9 X 108 cpm/ug) of 21° end-labeled oligonucleotide probe can then added to the solution. After 12-16 . hours of incubation, the membrane is washed for 30 minutes zat room temperature (RT) in 1X SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na;EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh 1X SET at Tm-10°C for the
( - { : ’ : ® WO 2004/033668 PCT/US2003/032819 oligonucleotide probe. The membrane is then exposed to auto-radiographic film for detection of hybridization signals. .
By varying the stringesncy of the hybridization conditions used to identify nucleic acids, such as cDNAs or gen omic DNAs, which hybridize to the detectable probe, nucleic acids having different levels of homology to the probe can be identified and isolated. Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes. The melting température,
Tm, is the temperature (under define d ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly cosmplementary probe. Very stringent conditions are selected to be equal to or about 5°C Mower than the Tm for a particular probe. The melting temperature of the probe ma-y be calculated using the following exemplary formulas. For probes between 14 and 70 nucleotides in length the melting temperature (Tm) is calculated using the formula= Tm=81.5+16.6(log [Na+])+0.41 (fraction G+C)- (600/N) where N is the length of the probe. If the hybridization is carried out in a 16 solution containing formamide, the nnelting temperature may be calculated using the equation: Tm=81.5+16.6(log [Na+])—+0.41 (fraction G+C)-(0.63% formamide)-(600/N) where N is the length of the probe. Prehybridization may be carried out in 6X SSC, 5X
Denhardt's reagent, 0.5% SDS, 100px denatured fragmented salmon sperm DNA or 6X
SSC, 5X Denhardt's reagent, 0.5% SIDS, 100pg 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. Where 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. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25°C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5-10°C below the Tm. In one aspect, hybridizations in 6X SSC are conducsted at approximately 68°C. In one aspect, hybridizations in 50% formamide corataining solutions are conducted at approximately 42°C. All of the foregoing hybridizations would be considered to be under conditions of high stringency. :
Following hybridization, the filter- is washed to remove any non- specifically bound detectable probe. The stringemncy used to wash the filters can also be varied depending on the nature of the nucleic acicds 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); 0.1X SSC, 0:3% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1X SSC, 0.5% SDS for 15 to 30 minutes at between the hybridization. temperature and 68°C (high 1» stringency); and 0.15M NaCl for 15 minutes at 72°C (very high stringency). A final low stringency wash can be conducted in 0.1X SSC a® room temperature. The examples above are merely illustrative of one set of conditieons that can be used to wash filters. One of skill in the art would know that there are nume rous recipes for different stringency washes.
Nucleic acids which have hybridizzed to the probe can be identified by autoradiography or other conventional techniquess. 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. For example, the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°CC in a hybridization buffer having a Na+ concentration of approximately 1M. Following hybridization, 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°C and “low” conditions below 50°C.
An example of “moderate” hybridization conditio ns 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.
Alternatively, the hybridization may be carried out in buffers, such as 6X
SSC, containing formamide at a temperature of 42°C. In this case, the concentration of formamide in the hybridization buffer may be redwiced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe. Following hybridization, the filter may be washed with 6X S SC, 0.5% SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide. A specific example of “moderate” hybridization conditions is when the above hybridization is conclucted at 30% formamide. A specific
Ne | / ® WO 2004/033668 PCT/US2003/032819 . example of “low stringency” hybridization ceonditions is when the above hybridization is : conducted at 10% formamide.
These probes and methods of the invention can be used to isolate nucleic - acids having a sequence with at least about 39%, 98%, 97%, at least 95%, at least 90%, at § least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% homology to a nucleic zcid sequence of the invention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 407, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive bases thereof, and the sequences complementary thereto. Homology may be measured using an alignment algorithm, as discussed herein. For example _, the homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic var-iants may have a substitution, deletion or addition of one or more nucleotides when co mpared to a nucleic acid of the ifivention.
Additionally, the probes and rmethods of the invention can be used to i isolate nucleic acids which encode polypepti des having at least about 99%, at least 95%, at least 90%, at least 85%, at least 80%, at le ast 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence -identity (homology) to a polypeptide of the invention comprising at least 5, 10, 15, 20, 2 5,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids, as determined using a sequences alignment algorithm (e.g., such as the
FASTA version 3.0t78 algorithm with the de=fault parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein).
Inhibiting Expression of Protease
The invention provides nucleic acids complementary to (e.g., antisense sequences to) the nucleic acids of the inventi_on, e.g., protease-encoding nucleic acids.
Antisense sequences are capable of inhibiting the transport, splicing or transcription of protease-encoding genes. The inhibition can_ be effected through the targeting of genomic
DNA or messenger RNA. The transcription -or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage. One particularly useful set of inhibitors provided by the present invention Hncludes oligonucleotides which are able to either bind protease gene or message, in either case preventing or inhibiting the production or function of protease. The asso ciation can be through sequence specific hybridization. Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of protease message The oligonucleotide can have enzyme activity which causes such cleavage, sucha as ribozymes. The oligonucleotide can be chemically modified or conjugated to an e=nzyme or composition capable of cleaving the complementary nucleic acid. A pool of many different such oligonucleotides can be screened for those with the desired activi€y. Thus, the invention provides various § compositions for the inhibition of protease expression on a nucleic acid and/or protein level, e.g., antisense, iRNA and ribozyme=s comprising protease sequences of the invention and the anti-protease antibodies of the invention. ==
Inhibition of protease expression can have a variety of industrial ~ applications. For example, inhibition of protease expression can slow or prevent spoilage. Spoilage can occur when polyp-eptides, e.g., structural polypeptides, are enzymatically degraded. This can lead to the deterioration, or rot, of fruits and vegetables. In one aspect, use of compositions of the invention that inhibit the expression and/or activity of proteases, e.g., antibodi es, antisense oligonucleotides, ribozymes and .
RNAI, are used to slow or prevent spoilage. Thus, in one aspect, the invention provides methods and compositions comprising ap plication onto a plant or plant product (e.g, a fruit, seed, root, leaf, etc.) antibodies, ant sense oligonucleotides, ribozymes and RNAi of the invention to slow or prevent spoilage. These compositions also can be expressed by the plant (e.g., a transgenic plant) or anotlaer organism (e.g., a bacterium or other microorganism transformed with a protease gene of the invention).
The compositions of the in vention for the inhibition of protease expression . (e.g., antisense, iRNA, ribozymes, antiboclies) can be used as pharmaceutical compositions, e.g., as anti-pathogen agents or in other therapies, e.g., anti-inflammatory or skin or digestive aid treatments. For example, proteases are attractive antimalarial targets because of their indispensable role s in parasite infection and development, especially in the processes of host erythro cyte rupture, invasion and hemoglobin degradation, see, e.g., Wu (2003) Genome= Res.13:601-616. Selective inhibition of the mosquito angiotensin-converting enzyme (ACE) (a dipeptidyl carboxypeptidase) ) involved in the activation/inactivation of & peptide regulating egg-laying activity can be an effective anti-mosquito method; see, e. g., Ekbote (2003) Comp. Biochem. Physiol. B.
Biochem. Mol. Biol. 134:593-598. Inhibi tion of matrix metalloproteases (e.g., metalloproteinases) and collagenases, whi_ch can degrade extracellular matrices and promote cancer cell migration and metastases, can be used to treat ot ameliorate these conditions; see e.g., Elnemr (2003) Gastriac Cancer 6:30-38.
o WO 2004/033668 ) PCT/US2003/032819
Antisense Oligonucleotides
The invention provides antisense oligonucleotides capable of binding ’ protease message which can inhibit proteolytic activity by targeting mRNA. Strategies for designing antisense oligonucleotides are well desczribed in the scientific and patent : 5 literature, and the skilled artisan can design such protease oligonucleotides using the : novel reagents of the invention. For example, gene w-alking/ RNA mapping protocols to screen for effective antisense oligonucleotides are welll known in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183, describing arm RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection. See also Smith (2000) Eur. J. Pharm. Sci. 11:191-198.
Naturally occurring nucleic acids are ussed as antisense oligonucleotides.
The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 300, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concemtration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non- naturally occurring nucleotide and nucleic acid analogies are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can_ be used. 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, methylphosphonzate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(me-thylimino), 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
Bo antisense protease sequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
Inhibitory Ribozymes
The invention provides ribozymes capable of binding protease message.
These ribozymes can inhibit protease activity by, e.g., targeting mRNA. Strategies for designing ribozymes and selecting the protease-specific antisense sequence for targeting are well described in the scient:ific 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 IRINA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the . ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA.
Cleavage of a target RNA in suach 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 tar get, it can be released from that RNA to bind and cleave new targets repeatedly. :
In some circumstances, the enzymatic nature of 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 molec ule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide.
This potential advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is abr le to cleave many molecules of target RNA. In addition, 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 cleavages 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 ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule, can be formed in a hammerhead motif, a hairpin motif, as a hepatitis delta virus motif, a group I intron motif arx d/or an RNaseP-like RNA in association with an RNA
Co guide sequence. Examples of lnammerhead motifs are described by, e.g., Rossi (1992)
Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989)
Biochemistry 28:4929, and Harmpel (1990) Nuc. Acids Res. 18:299; the hepatitis delta
; i @r WO 2004/033668 PCY/US2003/032819
Virus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif by Guerrier-Takada : (1983) Cell 35:849; and the group [intron by Cech U.S. Pat. No. 4,987,071. The recitation of these specific motifs is not intencled to be limiting. Those skilled in the art . will recognize that a ribozyme of the invention, e.g., an enzymatic RNA molecule of this invention, can have a specific substrate binding site complementary to one or more of the target gene RNA regions. A ribozyme of the invention can have a nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule. '
RNA interference (RNAi)
B In one aspect, the invention provides an RNA inhibitory molecule, a so- called “RNAI” molecule, comprising a protease sequence of the invention. The RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAI can inhibit © 15 expression of a protease gene. In one aspect, the RNAI is about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar cor identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAI). A possible basic mechanism behind RNAI is the breaking of a double-stranded
RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In one aspect, the RNAI’s of the invention are used in gene-silencing therapeutics, see, e.g.,
Shuey (2002) Drug Discov. Today 7:1040-104:6. In one aspect, 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. In one aspect, the RNAI molecules of the invention can be used to generate a loss-of-fun ction mutation in a cell, an organ or an animal. Methods for making and using RNAi molecules for selectively degrade RNA 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. . -
Modification of Nucleic Acids
The invention provides methods of generating variants of the nucleic acids of the invention, e.g., those encoding a protease. These methods can be repeated or used in various combinations to generate proteases having an altered or different activity or an : altered or different stability from that of a protease 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. In another aspect, the genetic composition: of a cell is altered by, e.g., modificatien of a homologous gene ex vivo, followed by its reinsertion into the cell.
A nucleic acid of the inwention 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. For example, 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 mutag ens 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.
Any technique in molecular biology can be used, e.g., random PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471; or, combinatorial multiple cassette mutagemesis, see, e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleic acids, e.g., genes, can be reassembled after random, or “stochastic,” fragmentation, see, e.g., (F.S. Patent Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811 ,238; 5,605,793. In alternative aspects, 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 saturated mutagenesis (GSSM), synthetic ligatiora reassembly (SLR), recombination, recursive sequence recombination, phosphothioat €-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis,
UC ) WO 2004/033668 PCT/US2003/032819 repair-deficient host strain mutagenesis, chemical xnutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid oo multimer creation, and/or a combination of these amd other methods. ’
The following publications describe a variety of recursive recombination procedures and/or methods which can be incorporated into the methods of the invention:
Stemmer (1999) "Molecular breeding of viruses for targeting and other clinicak- properties” Tumor Targeting 4:1-4; Ness (1999) N ature Biotechnology 17:893-896;
Chang (1999) "Evolution of a cytokine using DNA family shuffling” Nature
Biotechnology 17:793-797; Minshull (1999) "Prote in evolution by molecular breeding"
Current Opinion in Chemical Biology 3:284-290; Christians (1999) "Directed evolution of thymidine kinase for AZT phosphorylation usings DNA family shuffling" Nature
Biotechnology 17:259-264; Crameri (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391:288-291; Crameri (1997) "Molecular evolution of an arsenate detoxification Pathway by DNA shuffling," Nature
Biotechnology 15:436-438; Zhang (1997) "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening" Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Vaccines" Current Opinion in Biotechnology 8:724-733; Crameri et al. (1996) : "Construction and evolution of antibody-phage libra ries by DNA shuffling" Nature
Medicine 2:100-103; Gates et al. (1996) "Affinity seslective isolation of ligands from peptide libraries through display on a lac repressor ‘headpiece dimer" Journal of
Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and Assembly PCR" In:
The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp.447-457, : Crameri and Stemmer (1995) "Combinatorial multip le cassette mutagenesis creates all the permutations of mutant and wildtype cassettes" BioT echniques 18:194-195; Stemmer et al. (1995) "Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxyribonucleotides" Gene, 164:49-53; Stemmer (1995) “The Evolution of
Molecular Computation” Science 270: 151] 0; Stemmesr (1995) “Searching Sequence
Space" Bio/Technology 13:549-553; Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370:389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution.” © Proc. Natl. Acad. Sci. USA 91:10747-10751.
* 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; IDale et al. (1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method" Methods Mol. 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. 237:1-7; and Kunkel (1 987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular
Biology (Eckstein, F. and Lilley, ID. M. J. eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci. USA 82:488-492;
Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis without phenotypic ’ selection Methods in Enzymol. 1 54, 367-382; and Bass et al. (1988) "Mutant Trp .. repressors with new DNA-binding specificities" Science 242:240-245); oligonucleotide- directed mutagenesis (Methods in. Enzymol. 100: 468-500 (1983); Methods in. Enzymol. 154: 329-350 (1987); Zoller (19822) "Oligonucleotide-directed mutagenesis using M13- derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983) "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors”
Methods in Enzymol. 100:468-500; and Zoller (1987) Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded
DNA template" Methods in Enzyrmol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764; Taylor (1985) "The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified IDNA" Nucl. Acids Res. 13: 8765-8787 (1985);
Nakamaye (1986) "Inhibition of restriction endonuclease Nei I cleavage by phosphorothioate groups and its agoplication to oligonucleotide-directed mutagenesis"
Nucl. Acids Res. 14: 9679-9698; Sayers (1988) "Y-T Exonucleases in phosphorothioate- based oligonucleotide-directed muxtagenesis" Nucl. Acids Res. 16:791-802; and Sayers et al. (1988) "Strand specific cleavagge of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide" Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “The gapped duplex DNA approach to oligonucleotide-directed mutation construction" Nucl. Acids
[ WC 2004/033668 | PCT/US2003/032819
Rens. 12: 9441-9456; Kramer & Fritz (1987) Methods in Erazymol. "Oligonucleotide- directed construction of mutations via gapped duplex DNA" 154:350-367; Kramer (1988) "Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oli gonucleotide-directed construction of mutations" Nucl. Acids Res. 16; 7207; and Fritz (1%988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA preocedure without enzymatic reactions in vitro™ Nucl. Acids Res. 16: 6987-6999).
Additional protocols that can be used to practice the invention Ticlude 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 vectoxs" Nucl. Acids Res. 13: 4431- 44-43; and Carter (1987) "Improved oligonucleotide-directe«d mutagenesis using M13 vectors" Methods in Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh oo (19286) "Use of oligonucleotides to generate large deletions’™ Nucl. Acids Res. 14: 5115), res-triction-selection and restriction-selection and restriction —purification (Wells et al. (19°86) "Importance of hydrogen-bond formation in stabilizk ng the transition state of subetilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning of a gene coding for the : ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana (1988) "Total syn thesis and expression of a gene for the a-subunit of bovire rod outer segment guanine nuc-leotide-binding protein (transducin)” Nucl. Acids Res. 14: 636 1-6372; Wells et al. (19385) "Cassette mutagenesis: an efficient method for generation of multiple mutations at defi_ned sites” Gene 34:315-323; and Grundstrom et al. (198 5) "Oligonucleotide-directed mutagenesis by microscale ‘shot-gun' gene synthesis" Nucl. Acids Res. 13: 3305-33 16), dourble-strand break repair (Mandecki ( 1986); Arnold (1993) "Protein engineering for . unussual environments" Current Opinion in Biotechnology 4: 450-455. "Oligonucleotide- direscted double-strand break repair in plasmids of Escherichia coli: a method for site- specsific mutagenesis” Proc. Natl. Acad. Sci. USA, 83:7177-718 1). Additional details on man_y of the above methods can be found in Methods in Enzy/mology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis metkaods.
Protocols that can be used to practice the invemtion are described, e.g., in
U.S. Patent Nos. 5,605,793 to Stemmer (Feb. 25, 1997), "Methods for In Vitro
Recommbination;" U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 22, 1998) "Methods for
Genesrating Polynucleotides having Desired Characteristics by Iterative Selection and
Recombination;" U.S. Pat_ No. 5,830,721 to Steramer et al. (Nov. 3, 1998), "DNA_
Mutagenesis by Random Fragmentation and Reassembly;" U.S. Pat. No. 5,834,252 to
Stemmer, et al. (Nov. 10, 1998) "End-Complementary Polymerase Reaction;" U.SS. Pat.
No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), "Methods and Compositions fomr § Cellular and Metabolic Eragineering;" WO 95/22625, Stemmer and Cramer, "Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207 by Stexmmer and Lipschutz "End Complementary Polymerase Chain Reaction," WO 97/20078 by
Stemmer and Crameri "M ethods for Generating Polynucleotides having Desired
Characteristics by Iterative Selection and Recombination," WO 97/35966 by Minsshull and Stemmer, "Methods a nd Compositions for Cellular and Metabolic Engineerin_g;" WO 99/41402 by Punnonen et al. "Targeting of Genetic Vaccine Vectors;" WO 99/41 383 by
Punnonen et al. "Antigen Library Immunization;* WO 99/41369 by Punnonen et al. "Genetic Vaccine Vector Engineering;” WO 99/41368 by Punnonen et al. “Optimization of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by Stemmer &and
Crameri, "DNA Mutagenesis by Random Fragmentation and Reassembly;" EP (9»32670 by Stemmer "Evolving Cellular DNA Uptake by Recursive Sequence Recombination;"
WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and Host Range by
Viral Genome Shuffling;'® WO 99/21979 by Apt et al., "Human Papillomavirus V~ ectors;"
WO 98/31837 by del Carclayre et al. "Evolution of Whole Cells and Organisms by
Recursive Sequence Recombination;” WO 98/27230 by Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer etal _, "Methods for Optimizatiosn of Gene Therapy by Recursive Sequence Shuffling amd
Selection,” WO 00/00632, "Methods for Generating Highly Diverse Libraries," W/O 00/09679, "Methods for Obtaining in Vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832 by Arnold et al., "Recombination of
Polynucleotide Sequences Using Random or Defined Primers," WO 99/29902 by Amold et al,, "Method for Creating Polynucleotide and Polypeptide Sequences," wo 98741653 by Vind, "An in Vitro Method for Construction of a DNA Library," WO 98/4162 2 by
Borchert et al., "Method or Constructing a Library Using DNA Shuffling," and "VO 98/42727 by Pati and Zar-ling, "Sequence Alterations using Homologous Recombmination.”
Protocols that can be used to practice the invention (providing details regarding various diversi €y generating methods) are described, e.g., in U.S. Paterx.t application serial no. (US SN) 09/407,800, "SHUFFLING OF CODON ALTERE-D
GENES" by Patten et al. filed Sep. 28, 1999; "EVOLUTION OF WHOLE CELL.S AND
® WO 2004-/033668 PCT/US2003/032819
ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION" by del Cardayre et al., United States Patent No. 6,3 79,964; "OLIGONUCLEOTIDE MEDIATED NUCLEIC
ACID RECOMBINATION" by Crameri et al, United States Patent Nos. 6,319,714; 6,368,861; 6,376,246; 6,423,542; 6,426,224 and PCT/US00/012203; "USE OF CODON- § VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by
Welch eet al., United States Patent No. 6,436,675; "METHODS FOR MAKING
CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING
DESIRIED CHARACTERISTICS" by Selifonov et al., filed Jan... 18, 2000, (PCT/UI'S00/01202) and, e.g. "METHODS FOR MAKING CHARACTER STRINGS,
POLYMNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED
CHARACTERISTICS" by Selifonov et al., filed Jul. 18,2000 (XJ.S. Ser. No. 09/618, 579); "METHODS OF POPULATING DATA STRUCT URES FOR USE IN
EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer, filed Jan. 18, 2000 (PCT/Ur S00/01138); and "SINGLE-STRANDED NUCLEIC ACID TEMPLATE-
MEDIA.TED RECOMBINATION AND NUCLEIC ACID FRA_GMENT ISOLATION" by Affh olter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and United States Patent Nos. 6,177,263; 6,153,410. }
Non-stochastic, or “directed evolution,” methods include, e.g., saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a <ombination thereof are used to mrodify the nucleic acids of the invention to generate proteases with new or altered properties (e.g., activity under highly acidic or alkaline conditions, high or low 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, US.
Patent Nos. 6,361,974; 6,280,926; 5,939,250.
Saturation mutagenesis, or, GSSM
In one aspect, codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, e.g., a protease or an antibody of the in—vention, so as to generate a set of progeny polypeptides i n which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amina acid resiclue in an enzyme active site or ligand binding site targeted to be modified. .
These old gonucleotides 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,IN,G/T sequence includes codons for all 20 amino acids. In one aspect, 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. In another aspect, at least two degenerate cassettes : are used — either in thie same oligonucleotide or not, for subjecting at least twro-eriginal codons in a parental polynucleotide template to a full range of codon substitistions. For example, more than ane 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). In another aspect, oligonucleotides serviceable for introducing zadditions and deletions can be used either alone or in combination with the codons contain¥ng an :
N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.
In one aspect, simultaneous mutagenesis of two or more conti guous amino acid positions is done using an oligonucleotide that contains contiguous N,N, G/T triplets, i.e. a degenerate (N,NI,G/T)n sequence. In another aspect, degenerate cassettes having less degeneracy than the N,N,G/T sequence are used. For example, it may be desirable in some instances to use (e.g. in an oligonucleotide) a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet. Any other bases including any combinations and permutations thereof can be usec® in the remaining two positions of the triplet. Alternatively, it may be desirable in some instances to use (e.g. In an oligo) a degenerate N,N,N triplet sequence.
In one aspect, use of degenerate triplets (e.g., N,N, G/T 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 Cin alternative aspects, the methods also include generation of less than all possible substitutions per amimo acid residue, or codon, position). For example, fora 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated. Through the use of an oligonucleoticle or set of oligonucleotides containing a degenerate N,N,G/T triplet, 32 individual sequesnces can code for all 20 possible natural amino acids. Thus, in a reaction vessel in whi.ch a parental polynucleotidle sequence is subjected to saturation mutagenesis usings at least one
[| WO 2004/03366a8 PCT/ US2003/032819 such oligonucleotide, there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides. In contrast, the use of a non-degenerate oligonucleotide in site- directed mutzagenesis leads to only one progeny polypeptide product per resaction vessel.
Nondegencraate oligonucleotides can optionally be used in combination with degenerate primers discl osed; for example, nondegenerate oligonucleotides can be us ed to generate : specific poin@ mutations in a working polynucleotide. This provides one rneans to : generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codonss and the correspondin_g expression of polypeptide fragments.
In one aspect, each saturation mutagenesis reaction vessel Contains polynucleoticies encoding at least 20 progeny polypeptide (e.g., proteases) molecules such that all 20 nasural amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide (other aspects use le=ss than all 20 natural combinations). The 32-fold degenerates progeny polypeptides generated from each saturation mutagenesis reaction vessel c=an be subjected . to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression ve=ctor) and subjected to expression screening. When an indiviedual progeny polypeptide iss identified by screening to display a favorable change in progperty (when compared to the parental polypeptide, such as increased proteolytic activit—y under alkaline or acidic conditions), it can be sequenced to identify the correspondingly fZavorable amino acid substitution contained therein.
In one aspect, upon mutagenizing each and every amino acid positionin a parental polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes mmay be identified at more than one amino acid position. One= or more new progeny mole-cules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if 2 specific favorable armino acid changes are identified in each of 3 amino acid positions in a polypeptide, tEae permutations &nclude 3 possibilities at each position (no change from the omriginal amino acid, and each. of two favorable changes) and 3 positions. Thus, there are 3 x3 x 3 or 27 30 . total possibilities, including 7 that were previously examined - 6 single poimt mutations (i.e. 2 at each «of three positions) and no change at any position.
In another aspect, site-saturation mutagenesis can be used together with another stochaestic or non-stochastic means to vary sequence, e.g., synthetics ligation reassembly (sese below), shuffling, chimerization, recombination and other mutagenizing processes axad mutagenizing agents. This invention provides for the use of any mutagenizirag process(es), including saturation mutagenesis, in an iterati—~ve manner.
Synthetic Ligation Reassembly (SLR)
The invention provides a non-stochastic gene modificatio n system termed “synthetic li gation reassembly,” or simply “SLR,” a “directed evolution process,” to generate poRypeptides, e.g., proteases or antibodies of the invention, witka new or altered properties. SLR isa method of ligating oligonucleotide fragments togetkher non- stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid. building blocks are not shuffled, concatenated or chimerizecl randomly, but rather are assembled non-stochastically. See, e.g., U.S. Patent Application Serial No. (USSN) 09/332,835 entitled “Synthetic Ligation Reassembly in Directed Evolution” and filed on June 14, 1999 (“USSN 09/332,835™). In one aspect, SLR comprises the following steps: (a) providing a template polynucleotide, wherein the termplate N polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of ¥building block polynucleotides, wherein the building block peolynucleotides are designed to cross-over reassemble with the template polynucleotide eat a predetermin<ed 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 bslock polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over rezassembles with the template polynucleotide to generate pols/nucleotides comprising Fnomologous gene sequence variations. )
SLR does not depend on the presence of high levels of ho mology between polynucleoti_des to be rearranged. Thus, this method can be used to non- stochastically generate libraries (or sets) of progeny molecules comprised of over 1010 O different chimeras. SLR can be used to generate libraries comprised of over 1010400 different progeny chirmeras. Thus, aspects of the present invention include non-stochastic methods of producings a set of finalized chimeric nucleic acid molecule shaving ara overall assembly orcJer 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 | Agatable ends, and assembling these nucleic acid building blocks, such that a designed ove=rall assembly order is achieved.
EE
¢ WO 2004/033668 PCT/US2003/032819
The mutually compatible ligatable ends of the nucleic acid building belocks to be assembled are «considered to be “serviceable” for this type of ordered assembly if : they enable the buildling blocks to be coupled in predetermined orders. Thus, the ov=erall assembly order in which the nucleic acid building blocks can be coupled is specified. by the design of the ligemtable ends. If more than one assembly step is to be used, then thhe overall assembly ord er in which the nucleic acid building blocks can be coupled is a@so specified by the sequmential order of the assembly step(s). In one aspect, the amrealecd building pieces are treated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent borx ding of the building pieces.
In ones aspect, the design of the oligonucleotide building blocks is obt=ained 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 temp lates 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., chimerizead or shuffled. In one aspect of this method, the sequences of a plurality of parental nucle ic acid templates are ali gned 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 wor more nucleotides. Thaese 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 time parental polynucleoticles. The demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the fimnal chimeric progeny moRecules. A demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequeraces. Alternatively, 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 arm 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 sequeraces, 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.
In one aspect, a ligation reassembly process is performed exhaustively in order to generate an exchaustive library of progeny chimeric polynucleotides. In other
W~O 2004/033668 PCT/US2003/032819 ( LJ words, all possible ordered combinations of the nucleic acid but 1ding blocks are TT represented in the set of finalized chimeric nucleic acid molecul es. At the same time, in another aspect, the assembly order (i.e. the order of assembly of each building block in tthe 5’ to 3 sequence of each finalized chimeric nucleic acid) in each combination is by design (or non-stochastic) as described above. Because of the n-on-stochastic nature of thhis invention, the possibility of unwanted side products is great1y reduced.
In another aspect, the ligation reassembly method is performed" systematically. For example, 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. In other words this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, c-oupled 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 16 tO be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups. Because of its ability to perform chimerizations in a manner that is highly flexible yet exhaustive and systematic ass well, particularly when there is a low level of homology among the progenitor molecules, these methods provide for the generation of a library (or set) comprised of a laarge number of progeny molecules. Because of the non-stochastic nature of the instant li gation reassembly invention, 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 clirected evolution nz.ethods also can be used to generate different progeny molecular species. It is apopreciated that the invention provides freedom of choice and co ntrol regarding the se=lection of demarcation points, the size and number of the nucle=ic acid building blocks, arad the size and design of the couplings. It is appreciated, furthermore, that the re-quirement for intermolecular homology is highly relaxed for the operability of this in~vention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobbKe, i.e. the degeneracy of co-dons, nucleotide substitutions can be introduced into nucleic acid building blocks “ without altering the amino acid originally encoded in the corresponding progenitor termplate. Alternatively, a codon can be altered such that the coding for an originally arrino acid is altered. This invention provides that such substitut ions can be introduced
( ® WO 2004/033668 PCT/US2003/032819 into the nucleic acid building black 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 blaecks, which in turn allows a greater number of progeny chimeric molecules to be generated. | . [ In another aspect, 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 sequehces) 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 thes gene splicing ability of a host arganism). Itis appreciated that in many instance=s the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creatinga serviceable demarcation point.
In one aspect, a numcleic acid building block is used to introduce an intron.
Thus, functional introns are introcluced into a man-made gene manufactured according to the methods described herein. Time 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.
Optimized Directed Evoluation System
The invention prov-ides a non-stochastic gene modification system termed “optimized directed evolution system” to generate polypeptides, e.g., proteases or antibodies of the invention, with ncew 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 predeterrmined number of crossover events,
A crossover event iss 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 za predetermined number of crossover events,
In addition, this method provides a convenient means for exploring a tremendous amount of the possible protein variant sgoace in comparison to other systems.
Previously, if one generated, for example, 10" chim eric molecules during a reaction, it would be extremely difficult to test such a high num ber of chimeric variants for a particular activity. Moreover, a significant portion of the progeny population would have a very high number of crossover events which result ed in proteins that were less likely to have increased levels of a particular activity. By usi ng these methods, the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events. Thus, although one can still generate 10 chimeric molecules during a reaction, cach of the molecules chosen for further anx alysis most likely has, for example, only three crossover events. Because the resulting progeny population can be skewed to have a predetermined number of crossover events, thae 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 affectinga particular trait.
One method for creating a chimeric p rogeny polynucleotide sequence is to create oligonucleotides corresponding to fragments or portions of each parental sequence.
Each oligonucleotide preferably includes a unique resgion of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. Additional informati on can also be found, e.g., in USSN : 09/332,835; U.S. Patent No. 6,361,974.
The number of oligonucleotides genemrated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created. For example, three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to firad a chimeric variant having, for example, greater activity at high temperature. As one example, 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. If each oligo nucleotide fragment is present in the . ligation reaction in the same molar quantity it is.likel y 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
@o WO 2004/033668 PCT/US2003/032819 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 prod-uce no CrossOver,
Accordingly, a probabil ity density function (PDF) 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 eath step in the ligation reaction. The statistics and. mathematics behind determining the PDF is described below. By utilizing these methods, one can calculate such a probability density function, and thus enrich the chimeric Progeny population for a predetermined number of crossover events resulting from a partic ular ligation reaction. Moreover, 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 ©f 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.
In addition, these methods provide a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.
By using the methods described herein, the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events. Thus, although one can still generate 10" chimeric molecules during a reaction, each of the molecules chosen for further analysis mo st likely has, for example, only three crossover events. Because the resulting progeny population can be skewed to have a predetermined number of 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. oo
In one aspect, the method creates a chimeric progeny polynucleotide sequence by creating oligonucleotides corresponding to fragments or portions of each parental sequence. Each oligonucleotide preferably includes a unique region efoverlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. See also USSN 09/332,835.
Determining 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. 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 MATLAB™ (The Mathworks, Natick,
Massachusetts) a programming language and development environment for technical computing.
Iterative Processes
In practicing the invention, these processes can be iteratively repeated.
For example, a nucleic acid (or, the nucleic acid) responsible for an altered or new protease 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, e.g., epoxide hydrolysis activity.
Similarly, if it is determined that a particular oligonucleotide has no affect at all on the desired trait (e.g., a new protease phenotype), it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed.
Since incorporating the sequence within a larger sequence prevents any crossover events, there will no longer be any variation of this sequence in the progeny polynucleotides.
This iterative practice of determining which oligonucleotides are most related to the 10a
‘ desired trait, and which are unrelated, allows snore efficient exploration all of the possible protein variants that might be provide a particular trait or activity.
In vivo shuffling : In vivo shuffling of molecules iis use in methods of the invention that provide variants of polypeptides of the invent& on, e.g., antibodies, proteases, and the like.
In vivo shuffling can be performed utilizing th e natural property of cells to recombine multimers. While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination rermains 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 recombinecl molecules, The formation of the chiasma requires the recognition of homologous sequences.
In one aspect, the invention prosvides a method for producing a hybrid polynucleotide from at least a first polynucleotide (e.g, a protease of the invention) and a second polynucleotide (e.g., an enzyme, such as a protease of the invention or any other pratease, or, a tag or an epitope). 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. The term “hybrid polynucleotide”, as used herein, is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences.
Such hybrid polynucleotides can result from imtermolecular recombination events which promote sequence integration between DNA molecules. In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.
Producing sequence variants
The invention also provides add itional methods for making sequence variants of the nucleic acid (e.g., protease) sequiences of the invention. The invention also provides additional methods for isolating proteases using the nucleic acids and polypeptides of the invention. In one aspect, thae invention provides for variants of a protease coding sequence (e.g., a gene, cDNA or message) 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 chemnical mutagenesis, Exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives may be created wising 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 havimg 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 amimo acid changes with respect to the polypeptides encoded by the nucleic acids from the matural isolates.
For example, variants may be created using error prone PCR. In error prone PCR, PCR is performed under co nditions 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. Briefly, in such procedigres, nucleic acids to be mutagenized are mixed with PCR primers, reaction buffer, MgCl, MnCl,, Taq polymerase and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction may be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmo Xe of each PCR primer, a reaction buffer comprising 50mM KCl, 10mM Tris HC 1 (pH 8.3) and 0.01% gelatin, 7mM MgCI2, : 0.5mM MnCl, 5 units of Taq polymerase, 0.2mM dGTP, 0.2mM dATP, ImM dCTP, and 1mM dTTP. PCR may be performed fox 30 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min. However, it will bes 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 are 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-QOlson (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.
Another method for generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixtures of small DNA fragments. A large ‘ . number of different PCR reactions occur in parallel in the same vial, with the ptoducts of one reaction priming the products of another reaction. Assembly PCR is described in, e.g., U.S. Patent No. 5,965,408. 108 Still another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different but highly related DNA sequences 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 P<CR mixture. PCR is conducted under conditions which facilitate recombination between the nucleic acid fragments. For example, PCR may be performed by resuspending the paurified fragments at a concentration of 10-30ng/pl in a solution of 0.2mM of each dNTP, 2.2mM MgCl, 50mM
KCL, 10mM Tris HCI, pH 9.0, and 0.1% Triton X-100. 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°C for 30 seconds, 72°C for 30 seconds (30-45 times) and 72°C for 5 minutes. However, it will be appreciated that these parameters may be varied as appropriate. In some aspects, oligonucleotides may be ’ included in the PCR reactions. In other aspects, the Klerow 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 sequaences are isolated and the activities of the polypeptides they encode are assessed.
Variants may also be created by in vivo nmautagenesis. In some aspects, 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, whi ch carries mutations in one or more of the DNA repair pathways. Such “mutator” straims have a higher random mutation rate than that of a wiEd-type parent. Propagating the DNA in one of these strainss ) will eventually generate randosm 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 al so be generated using cassette mutagenesis. In 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 ensernble 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 amin« 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.
In some aspects, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating ) combinatorial libraries with a nigh percentage of unique and functional mutants, wherein small groups of residues are ramdomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described, e.g., in Delegrave (1.993) Biotechnology Res. 11:1548-1552. Random and site-directed mutagenesis are described, e.g., in Amold (1993) Current Opinion in
Biotechnology 4:450-455. E
In some aspects , 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, ¢ .g., U.S. Patent Nos. 5,965,408; 5,939,250 (see also discussion, above).
The invention also provides variants of polypeptides of the invention (e.g., proteases) comprising sequences in which one or more of the amino acid residues (e.g., off an exemplary polypeptide of thae invention) are substituted with a conserved or non- conserved amino acid residue (Ce.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. Thus, polypeptides of the invention include those with conservative substitutions of seequences of the invention, e.g., the exemplary polypeptides
: CJ WO 2004/033668 PCT/US2003/032819 of the invention, including but not limited to the following replacements: replacements of an aliphatic amino acid such as Alanine, Valine, Leucine and Issoleucine 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 bassic residue such as Lysine &|nd Arginine with another basic residue; and replacement of an aromatic residue such as
FPhenylalanine, Tyrosine with another aromatic residue. Other v=ariants are those in which ©ne or more of the amino acid residues of the polypeptides of th e invention includes a substituent group.
Other variants within the scope of the invention are those in which the
Polypeptide is associated with another compound, such as a corrapound to increase the half-life of the polypeptide, for example, polyethylene glycol.
Additional variants within the scope of the invenftion are those in which 16 additional amino acids are fused to the polypeptide, such as a lesader sequence, a secretory sequence, a proprotein sequence or a sequence which facilitates —purification, enrichment, oor stabilization of the polypeptide.
In some aspects, the variants, fragments, derivatiwes and analogs of the peolypeptides of the invention retain the same biological function. or activity as the exemplary polypeptides, e.g., protease activity, as described herein. In other aspects, the v-ariant, 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.
Optimizing codons to achieve high levels of protein expre=ssion in host cells
The invention provides methods for modifying preotease-encoding nucleic acids to modify codon usage. In one aspect, the invention provides methods for mmodifying codons in a nucleic acid encoding a protease to increase or decrease its expression in a host cell. The invention also provides nucleic acids encoding a protease modified to increase its expression in a host cell, protease so modified, and methods of making the modified proteases. The method comprises identifyirg a “non-preferred” or a . “less preferred” codon in protease-encoding nucleic acid and repl_acing one or more of "theese non-preferred or less preferred codons with a “preferred cocdon” encoding the same amnino acid as the replaced codon and at least one non-preferred owr less preferred codon in the nucleic acid has been replaced by a preferred codon encoding the same amino acid. A : preferred codon is a c-odon over-represented in coding sequences in genes in the host: cell ‘ and a non-preferred o r less preferred codon is a codon under-represented in coding sequences in genes in_ the host cell. :
Host ceells for expressing the nucleic acids, expression cassettes and vectors of the invention include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Th us, the invention provides methods for optimizing codonusage in all of these cells, codon-altered nucleic acids and polypeptides made by the codon-al tered nucleic acids. Exemplary host cells include gram negative bacteria, such as Escheric=hia coli and Pseudomona.s fluorescens; gram positive bacteria, such as Streptomyces divezrsa,
Lactobacillus gasseri ., Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis. } ,
Exemplary host cells also include eukaryotic organisms, e.g., various yeast, such as
Saccharomyces sp., itacluding Saccharomyces cerevisiae, Schizosaccharomyces pom be,
Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines. Thus, the invention also includes nucleic acids and polypeptides optimized for expression in these organisms and species. ;
For example, the codons of a nucleic acid encoding a protease isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell differen® from the bacteria from which the protease was derived, ayeast, a fungi, a plant cell, an insect cell or a mammalian cell. Methods for optimizing codoras 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 (19 98) Protein Expr. Purif. 12:185-188; Narum (2001) Infect. Immun. 69:7250-7253. See aliso Narum (2001) Infect. Immun. 69:7250-7253, describing optimizing codons in mouse systems; Outchkourov (2002) Protein Expr. Purif. 24:188-24, describing optimizings codons in yeast; Feng (2000) Biochemistry 39:15399-15409, describing optimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif, 20:2. 52- 264, describing optimizing codon usage that affects secretion in E. coli.
Transgenic non-human animals
The in~vention provides transgenic non-human animals comprising a nucleic acid, a polype=ptide (e.g., a protease), an expression cassette or vector or a transfected or transformed cell of the invention. The invention also provides methods of making and using thesse transgenic non-human animals.
The transgenic non-human animals can be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice, comprising the nucleic acids of the invention. Thesse animals can be used, e.g, as in vivo models to study protease activity, or, as models to sscreen for agents - that chamge the protease activity in vivo. The coding sequences for the Polypeptides to be expressexd in the transgenic non-human animals can be designed to be camnstitutive, or, under thie 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. 6,211,428; 6,187,992; 6,156,952; oo 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,8880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and usimmg transformed cells andl eggs and transgenic mice, rats, rabbits, sheep, pigs and cows. See also, eg,
Pollock (1999) J. Immunol. Methods 231 1147-157, describing the production of recombinant proteins in the milk of transgenic dairy animals; Baguisi (1599) Nat. } Biotechmaol. 17:456-461, demonstrating the production of transgenic goat=s. U.S. Patent
No. 6,21 1,428, describes making and using transgenic non-human mamn—als which express in their brains a nucleic acid construct comprising a DNA sequermce. 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 fe=males, and growing ‘to term transgenic mice whose cells express proteins related to tkhe pathology of
Alzheimers disease. U.S. Patent No. 6,187,992, describes making and ussing a transgenic mouse whose genome comprises a disruption of the gene encoding amyloid precursor protein (APP). “Knockout animals” can also be used to practice the methods of the invention. For example, in one aspect, the transgenic or modified animals of the invention comprise a “knockout animal,” e.g., a “knockout mouse,” engimeered not to express an endogenous gene, which is replaced with a gene expressing a porotease of the invention, or, a fusion protein comprising a protease of the invention.
Transgeni c Plants and Seeds
The invention provides transgenic plants and seeds comprissing a nucleic acid, a polypeptide (e.g., a protease), an expression cassette or vector ora transfected or transformed cell of the invention. The invention also provides plant prodiacts, e.g., oils, seeds, leawes, extracts and the like, comprising a nucleic acid and/or a polypeptide (e.g., a protease) of the invention. The transgenic plant can be dicotyledonous (a edicot) or monocotyledonous (a monocot). The invention also provides methods of making and using these transgenic plants and seeds. The transgenic plant or plant cell excpressing a polypeptide Of the present invention may be constructed in accordance with any method known in thes 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. For example, nucleic acids or expression corastructs can be introduce into the genome of a desired plant host, or, the nucleic acids ox expression : constructs can be episomes. Introduction into the genome of a desired plant can be such that the host® s protease production is regulated by endogenous transcriptional or translational control elements. The invention also provides “knockout plants where insertion of g=ene sequence by, e.g., homologous recombination, has disrupted the expression off the endogenous gene. Means to generate “knockout” plants are well-known oo 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 nucleic acids of the invention can be used to confer desired traits on essentially ary plant, e.g., on starch-producing plants, such as potato, wheat, rice, barley, and the like. Nucleic acids of the invention can be used to manipulate metabolic pathways of =a plant in order to optimize or alter host’s expression of protease. The can change protease activity in a plant. Alternatively, a protease of the invention: can be used in productiorm of a transgenic plant to produce a compound not naturally proctuced by that plant. This c an lower production costs or create a novel product.
In one aspect, the first step in production of a transgenic plant involves making an exxpression 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 facilitatin_g efficient binding of ribosomes to mRNA and selecting the appropriate gene terminator sequences. One exemplary constitutive promoter is CaMV3 5S, from the : cauliflower mosaic virus, which generally results in a high degree of expression in plants.
Other promo ®ers 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.
In one aspect, the nucleic acid is modified to achieve greater expression in a plant cell. ¥For example, a sequence of the invention is likely to have a higher percentage off A-T nucleotide pairs compared to that seen in a plant, some of which prefer .
G-C nucleotide pairs. Therefore, 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 plaant cells or tissues that have successfully integrated the transgene. This may be necessary bmecause achieving incorporation and expression of genes in plant c-ells is a rare event, occu tring in just a few percent of the targeted tissues or cells. Selectalole marker genes encocle proteins that provide resistance to agents that are normally toxic To plants, such as ant biotics or herbicides. Only plant cells that have integrated the sele=ctable marker genes will survive when grown on a medium containing the appropriate antibiotic or herbicides. As for other inserted genes, marker genes also require promoter and termination sequences for proper function.
In one aspect, making transgenic plants or seeds comprises inc=orporating sequences omf the invention and, optionally, marker genes into a target express _ion ‘ ’ construct (e .g., a plasmid), along with positioning of the promoter and the terrminator sequences. This can involve transferring the modified gene into the plant through a suitable me€hod. For example, a construct may be introduced directly into thes genomic
DNA of the plant cell using techniques such as electroporation and microinjection of plant cell pr-otoplasts, or the constructs can be introduced directly to plant tisstae using ballistic me€hods, such as DNA particle bombardment. For example, see, e.g. , Christou (1997) Plant Mol. Biol. 35:197-203; Pawlowski ( 1996) Mol. Biotechnol. 6:17 -30; Klein (1987) Natuare 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discu=ssing use of particle borrmbardment to introduce transgenes into wheat; and Adam (1997) supra, for use of particle beombardment to introduce YACs into plant cells. For example, Rimehart (1997) supram, used particle bombardment to generate transgenic cotton plants. Apparatus for accelerat_ ing particles is described U.S. Pat. No. 5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000 particle acceleration instrument; see also, John, : U.S. Patent Io. 5,608,148; and Ellis, U.S. Patent No. 5, 681,730, describing paarticle- mediated tramsformation of gymnosperms.
In one aspect, protoplasts can be immobilized and injected witha 2 nucleic acids, e.g., ara expression construct. Although plant regeneration from protoplasts is not easy with cer-eals, plant regeneration is possible in legumes using somatic emb xyogenesis from protoplast derived callus. Organized tissues can be transformed with nak-ed DNA . using gene gen technique, where DNA is coated on tungsten microprojectiles, shot 1/100th the s3ze of cells, which carry the DNA deep into cells and organelles.
Transfommed tissue is then induced to regenerate, usually by somatic embryogenesis. This techniquae has been successful in several cereal species including maize axnd rice.
Nucleic acids, e.g., expression constructs, can also be intrapduced in to + plant cells using recombinant viruses. Plant cells can be transformed usitag viral vectors, suchas, e.g. tobacco mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use of viral replicons for the expression of genes in plants,” Mol. Biotechnol. 5:209-221. —-
Alternatively, nucleic acids, e.g., an expression construct, «can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefac&ens host vector. The virulence functions of the Agrobacterium famefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when thes cell is infected by the bacteria. Agrobacterium tumefaciens-mecliated transformation techniques, including disarming and use of binary vectors, are well describe d in the scientific literature. See, e.g., Horsch (1984) Science 233 1496-498;
Fraley (983) Proc. Natl. Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants,
Potrykuss, ed. (Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell is contained in the bacterial chromosome as well as in another structure kno~wn as a Ti (tumor-imducing) 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 sseries of vir (virulence) genes that direct the infection process. A. tumefaciens can onlsy infect a plant through wounds: when a plant root or stem is wounded it gives off certaire. chemical signals, in response to which, the vir genes of 4. tumefaciens become activated and direct a series Of events necessary for the transfer of the T-DNA from the Ti plassmid to the plant's ciromosome. 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 replicatesd or transcrib ed, then inserts itself into the exposed plant DNA. In order to uses A. tumefaciens as a transgene vector, the tumor-inducing section of T-DNA have to be reamoved, 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 ceE1 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 Fiei (1997) Plant :
Mol. Bio 1. 35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley (1983)
Proc. Na#tl. Acad. Sci USA 80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.
C WO 2004/033668 PCT/US2003/03281=3 32:1135-1148, discussing T-DNA integration into genomic DNA. See also D'Halluin,
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. Co
Ix one aspect, 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 iii'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 fronm cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture,
Handbook of Plarit 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 thrercof. 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 im tmature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. On.ce whole plants are generated and produce seed, evaluation of the progeny begins. :
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. Thus, 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 (e.g., expression of the polypeptides of the invention to produce a plant in which flowering behavior is altered) can be enhanced when both parental plants express the polypeptides (e.g., a protease) of . theinvention. The desired effects can be passed to future plant generations by standard propagation means. oo The nucleic acids and polypeptides of the invention are expressed in or inserted in any plant or seed. Transgenic plants of the invention can be dicotyledonous or monocotyledonous. Examples of 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 (com). Examples of 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. Thus, the transgenic plants and seeds of the imvention 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, OZ ea, Oryza, Panieum,
Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, R aphanus, Ricinus,
Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, TrigoneZ la, Triticum, Vicia,
Vitis, Vigna, and Zea.
In alternative embodiments, the nucleic acids of the i nvention 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. In alternative embodiments, 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., a protease or antibody) of the invention. For example, see 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 (mas1',2") promoter with
Agrobacterium tumefaciens-mediated leaf disc transformation methods).
Using known procedures, one of skill 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 wekl known in the art.
Polypeptides and peptides
In one aspect, the invention provides isolated or recombinant polypeptides having a sequence identity (e.g., at least about 50%, 51%, 52%, 53%4%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 13%, 74%, 15%, 7 6%, 11%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, : 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity) to an exemplary polypeptide (amino acid) sequence ofthe invention, e.g., proteins having a sequence as set forth in 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 N24; SEQ
ID NO:26; SEQ ID NO:228; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID
NO:36; SEQ ID NO:38; SEQID 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 ID NO:72; SEQ ID NO:74; SEQ ID
NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID
NO:86; SEQ ID NO:88; SEQ ID N0:90; SEQ ID NO:92; SEQ ID NO:94; SEQ ID
NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID
NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:1 12; SEQ ID NO:114; SEQ ID
NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ ID
NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID
NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144; SEQ ID
NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID
NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ ID
NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID
NO:242; SEQ ID NO:249 or SEQ ID NO:255, or the polypeptide encoded by SEQ ID
NO:145. In one aspect, the polypeptide has a protease activity, including proteinase and/or peptidase activity, e.g., the ability to hydrolyze a peptide bond. The protease activity can comprise a peptidase activity, e.g., a carboxypeptidase activity, a dipeptidylpeptidase or an oligopeptidase activity, or an aminopeptidase activity. The protease activity can comprise a serine proteinase activity, a metalloproteinase activity, a cysteine protease activity and/or an aspartic protease activity, or, the same or similar ) activity to a chymotrypsin, a trypsin, an elastase, a kallikrein and/or a subtilisin.
Exemplary protease activities are set forth in Table 1, Table 2 and Table 3.
Assays are described in destail in Examples, below. Assays were developed to determine ’ pratease activity on a variety of pNA (para-nitroanalide) linked small peptide substrates “as well as protein substrates, such as casein, gelatin, corn zein, soybean trypsin inhibitor,
soybean lectin, and wheat germ lectin. For the small peptide substrate assays, hydrolysis of the terminal peptide bond liberates the pNA group and causes an increase in absorbance at 410nm. To monitor activity on the protein substrates, incubation of the : + protease and substrate at 370C was followed by monitoring the increase in fluorescence from an intramolecularly quenched substrate, by O-pthaldialdehyde (OPA) analysis, where in the presence of BME, OPA reacts with free amino ends to produce a fluorescent imidazole that can be detected using a standard fluorescence plate reader, or bySDS- )
PAGE analysis, where protease activity is indicated by the reduction or disappearance of substrate band(s).
Proteinase activity on casein, gelatin, or corn zein was also determined . using zymograms: zymogram gels contain the enzyme substrate (e.g., alpha-zein) embedded within the gel matrix. Ifa protease has activity om the zein in the gel, a clearing zone will be produced within an otherwise blue background following electrophoresis, renaturation, development, and staining steps. The clearing zone corresponds to the position of the protease in the gel.
Table 1, below, describes exemplary polypeptides having proteinase activity.
@® WO 2004/033668 PCT/US2003/032819
SEQIDNOS: Casein Gelatin AAA AAPF BAPNA GGF IEGR PFR
EH TER I EE I SE
Il XS RT A
IH WR I A A EE I A EN
IRN I I A A
TXT IE ET Er A NE
ICE I I I I I a
EA YER NE I I I EE I
IHN I I A I I
7 7A I RE I I
Ores [+ | +
ELE I I
EXT MN EE I A I
3 SC I IE I I EE I a rE
Mhasaas | |e Ie
EZR TI EE EE EE EN EE
CETTE MN NS I A A I I I
EA: SVYRN I I I A I
ICL TI NN I A I I I
Br EE EAE EE TA
EYE EE EE I I
+ Indicates activity was de tected on this substrate, - indicates that activity was not detected on this substrate using the conditions tested, and a blank box indicates that activity on the corresponding subs trate has yet to be determined. (AAPF = N-Suc-
Alanine-Proline-Phenylalanine- pNA, AAA = N-Suc-Alanine-Alanine-Alanine-pNA,
BAPNA =N-BZ=D,L-Arginine-pINA, GGF = N-Suc-Glycine-Glycine-Phenylalanine- pNA, IEGR = N-Suc-Isoleucine-G-lutamate-Glycine-Arginine-pNA, PFR = N-Suc-
Proline-Phenylalanine-Arginine-pINA).
Tables 2 and 3, below, describes exemplary polypeptides having peptidase activity, and summarizes their protease activities. a 10 Table 2:
Table 3: Activity summary
SEQID | Zein |SBTI | SB WG | Ze SBTI | SB WG AquaZe
NOS: Lectin | Lectin Lectin | Lectin | in
Zymo- gram (7,8 [138 112 [198 [116 [Ves | | | [ve 69,70 [191 108 061 [079 |Yes | M _ |[M |Ves [ND 65, 66 3.59 057% { 0.48 0.88 Yes ND ND ND ND
SEQIDNOS: Casein Gelatin AAA AAPF BAPNA GGF IEGR PFR (SEH I NC I IR I EE 0 I
RCH INE AR EN NE IE IE EN
ALE IE BE EN NE EN ER
[iS I IE I I Ea I ER
XSI IC I I NE IE a I
EC CO CC a 73, 74 1.73 0.5 0.23 Yes and 87, 88 29,30 1203 [119 J023 ]052 |¥es | M [ND |Yes [Yes (23,24 [161 [139 [037 093 [Ves | M [ND |M _ [Yes 49,50 1138 1088 [045 1092 [Ves [| | | [Ves 93,94 1.49 0.24 0.95 Yes M and 101, 102 103,104 3.05 1098 [264 1086 {Yes | | | ves [41,42 [164 1064 1067 081 Yes | | | Ives | - [19.20 [234 [071 [076 [08 |M | | | [ves 77,78 [158 Jo6 [109 Joo [mM | | 1 Tm 31,32 [215 1068 [058 081 [M | ND [ND [ND [Yes 67,68 1165 146 {077 1099 |M | ND |ND |ND [Yes [61,62 1171 [08 016 [077 [Yes | ND |ND [ND [ND : 121,22 144 [09 [047 |093 |™M | [| IwND 41,142 1178 [104 [071 [107 ND | |" 1ves
Lz 1 1 TT Nes |] (43,44 [159* [086* [043* [08 [ND | ND [ND [ND [Yes
ND = no detectable activity under the conditions tested, M = Maybe (slight activity under the conditions tested) * - Data from 48 hr time point
Corresponding negative controls were analy zed and shown to have no detectable activity
® WO 2004/033668 PCT/US2003/032819
OPA data is expressed as the ratio off the fluorescence (FL) of the enzyme and substrate reaction divided by the sum of the corresponding enzyme only and substrate - only controls,
Activity Ratio = © _FL substrate and enzyme preparation reaction (FL substrate alone) + (FL enzyme preparation alone)
A fluorescence ratio of 1 indicates no activity above background. A fluorescence ratio above 1 indicates the presence of free amino ends created by proteolysis of the substrate by the protease. An FL ratio below | may indicate that the protease is inhibited by the substrate such that the hydrolysis of background proteins in the enzyme preparation occurs to a greater extent in the absence of the substrate than it does in the presence of substrate. In this case, the FL background fluorescence in the enzyme only control would be inflated relative to the back ground component of the enzyme and substrate sample. -
The polypeptides of the invention include proteases in an active or inactive form. For example, the polypeptides of the irvention include proproteins before “maturation” or processing of prepro sequences, €.g., by a proprotein-processing enzyme, such as a proprotein convertase to generate ar “active” mature protein. The polypeptides of the invention include proteases inactive for other reasons, e.g., before “activation” by a post-translational processing event, e.g., an erado- or exo-peptidase or proteinase action, a : 20 phosphorylation event, an amidation, a glycosylation or a sulfation, a dimerization event, and the like.
The polypeptides of the invent&on include all active forms, including active subsequences, e.g., catalytic domains or actives sites, of the protease. In one aspect, the invention provides catalytic domains or active= sites as set forth below. In one aspect, the invention provides a peptide or polypeptide comprising or consisting of an active site domain as set forth below (the domains were predicted through use of the database, Pfam, which is a large collection of multiple sequenc=e 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,
KL. Howe, M. Marshall, and E.L.L. Sonnharme: mer, Nucleic Acids Research, 30(1):276- 280, 2002):
SEQ ID NO: Domains (AA = Amino Acid) : 248, 249 AA(104)...(500) :
Eukaryotic aspartyl protease
AA(112)..(317) 218, 219 Zinc carboxypeptidase
AA(116)...(325) : 178, 180 Zinc carboxypeptidase
AA(117)..(321) 241, 242 Zinc carboxypeptidase N
AA(121)..(228) ” . PA (protease associated) d «omain;
AA(234)...(468) 193, 194 Peptidase family M28
AA(124)...(340) : 204, 205 Zinc carboxypeptidase
AA(124)...(344) 199, 200 Zinc carboxypeptidase ’
AA(128)...(378) 164, 165 Peptidase family M28
AA(156)...(426)
Subtilase family; :
AA(74)...(142) 187, 188 Subtilisin N-terminal Regior
AA(234)...(471)
Peptidase family M28;
AA(115)...(224) 222, 223 PA (protease associated) d omain
AA(239)...(439) ’ ' 171, 172 Peptidase family M48
AA(35)...(120)
Subtilisin N-terminal Reglor; AA(134)...(397) 229, 230 Subtilase family
AA(5)...(389) 150, 151 Eukaryotic aspartyl protease
AA(52)...(494) 210, 211 Serine carboxypeptidase
AA(74)...(522) 254, 255 Serine carboxypeptidase
Co AA(96)...(532) 158, 159 Serine carboxypeptidase
(J WO 2004/033668 PCT/US2003/032819
For example, the invention provides a peeptide or polypeptide comprising or consisting of an active site domain as set forth in residues 104 to 500 of SEQ ID
NO:249 (as encoded by SEQ ID NO:248), wherein the= active site has an aspartyl protease activity. In another aspect, the invention provides a pe=ptide or polypeptide comprising or 6 consisting of an active site domain as set forth in residmues 112 to 317 of SEQ ID NO:219 ) (as encoded by SEQ ID NO:218), wherein the active sEte has a zinc carboxypeptidase activity, etc. =
Methods for identifying "prepro™ domai n sequences and signal sequences : are well known in the art, see, e.g., Van de Ven (1993) Crit. Rev. Oncog. 4(2):115-136.
For example, to identify a prepro sequence, the protein is purified from the extracellular space and the N-terminal protein sequence is determined and compared to the unprocessed form.
The invention includes polypeptides with or without a signal sequence and/or a prepro sequence. The invention includes poly peptides with heterologous signal sequences and/or prepro sequences. The prepro sequence (including a sequence of the invention used as a heterologous prepro domain) can bes located on the amino terminal or the carboxy terminal end of the protein. The invention also includes isolated or recombinant signal sequences (e.g., see Table 4), prepreo sequences and catalytic domains (e.g., “active sites™) comprising sequences of the invention. ee So
The percent sequence identity can be oveer the full length of the polypeptide, or, the identity can be over a region of at leeast 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. In alternative aspects, the invention provides polypeptides (peptides, fragments) ranging in size between about 5 and the full length of a polypeptide, e.g., an } enzyme, such as a protease; exemplary sizes being of about 5, 10, 185, 20, 25, 30, 35, 40, 4s, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 1755, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of an exemplary : protease of the invention.
Peptides of the invention (e.g., a subsequence of an exemplary polypeptide of the invention) can be useful as, e.g., labeling probes, antigens, toleragens, motifs, protease active sites (e.g., “catalytic domains”), signal sequences and/or prepro domains.
Polypeptides and peptides of the inventicon 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 a3so be synthesized, whole or in part, using chemical :
Co 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. 225-232; Banga, AK., Therapeutic
Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic
Publishing Co., Lancaster, PA. For example, peptide synthesis can be perforriied using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289 :3-13) and automated synthesis may be achieved, e.g., using the ABI 431A Peptide Ss/mthesizer (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, whemein 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, as defined above, include all “mimetic” and “peptidomimnetic” forms. The terms “mimetic” and “peptidomimetic™ refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of thee 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 imcorporate any amount of natural amino acid conservative substitutions as long as such siabstitutions also do not substantially alter the mimetic’s structure and/or activity. As with polypeptides of the invention which are conservative variants, routine experimentatieon will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, in one aspect, a mimetic composition is within the scope of the invention if it has a protease activity.
Polypeptide mirmetic compositions of the invention can contain any combination of non-natural streactural components. In alternative aspect, mimetic ' compositions of the invention # nclude 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 off naturally occurring amino acid residues; or ¢) residues
@® WO 2004/033668 PCT/US2003/032819 which induce secondary structural mimicry, i.e., to induce or- stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha heli~ conformation, and the like.
For example, a polypeptide of the invention can be character#ized as a mimetic when all or : some of its residues are joined by chemical means other than natural peptide bonds. oo § Individual peptidomimetic residues can be joined by peptide Bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxys-uccinimide esters, bifunctional maleimides, N,N’-dicyclohexylcarbodiimide (DCC) or NN*- diisopropylcarbodiimide (DIC). Linking groups that can be zn alternative to the traditional amide bond (“peptide bond”) linkages include, e. g£-, ketomethylene (e.g. - 80 C(=0)-CH,- for -C(=0)-NH-), aminomethylene (CH,-NH), exthylene, olefin (CH=CH), ether (CH;-O), thioether (CHz-S), tetrazole (CN), thiazole, etroamide, thioamide, or ester (see, €.g., Spatola (1983) in Chemistry and Biochemistry= of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker,
NY).
A polypeptide of the invention can also be characterized as a mimetic by containing all or some non-natural residues in place of natural dy 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 arormatic 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-1, -2, 3, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- omr L-(2-pyrazinyl)-alanine;
D- or L-(4-isopropyl)-phenylglycine; D~(trifluoromethyl)-phemmylglycine; D- (trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p- biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalarine; D- or L-2- indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl caan be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of a no n-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, maphthy), 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 chargge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or gluta myl) can also be selectively modified by reaction with carbodiimides (R’-N-C-N—R’) suchas, e.g, 1-
cyclohexyl-3(2-morpholinyl<(4-ethyl) carbodiimide or 1-ethyl-3(4-azonia- 4,4- dimetholpentyl) carbodi®mide.
Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues "by reaction with ammonium jons, 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 aboove.
Nitrile derivative (e.g. containing the CN-moiety in place . of COOH) can be substituted for asparagine or glutamine.
Asparaginyl and glittaminyl residues can be deaminatted to the corresponding aspartyl or glutamyl residues.
Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, iracluding, 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 ane 3-nitro derivatives, respectively.
Cysteine residue mimetics can be generated by reacting cysteinyl residues with, eg. alpha-haloacetates such as 2- chloroacetic acid or chlo xoacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
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-pyrid yl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7—nitrobenzo-oxa-1,3-diazole.
Lysine mimetics can be generated (and amino terminal resiclues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid ankaydrides.
Lysine and other alpha-amino-containing residue mimetics can also be gerx erated by reaction with imidoesters, such as methyl picolinimidate, pyridoxaE phosphate, pyridoxal, chloroborohydride, trinitro- benzenesulfonic acid, O-amethylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylatez.
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- knydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,- dimethylproline.
Histidire residue mimetics can be generated by reacting histidy! with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
Other mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyy/1 residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain
{ @® ‘WO 2004/033668 P«CT/US2003/032819 amide residues or substitution with N-methyl amino acids; or amidatiosn of C-terminal carboxyl groups. :
A residue, e.g., an amino acid, of a polypeptide of the iravention can also be replaced by an amino acid (or peptidomimetic residue) of the oppos ite chirality. Thus, § any amino acid naturally occurring in the L-configuration (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 peptidomimet&c, but of the opposite chirality, referred to as the D- amino acid, but also can be refe=rred to as the R- or
S- form. :
The invention also provides methods for modifying the gpolypeptides of the invention by either natural processes, such as post-translational process ing (e.g., phosphorylation, acylation, etc), or by chemical modification technique=s, 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 bose present in the : same or varying degrees at several sites in a given polypeptide. Also a ggiven polypeptide may have many types of modifications. Modifications include acetylati .on, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent atetachment of a
Theme moiety, covalent attachment of a nucleotide or nucleotide derivati—ve, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosplnatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formmation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylat=ion, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodimation, methylation, myristolyation, oxidation, pegylation, proteolytic processin_g, . 26 phosphorylation, prenylation, racemization, selenoylation, sulfation, and_ transfer-RNA mediated addition of amino acids to protein such as arginylation. See, e_g., Creighton,
T .E., Proteins — Structure and Molecular Properties 2nd Ed., W.H. Freeman and
Company, New York (1993); Postiranslational Covalent Modification of Proteins, B.C.
Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).
Solid-phase chemical peptide synthesis methods can also the used to synthesize the polypeptide or fragments of the invention. Such method mave been known im 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. D., Solid Phase Peptide Symthesis, 2nd Ed.,
Pierce Chemical Co., Rockford, Ill. pp. 11-12)) and have recently been e_mployed in commercially available laboratory peptide design and synthesis kits (Cambridge Research
Biochemicals». Such commercially available laboratory kits have generally utilized the teachings of HL. M.. Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for synthesizirag peptides upon the tips of a multitude of “rods” or “pins” all of which. are connected to am single plate. When such a system is utilized, a plate of rods or pins is . inverted and ixaserted 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 xod's tips. By repeating such a process step, i.e., inverting and inserting the rod's and pin's tips into appropriase solutions, amino acids are built into desired peptides. In addition, a number of available FMOC peptide synthesis systems are available. For example, assembly ofa polypeptide or fragment can be carried out on a solid support using an
Applied Biosy~stems, Inc. Model 431A™ automated peptide synthesizer. Such equipment provides ready~ access to the peptides of the invention, either by direct synthesis or by synthesis of a sseries of fragments that can be coupled using other known techniques.
The invention includes proteases of the invention with and without sigmal.
The polypepticle comprising a signal sequence of the invention (e.g., see Table 4) can bea protease of thes invention or another protease or another enzyme or other polypeptide.
The invention includes immobilized proteases, anti-protease antibodies and fragments thereof, The invention provides methods for inhibiting protease activity, e.g, using dorxainant negative mutants or anti-protease antibodies of the invention. The invention inclLades heterocomplexes, e.g., fusion proteins, heterodimers, etc., comprising the proteases c»f the invention.
Polypeptides of the invention can have a protease activity under various conditions, e.gz., extremes in pH and/or temperature, oxidizing agents, and the like. The invention prov-ides methods leading to alternative protease preparations with different catalytic efficiencies and stabilities, e.g., towards temperature, oxidizing agents and changing washa conditions. In one aspect, protease variants can be produced using techniques of site-directed mutagenesis and/or random mutagenesis. In one aspect, directed evolution can be used to produce a great variety of protease variants with alternative specificities and stability.
The proteins of the invention are also useful as research reagents to oo identify proteasse modulators, e.g., activators or inhibitors of protease activity. Briefly, test samples (compounds, broths, extracts, and the like) are added to protease assays to determine their= ability to inhibit substrate cleavage. Inhibitors identified in this way can
@® WO 2004/033668 PCT/US2 003/032819 be used fn industry and research to reduce or prevent undesired proteolysis. AAs with proteases, inhibitors can be combined to increase the spectrum of activity.
The enzymes of the invention are also useful as research reage-nts to digest proteins «or in protein sequencing. For example, the proteases may be used to break polypeptides into smaller fragments for sequencing using, e.g. an automated Ssequencer.
The invention also provides methods of discovering new proteases using : the nucle-ic acids, polypeptides and antibodies of the invention. In one aspectz-phagemid libraries are screened for expression-based discovery of proteases In another aspect, lambda phage libraries are screened for expression-based discovery of proteasses.
Screening of the phage or phagemid libraries can allow the detection of toxic aclones; improvedl 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 clones densities. Screening of phage or phagemid libraries can be in liquidll phase or in solid phasse. In one aspect, the invention provides screening in liquid phase. Whis 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 arad nucleic acids of thie invention and robotic automation to enable the execution of many thousands of biocata lytic reactions and screening assays in a short period of time, e.g., pesr day, as well as ensuring a high level of accuracy and reproducibility (see discussion off arrays, below). Aas a result, a library of derivative compounds can be produced in a matter of weeks. For further teachings on modification of molecules, including small meolecules, see PCT/UJS94/09174.
The present invention includes protease enzymes which are non_-naturally occurring «carbonyl hydrolase variants (e.g., protease variants) having a differert proteolytic activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the precursor carbonyl hydrolase from which the amino acid sequence Of the variant is derived. Specifically, such protease variants have an amino acid sequemnce not found in nature, which is derived by substitution of a pluralitzy of amino . acid residu es of a precursor protease with different amino acids. The precursor protease may be a n_aturally-occurring protease or a recombinant protease. The useful preotease variants encompass the substitution of any of the naturally occurring L-amino a_cids at the designated amino acid residue positions.
Protease signal sequences, prepro and catalytic domains :
The invention provides protease signal sequences (e.g., signal peptides (SPs)), porepro domains and catalytic domains (CDs). 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 sacids 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).
In one aspect, the invention provides a signal sequence comprising a peptide ~comprising/ consisting of a sequence as set forth in residues 1 to 12, 1to 13, 1 to 14,1t0 15,1t016,1t017,1t018,1t019,11t020,1t021,1t022,1t023,1t024,1to 25,1t0 26,1t027,1t028,1t028,1t030,1t031,1t032,1t033,1to34,1t035,1t0 : 36,1to 37,11t038,11039,1t040,1to41,1to42,1to 43,1 to 44 (or a lo nger peptide) of a pol=ypeptide of the invention.
In an alternative aspéct, the invention provides a signal sequence : comprissing a peptide comprising/ consisting of a sequence as set forth in Ta ble 4, below:
SEQID BNO: Signal (AA) 1,2 1-37 -101, 102 1-22 -111, 112 1-36 -113, 114 1-32 —115, 116 1-33 121, 122 1-25 —123, 124 1-56 -127, 128 1-27 13, 14 1-33 -131, 132 1-21 —133, 134 1-27 =139, 140 1-38 —141, 142 1-25 143, 144 1-35 15, 16 1-31 164, 165 1-17 -179, 180 1-21 19, 20 1-39
I : | .
: © 193, 194 1-19 : 199, 200 1-18 21,22 1-22 © 210,211 1-19 222,223 1-15 229, 230 1-21 23,24 1-23 241, 242 1-20 = 254, 265 1-18 27,28 1-27 29, 30 1-24 3,4 1-36 31,32 1-26 35, 36 1-27 37,38 1-37 . : 41, 42 1-22 43, 44 1-25 45, 46 1-26 47,48 1-24 . 49, 50 1-30 5,6 1-32 51, 52 1-27 53, 54 1-32 55, 56 1-27 57, 58 1-31 61, 62 1-40 NB 67, 68 1-27 69, 70 1-32 71,72 1-25 : 73,74 1-28 75,76 1-25 81,82 1-20 83, 84 1-22 85, 86 1-20 87, 88 1-35 89, 90 1-32 9,10 1-28 93, 94 1-36 gs, 96 1-24
The protease signal sequences (SPs) and/or prepro sequences of the invention can be isolated peptides, or, sequences joined to another protease or a non- protease polypeptide, e.g., as a fusion (chimeric) protein. In one aspect, the invention provides polypeptides comprising protease signal sequences of the in vention. In one aspect, polypeptides comprising protease signal sequences SPs and/or prepro ef-the invention comprise sequences heterologous to a protease of the invem tion (e.g., a fusion protein comprising an SP and/or prepro of the invention and sequences from another protease or a non-protease protein). In one aspect, the invention prov-ides proteases of the invention with heterologous SPs and/or prepro sequences, e.g., sequemces with a yeast signal sequence. A protease of the invention can comprise a heterolo gous SP and/or prepro in a vector, e.g., a pPIC series vector (Invitrogen, Carlsbad, CA). : . .
In one aspect, SPs and/or prepro sequences of the inve ntion are identified following identification of novel protease polypeptides. The pathways by which proteins are sorted and transported to their proper cellular location are often res ferred 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 thee endoplasmic reticulum. More than 100 signal sequences for proteins in this group have been determined. The signal sequences can vary in length from 13 to 36 armino acid residues.
Various methods of recognition of signal sequences are known to thosse of skill in the art.
For example, in one aspect, novel protease 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).
It should be understood that in some aspects proteases ©f the invention may not have SPs and/or prepro sequences, or “domains.” In one aspect, the invention provides the proteases of the invention lacking all or part of an SP and/or a prepro domain. In one aspect, the invention provides a nucleic acid sequence encoding a signal
‘ {= @® WO 2004/033668 PCT/US2003/032819 sequence (SP) and/or prepro from one protease operably linked to a nucleic acid sequence of a different protease or, optiomally, a signal sequence (SPs) and/or prepro domain from a non-protease protein may be desired. © The invention al so provides isolated or recombinant polypeptides comprising signal sequences (S Ps), prepro domain and/or catalytic domains (CDs) of the invention and heterologous sequences. The heterologous sequences are sequences not : naturally associated (e.g., to a protease) 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 SPs, prepro domain and/or CD’s amino terminal end, carboxy terminal end, and/or on both ends of the SP arad/or CD. In one aspect, 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
Lo. invention with the proviso that i t is not associated with any sequence to which it is naturally associated (e.g., a protease sequence). Similarly in one aspect, the invention 16 provides isolated or recombinant nucleic acids encoding these polypeptides. Thus, in one aspect, the isolated or recombinant nucleic acid of the invention comprises coding sequence for a signal sequence (CSP), prepro domain and/or catalytic domain (CD) of the invention and a heterologous secjuence (i.e., a sequence not naturally associated with the a signal sequence (SP), prepro dormain 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.
Hybrid (chimeric) proteases and peptide libraries
In one aspect, the invention provides hybrid proteases and fusion proteins, including peptide libraries, comprising sequences of the invention. The peptide libraries ofthe invention can be used to isolate peptide modulators (e.g., activators or inhibitors) of targets, such as protease substrates, receptors, enzymes. 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. In one aspect, the invention provides chimeric proteins comprising a signal sequence (SP), prepro domain and/or catalytic domain (CD) of the invention or a combination thereof and a heterologous sequence (see above).
In one aspect, the fusion proteins of the invention (e-g., the peptide moiety) are conformationally stabilized (relative to linear peptides) to allow a higher binding affinity for targets. The inventiorn provides fusions of proteases of the invention and other peptides, including known and random peptides. They can be fused in such a manner that the structure of the proteases is 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 pressence within cells and its quantity.
Amino acid sequence variants of the imvention can be characterized bya predetermined nature of the variation, a feature that sets them apart from a naturally occurring form, e.g., an allelic or interspecies variatiom of a protease sequence="In one aspect, the variants of the invention exhibit the same qualitative biological activity as the naturally occurring analogue. Alternatively, the variamts can be selected for having modified characteristics. In one aspect, while the site or region for introducing an amino acid sequence variation is predetermined, the mutatior: per se need not be predetermined.
For example, in order to optimize the performance of & mutation at a given site, random mutagenesis may be conducted at the target codon or xegion and the expressed protease variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, as discussed herein for example, M13 primer anutagenesis and PCR mutagenesis.
Screening of the mutants can be done using assays of proteolytic activities. In alternative aspects, 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. To obtain a final derivative with the optimal properties, 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 proteases 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. In one aspect, the charge or hydrophobicity has been modified. In one aspect, 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 o f the alteration, for example a 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 invsention provides substitutions in polypeptide of the invention where (a) a hydrophilic residues, e. g. seryl or threonyl, is
® WO 2004/033668 PCT/US2003/032819 substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substi tuted 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. glutaamyl or aspartyl; or (d) a residue having a bulky : : 6 side chain, e.g. phenylalanine, is substitvated for (or by) one not having a side chain, e.g. glycine. The variants can exhibit the sarme qualitative biological activity (i.e. protease activity) although variants can be selected to modify the characteristics of the proteases as needed.
In one aspect, proteases of the invention comprise epitopes or purification tags, signal sequences or other fusion Sequences, etc. In one aspect, the proteases of the invention can be fused to a random peptide to form a fusion polypeptide. By "fused" or “operably linked" herein is meant that the random peptide and the protease are linked together, in such a manner as to minimize the disruption to the stability of the protease structure, e.g., it retains protease activity. The fusion polypeptide (or fusion 18 polynucleotide encoding the fusion polyp eptide) can comprise further components as well, including multiple peptides at multiple loops.
In one aspect, the peptides and nucleic acids encoding them are randomized, either fully randomized or th ey are biased in their randomization, e.g. in nucleotide/residue frequency generally or per position. "Randomized" means that cach nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. In one aspect, the nucleic ac ids 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 forrmation 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 su fficiently structurally diverse population of randomized expression products to affect a probabilistically sufficient range of cellular responses to provide one or more cells exhi biting a desired response. Thus, the invention provides an interaction library large enougha so that at least one of its members will have a structure that gives it affinity for some molecule, protein, or other factor, .
Screening Methodologies and “On-line” Monitoring Devices :
In practicing the methods of the invention, 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 fox: protease activity (e.g., assays such as hydrolysis of casein in zymograms, the relea se of fluorescence from gelatin, or the release of p-nitroanalide from various small peptide substrates), to screen compounds as potential modulators, e.g., activators or inhibitors, of & protease activity, 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. In addition to the array formats described in detail below for screening samples, alternative formats can also be used to practice the methods of the invention. Such formats include, for example, mass spectrometers, chromatogrraphs, e.g., high-throughput HPLC and other forms of liquid chromatography, and smaller formats, such as 1536-well plates, 384—well plates and so on. High throughput screenings apparatus can be adapted and used to practice the methods of the invention, see, e_g., U.S. Patent Application No. 20020001809.
Capillary Arrays
Nucleic acids or polypeptides 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, ete.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention. Capillary arrays, such as the GIGAMATRIX™, Diversa Corpooration, San Diego, CA; and arrays described in, e.g., U.S. Patent Application No. 200200080350 Al; WO 0231203 A; WO 0244336 A, provide an alternative apparatus for holding and screening samples. In one aspect, the capillary array includes 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 lumen may be cyliradrical, square, hexagonal or any other geometric shape so long as the walls forma 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 boumd together, by being fused (e.g., where the capillaries are made of glass), glued, bonded, or clamped side-by-side. Additionally, the capillary array can include interstitial mater3al disposed between adjacent capillaries in the array, thereby forming a solid planar dewice containing a plurality of through-holes.
A capillary array can be formed of ary number of individual capillaries, for example, a range from 100 to 4,000,000 capillarTes. Further, a capillary array having about 100,000 or more individual capillaries can be formed into the standard size and shape of a Microtiter® plate for fitment into standard laboratory equipment. The lumens are filled manually or automatically using either capillary action or microinjection using a thin needle. Samples of interest may subsequently toe removed from individual capillaries for further analysis or characterization. For examples, a thin, needle-like probeis positioned in fluid communication with a selected capillary to either add or withdraw material from the lumen. . 10 In a single-pot screening assay, the asssay components are mixed yielding a solution of interest, prior to insertion into the capillasry array. The lumen is filled by capillary action when at least a portion of the array i=s immersed into a solution of interest.
Chemical or biological reactions and/or activity in each capillary are monitored for detectable events. A detectable event is often referr=d to as a “hit”, which can usually be distinguished from “non-hit” producing capillaries b=y optical detection. Thus, capillary arrays allow for massively parallel detection of “hits™.
In a multi-pot screening assay, a polypeptide or nucleic acid, ¢.g., a ligand, can be introduced into a first component, which is instroduced into at least a portion of a capillary of a capillary array. An air bubble can then_ be introduced into the capillary behind the first component. A second component camn then be introduced into the capillary, wherein the second component is separated from the first component by the air bubble. The first and second components can then be mixed by applying hydrostatic pressure to both sides of the capillary array to collapsse the bubble. The capillary array is . then monitored for a detectable event resulting from r—eaction or non-reaction of the two components.
In a binding screening assay, a sample of interest can be introduced as a } first liquid labeled with a detectable particle into a cappillary of a capillary array, wherein the lumen of the capillary is coated with a binding mamterial for binding the detectable particle to the lumen. The first liquid may then be rermoved from the capillary tube, wherein the bound detectable particle is maintained within the capillary, and a second liquid may be introduced into the capillary tube. The capillary is then monitored for a detectable event resulting from reaction or non-reaction of the particle with the second liquid.
Arrays, or “Biochips”
Nucleic acids or peolypeptides of the invention can be immobilized to or applied to an array. Arrays can bee 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. For example, in one aspect of the invention, 2 momitored parameter is transcript expression of a protease 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 am cell, by hybridization to immobilized nucleic acids on an array, or “biochip.” By using an “array” of nucleic acids on a microchip, some or all of the transcripts of a cell can be simultaneously quantified. Alternatively, arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the mesthods of the invention. Polypeptide arrays” can also be used to simultaneously quantify za 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 generically a plurality— of “spots” or “target elements,” cach target element comprising a defined amount of ©ne 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., mRNA transcripts.
In practicing the rmethods of the invention, any known array and/or method of making and using arrays can I»e incorporated in whole or in part, or variations thereof, as described, for example, in U.SS. Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695 5 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 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 5,434,049; see also, e.g., WO 99/51773; WO 99/09217;
WO .97/46313; WO 96/17958; se=e also, e.g., Johnston (1998) Curr. Biol. 8:R171-R174;
Schummer (1997) Biotechniques 23:1087-1092; Kern (1997) Biotechniques 23:120-124;
Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999)
Nature Genetics Supp. 21:25-32. See also published U.S. patent applications Nos. 20010018642; 20010019827; 20 010016322; 20010014449; 20010014448; 20010012537; 20010008765. oo
Antibodies and Antibody-based sscreening methods
® WO 2004/033668 E>CT/US2003/032819 oo
The invention provides isolated or recombinant antiboclies that specifically bind to a protease of the invention. These antibodies can be used to iszolate, identify or quantify the proteases of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention or otlaer related proteases. The antibodies can be designed to bind to an active site of za protease. Thus, . the invention provides methods of inhibiting proteases using the antibendies of the invention (see discussion above regarding applications for anti-proteasse compasitions of the invention).
The invention provides fragments of the enzymes of thes invention, including immunogenic fragments of a polypeptide 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: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 ID NO :72; SEQ ID }
NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO :82; SEQ ID
NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO :92; SEQ ID
NO:94; SEQ ID NO:96; SEQ ID N0O:98; SEQ ID NO:100; SEQ ID N(O:102; SEQ ID
NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ IID NO:112; SEQ ID
NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ IID NO:122; SEQ ID
NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ II'™> NO:132; SEQ ID
NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ I'® NO:142; SEQ ID
NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ I NO:165; SEQ ID
NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ ID» NO:200; SEQ ID
NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID» NO:230; SEQ ID
NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or the- polypeptide encoded by SEQ ID NO:145. The immunogenic peptides of the invent-ion (e.g., the immunogenic fragments 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; SEQ ID NO:26; SEQ ID IN0:28; SEQ ID
NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO: 38; SEQID
NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:-48; SEQ ID
NO:50; SEQ IID NO:52; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO: 58; SEQID
NO:60; SEQ IID NO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQID
NO:70; SEQ IID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID
NO:80; SEQ IID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID
NO:90; SEQ IID NO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID
NO:100; SEQ MD NO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID
NO:110; SEQ MD NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO: 148; SEQ ID
NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID
NO:130; SEQ ED NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID
NO:140; SEQ ED NO:142; SEQ ID NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID
NO:159; SEQ ED NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID
NO:194; SEQ I'D NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID
NO:223; SEQ I'D NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ .
ID NO:255, or “the polypeptide encoded by SEQ ID NO:145) can further comprise adjuvants, carri -ers and the like. “The antibodies can be used in immunoprecipitation, staining, immunoaffinity~ columns, and the like. If desired, nucleic acid sequences encoding for specific antigerms can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array ofthe inventiora. Altematively, 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. Furthermore, the ability to make or modify antibodies can be a phenotype engimneered into a cell by the methods of the invention.
Fvfethods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, €.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY,
Wiley/Greene, INY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Med_ical Publications, Los Altos, CA (“Stites”); Goding, MONOCLONAL ’
ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York,
NY (1986); Koller (1975) Nature 256:495; Harlow (1988) ANTIBODIES, A
LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display li “braries, in addition to the traditional in vivo methods using animals. See,
o WO 24004/033668 PC_X/US2003/032819 - e.8., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Anu. Rev. Biophys.
Biomol. Struct. 26:27-45.
Polypeptides or peptides can be used to generate antibodi-es which bind specifically to the polypeptides, e.g., the proteases, of the invention. The resulting antibrodies may be used in immunoaffinity chromatography procedures te isolate or purify : the p olypeptide or to determine whether the polypeptide is present in a b3 ological sample.
In such procedures, a protein preparation, such as an extract, or a biologi: cal sample is contacted with an antibody capable of specifically binding to one of the polypeptides of the imavention.
In immunoaffinity procedures, 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 amtibody under conditions in which the antibody specifically binds to one of the polypeptides of the invention. After a wash to remove non-specifically b ound proteins, the specifically bound polypeptides are eluted.
The ability of proteins in a biological sample to bind to the antibody may be deEermined 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 dete=ctable label such as a fl uorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibowdy to the sample may be detected using a secondary antibody havin gsuch a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmmunoassays, 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 b-y administering the polypeptides to a non-human animal. The antibody so obtained will tkaen bind the polype=ptide itself. In this manner, even a sequence encoding only a fragmment of the polype=ptide can be used to generate antibodies which may bind to the whole native polype ptide. Such antibodies can then be used to isolate the polypeptide farom cells expressing that polypeptide.
For preparation of monoclonal antibodies, any technique whhich provides antibodlies produced by continuous cell line cultures can be used. Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma te=chnique, and the EB™V-hybridoma technique (see, e-g., Cole (1985) in Monoclonal Antitoodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (see, e.g., U.S. Pamtent No. 4,946,778) can be adapted to produce single chain antibod ies to the
Polypeptides of the invention. Altematively, 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 (e.g., proteases) from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody anc those polypeptides which specifically bind the antibody are detected. Any of the procedures described above may be used to detect antibody binding.
Kits
The invention provides kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, transgenic seeds or plants or plant parts, polypeptides (e.g., proteases) and/or antibodies of the invention. The kits also csan contain instructional material teaching the methodologies and industrial uses of” the invention, ass described herein.
Whole cell e ngineering and measuring metabolic parameters
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, €.g., a new or modified pramtease activity, by modifying the genetic composition of the cell. The genetic composition can be modified by addition to the cell of a nucleic acid of the inve=ntion, e.g., a coding sequence for an enzyme of the invention. See, e.g., W00229032;
WO0196551 .
To detect the new phenotype, at least one metabolic parameter of a modified cel@ is monitored in the cell in a “real time” or “on-line” time frame. In one aspect, a plurality of cells, such as a cell culture, is monitored in “real time” or “on-line.”
In one aspect, a plurality of metabolic parameters is monitored in “real time” or “on-line.”
Metabolic pa_rameters can be monitored using the proteases of the invention.
Metabolic flux analysis (MFA) is based on a known biochemistry framework. A linearly independent metabolic matrix is constructed based on th law of mass conserv-ation and on the pseudo-steady state hypothesis (PSSH) on the intracellular metabolites. In practicing the methods of the invention, metabolic networks are established, imcluding the: * identity of all pathway substrates, products and intermediary metabolite=s
@® WO 2004/033668 PCT/US2003/032819 * identity” of all the chemical reactions interconverting the pathway metabolites, the stoichiometry of the pathway reactions, + identity” of all the enzymes catalyzing the reactions, the enzyme reaction kinetics, : * the regulatory interactions between pathway components, e.g. allosteric interactions, enz-yme-enzyme interactions etc, * intracellular compartmentalization of enzymes or any other supramolecular organization of the enzymes, and, == : * the presence of any concentration gradients of metabolites, enzymes or effector molecules or diffusion barriers to their movement.
O nce the metabolic network for a given strain is built, mathematic presentation by matrix notion can be introduced to estimate the intracellular metabolic fluxes if the on-line metabolome data is available. 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. In one aspect of the methods of the invention, after the on-line MFA calculation, the dynamic behavior of the cells, their phenotype and other properties 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. Control of physiological state of cell cultures will become possible after the pathway analysis. 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. In practicing the methods of the invention, the MITA results can also be compared with transcriptome and proteome data to design experimen ts and protocols for metabolic engineering or gene shuffling, etc.
In practicing the methods of the invention, 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.
Monitoring expression of an mRNA transcript “In one aspect of the invention, the engineered phenotype comprises increasing or decreasing the expression of an mRNA transcript (e.g., a protease message)
or generating new (e.g., protease) transcripts in a cell. This increased or decreased expression can be traced by testing for the presence of a protease of the invention or by protease activity assays. mRNA transcripts, or messages, also can bes detected and quantified by any method known in the art, including, e.g., Northern blots, quantitative : 6 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?” (ses, e.g., Kreuzer (2001) Br. J. Haematol. 114:313 -318; Xia (2001)
Transplantation 72:907-914).
In one aspect of the invention, 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.
Thus, the expression of a transcript can be completely ablated or only decreased. :
In one aspect of the invention, the engineered phenotype comprises 16 increasing the expression of a homologous gene. This can be effected by knocking out of : a negative control element, including a transcriptional regulatory elerment 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 =0 to immobilized nucleic acids on an array.
Monitoring expression of a polypeptides, peptides and amino acids
In one aspect of the invention, the engineered phencty pe comprises increasing or decreasing the expression of a polypeptide (e.g., a protease) or generating new polypeptides in a cell. This increased or decreased expression can be traced by =65 determining the amount of protease present or by protease activity as says. Polypeptides, peptides and amino acids also can be detected and quantified by any xnethod 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), hyperdi fusion =0 chromatography, various immunological methods, e.g. immunoprecippitation, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, gel electrophoresis (e.g.,
SDS-PAGE), staining with antibodies, fluorescent activated cell sorter (FACS), pyrolysis
@® WO 2004/033668 PCT/US2003/032819 mass spectrometry, Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-
MS, and LC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, and the like. Novel bioactivities can also be screened using methods, or variations thereof, ’ described in U.S. Patent No. 6,057,103. Furthermore, as discussed below in detail, one or more, or, all the polypeptides Of a cell can be measured using a protein array.
Industrial Applications N Co
Detergent Compositioras C
The invention perovides detergent compositions comprising one or more polypeptides (e.g., proteases) of the invention, and methods of making and using these } compositions. The invention imcorporates all methods of making and using detergent compositions, see, e.g., U.S. Patent No. 6,413,928; 6,399,561; 6,365,561; 6,380,147. The detergent compositions can be a one and two part aqueous composition, a non-aqueous liquid composition, a cast solidl, a granular form, a particulate form, a compressed tablet, a gel and/or a paste and a slurrs form. The proteases of the invention can also be used as adetergent additive product in a solid ora liquid form. Such additive products are intended to supplement or boosst the performance of conventional detergent compositions and can be added at any stage of the cleaning process.
The invention al so provides methods capable of removing gross food soils, films of food residue and other minor food compositions using these detergent compositions. Proteases of the invention can facilitate the removal of stains by means of catalytic hydrolysis of proteins. Proteases of the invention can be used in dishwashing detergents in textile laundering detergents.
The actual active enzyme content depends upon the method of manufacture of a detergent com position and is not critical, assuming the detergent solution has the desired enzymatic activity. In one aspect, the amount of protease present in the final solution ranges froma about 0.001 mg to 0.5 mg per gram of the detergent composition. The particular enzyme chosen for use in the process and products of this invention depends upon the conditions of final utility, including the physical product form, use pH, use temperature, znd soil types to be degraded or altered. The enzyme can be chosen to provide optimum activity and stability for any given set of utility conditions.
In one aspect, the proteases of the present invention are active in the pH ranges of from about 4 to about 12 and in the te-mperature range of from about 20°C to about 95°C. The
® detergents of the invention can comprise cationic, senrai-polar nonionic or zwitterionic surfactants; or, mixtures thereof.
Proteases of the invention can be formualated into powdered and liquid detergents having pH between 4.0 and 12.0 at levels o f about 0.01 to about 5% . (preferably 0.1% to 0.5%) by weight. These detergent compositions can also include other enzymes such as proteases, cellulases, lipases or endoglycosidases, endo-beta.-1,4- glucanases, beta-glucanases, endo-beta-1,3(4)-glucanaases, cutinases, peroxidases, laccases, amylases, glucoamylases, pectinases, reductaases, oxidases, phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases, mannanases, xyloglucanases, xylanases, pectin acetyl esterases, rhamnogalacturonar acetyl esterases, polygalacturonases, thamnogalacturonases, galactanas es, pectin lyases, pectin methylesterases, cellobiohydrolases and/or transglutarminases. These detergent : .compositions can also include builders and stabilizers.
The addition of proteases of the inventi on to conventional cleaning 16 compositions does not create any special use limitatiora. In other words, any temperature and pH suitable for the detergent is also suitable for the compositions of the invention as long as the enzyme is active at or tolerant of the pH arx d/or temperature of the intended use. In addition, the proteases of the invention can be used in a cleaning composition without detergents, again either alone or in combinaticen with builders and stabilizers.
The present invention provides cleaningz compositions including detergent compositions for cleaning hard surfaces, detergent compositions for cleaning fabrics, dishwashing compositions, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning solutions.
In one aspect, the invention provides a enethod for washing an object comprising contacting the object with a polypeptide of the invention under conditions ~ sufficient for washing. A protease of the invention ma_y be included as a detergent additive. The detergent composition of the invention mnay, for example, be formulated as a hand or machine laundry detergent composition comprising a polypeptide of the invention. A laundry additive suitable for pre-treatment of stained fabrics can comprise a polypeptide of the invention. A fabric softener composition can comprise a protease of the invention. Alternatively, a protease of the invention can be formulated as a detergent composition for use in general household hard surface cleaning operations. In alternative aspects, detergent additives and detergent compositions of the invention may comprise one or more other enzymes such as a protease, a lipase. a cutinase, another protease, a
: { . @® WO 2004/033668 PCT/US2003/032819 carbohydrase, a cellulase, a pectinase, 2x mannanase, an arabinase, a galactanase, a
Xylanase, an oxidase, e.g., a lactase, and/or a peroxidase (sce also, above). The
Properties of the enzyme(s) of the inveration are chosen to be compatible with the selected detergent (i.e. pH-optimum, compatibil -ity with other enzymatic and non-enzymatic ingredients, ctc.) and the enzymes) is present in effective amounts. In one aspect, . protease enzymes of the invention are wmsed to remove malodorous materials from fabrics.
Various detergent compositions and me=thods for making them that can be usedin practicing the invention are described im, e.g., U.S. Patent Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038; 6,309,871; 6,204 232; 6,197,070; 5,856,164.
When formulated as commpositions suitable for use in a laundry machine washing method, the proteases of the in=vention can comprise both a surfactant and a * builder compound. They can additional 1y comprise one or more detergent components, €.8., organic polymeric compounds, blezaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispeersants, soil suspension and anti-redeposition 5 agents and corrosion inhibitors. Laundry compositions of the invention can also contain softening agents, as additional detergent components. Such compositions containing carbohydrase can provide fabric cleaningg, stain removal, whiteness maintenance, softening, color appearance, dye transfer= inhibition and sanitization when formulated as laundry detergent compositions.
The density of the laundrsy detergent compositions of the invention can range from about 200 to 1500 g/liter, or, about400 to 1200 g/liter, or, about 500 to 950 g/liter, or, 600 to 800 g/liter, of composition; this can be measured at about 20°C.
The "compact" form of la=undry detergent compositions of the invention is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventionzal ingredients of detergent compositions in powder form. In conventional detergent ~compositions, the filler salts are present in substantial amounts, typically 17% to 352% by weight of the total composition. In onc aspect of the compact compositions, the Filler salt is present in amounts not exceeding 15% of the total composition, or, not exceeding 10%, or, not exceeding 5% by weight of the composition. The inorganic filler salts can be selected from the alkali and alkaline- earth-metal salts of sulphates and chlorides, e.g., sodium sulphate. oo
Liquid detergent compositEans of the invention can also beina “concentrated form." In one aspect, the li =quid detergent compositions can contain a lower amount of water, compared to convention al liquid detergents. In alternative aspects, the water content of the concentrated liquid detergent is less than 40%, or, less than 30%, or, less than 20% by weight of the detergent composition. Detergent compounds of the - invention can comprise formulations as described in wo 97/01629,
Proteases, such as metalloprote=ases (MPs) and serine proteases, of the invention can be useful in formulating various cleaning compositions. A number of known compounds are suitable surfactants incRuding nonionic, anionic, cationic, or zwitterionic detergents, can be used, e.g., as di_sclosed in U.S. Patent Nos. 4,404;128; 4,261,868; 5,204,015. In addition, proteases can be used, for example, in bar or liquid soap applications, dish care formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile appolications, as fusion-cleavage enzymes in protein production, and the like. Proteases may provide enhanced performance in a detergent composition as compared to another detergent protease, that is, the enzyme group may increase cleaning of certain enzymes sensitive stains such as grass or blood, as determined by usual evaluation after a standarcd wash cycle. Metalloproteases, serine proteases (or other proteases of the invention) <an be formulated into known powdered and liquid detergents having pH between 6.5 a.nd 12.0 at levels of about 0.01 to about 5% (for example, about 0.1% to 0.5%) by weight. These detergent cleaning compositions can also include other enzymes such as known pro-teases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stab lizers. =20 Treating fibers and textiles
The invention provides method=s of treating fibers and fabrics using one or more proteases of the invention. The proteases can be used in any fiber- or fabric-treating method, which are well known in the art, see, e.g., U.S. Patent No. 6,261,828; 6,077,316; 6,024,766; 6,021,536; 6,017,751; 5,980,581; L=S Patent Publication No. 20020142438 =5 Al. For example, proteases of the invention czan be used in fiber and/or fabric desizing.
In one aspect, the feel and appearance of a fabric is improved by a method comprising contacting the fabric with a protease of the inveention in a solution. In one aspect, the fabric is treated with the solution under pressumre. For example, proteases of the invention can be used in the removal of stains. =0 In one aspect, proteases of the imavention are applied during or after the weaving of textiles, or during the desizing stag-e, or one or more additional fabric : processing steps. During the weaving of textiles, the threads are exposed to considerable mechanical strain. Prior to weaving on mechamical looms, warp yarns are often coated
® WO 2004/033668 PCT/US2003/032819 with sizing starch or starch derivatives in_ order to increase their tensile strength and to prevent breaking. The proteases of the irvention can be applied to remove these sizing starch or starch derivatives. After the textiles have been woven, a fabric can proceed to a desizing stage. This can be followed by ene or more additional fabric processing steps. Desizing is the act of removing “size” from textiles. After weaving, the size coating must : be removed before further processing the fabric in order to ensure a homogeneous and wash-proof result. The invention providezs a method of desizing comprising enzymatic treatment of the “size” by the action of proteases of the invention.
The enzymes of the inven€ion can be used to desize fabrics, including cotton-containing fabrics, as detergent additives, e.g., in aqueous compositions. The invention provides methods for producing a stonewashed look on indigo-dyed denim fabric and garments. For the manufactures of clothes, the fabric can be cut and sewn into clothes or garments. These can be finished before or after the treatment. In particular, for the manufacture of denim jeans, different enzymatic finishing methods have been developed. The finishing of denim garmemt normally is initiated with an enzymatic desizing step, during which garments are subjected to the action of amylolytic enzymes in order to provide softness to the fabric andl make the cotton more accessible to the } subsequent enzymatic finishing steps. Time invention provides methods of finishing denim garments (e.g., a "bio-stoning process"), enzymatic desizing and providing softness to fabrics using the proteases of the inven tion. The invention provides methods for quickly softening denim garments in a de=sizing and/or finishing process.
Other enzymes can be also be used in these desizing processes. For example, an alkaline and thermostable amylase and protease can be combined in a single bath for desizing and bioscouring. Amongg advantages of combining desizing and scouring in one step are cost reduction anad lower environmental impact due to savings in energy and water usage and lower waste production. Exemplary application conditions for desizing and bioscouring are about pH 8.5 to 10.0 and temperatures of about 40°C and up. Using a protease of the invention, loves enzyme dosages, e.g., about 100 grams (g) per a ton of cotton, and short reaction times, e=.g., about 15 minutes, can be used to obtain efficient desizing and scouring with out aclded calcium.
In one aspect, an alkaline a-nd thermostable amylase and protease are combined in a single bath desizing and bioscouring. Among advantages of combining desizing and scouring in one step are cost reduction and lower environmental impact due . to savings in energy and water usage and Eower waste production. Application conditions for desizing and bioscouring can be betweera about PH 8.5 to pH 10.0 and temperatures at about 40°C and up. Low enzyme dosages (e=.g., about 100 g per a ton of cotton) and short reaction times (e.g., about 15 minutes) can base used to obtain efficient desizing and scouring with out added calcium.
The proteases of the inventiom can be used in combination with other carbohydrate degrading enzymes, e.g., cellulase, arabinanase, xyloglucanase, pectinase, and the like, for the preparation of fibers or or cleaning of fibers. These can be used in combination with detergents. In one aspect, proteases of the invention can be used in treatments to prevent the graying of a textile .
The proteases of the inventiomn can be used to treat any cellulosic material, including fibers (e.g., fibers from cotton, hermp, flax or linen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yams, and towe Ring, made from cotton, cotton blends or natural or manmade cellulosics (e.g. originating from xylan-containing cellulose fibers . such as from wood pulp) or blends thereof. “Examples of blends are blends of cotton or 16 rayon/viscose with one or more companion rnaterial such as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibexs (e.g. rayon/viscose, ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocesll).
The textile treating processes of the invention (using proteases of the invention) can be used in conjunction with other textile treatments, e.g., scouring and bleaching. Scouring is the removal of non-cellulosic material from the cotton fiber, e.g., the cuticle (mainly consisting of waxes) and primary cell wall (mainly consisting of pectin, protein and xyloglucan). A proper wax removal is necessary for obtaining a high wettability. This is needed for dyeing. Removal of the primary cell walls by the processes of the invention improves wax removal and ensures a more even dyeing.
Treating textiles with the processes of the inwention can improve whiteness in the bleaching process. The main chemical used dn scouring is sodium, hydroxide in high concentrations and at high temperatures. Ble=aching comprises oxidizing the textile.
Bleaching typically involves use of hydrogera peroxide as the oxidizing agent in order to obtain either a fully bleached (white) fabric or to ensure a clean shade of the dye.
The invention also provides aE kaline proteases (proteases active under alkaline conditions). These have wide-rangirg applications in textile processing, degumming of plant fibers (e.g., plant bast fibers), treatment of pectic wastewaters, paper-
; i making, and coffee and tea fermentations, See, e.g, Hoondal (2002) Applied : ’
Microbiology and Biotechnology 59:409-418. . Treating foods and food processing | ’
The proteases of the invention have mnumerous applications in food processing industry. For example, in one aspect, th_e proteases of the invention are used to improve the extraction of oil from oil-rich plant rmaterial, e.g., oil-rich seeds, for example, soybean oil from soybeans, olive oil from olives, rapeseed oil from rapeseed and/or sunflower oil from sunflower seeds.
The proteases of the invention can bes used for separation of components of plant cell materials. For example, proteases of the i_nvention can be used in the separation of protein-rich material (e.g., plant cells) into compcorents, e.g., sucrose from sugar beet or starch or sugars from potato, pulp or hull fractiorms. In one aspect, proteases of the invention can be used to separate protein-rich or oil—rich crops into valuable protein and oil and hull fractions. The separation process may toe performed by use of methods known in the art.
The proteases of the invention can be= used in the preparation of fruit or vegetable juices, syrups, extracts and the like to increase yield. The proteases of the invention can be used in the enzymatic treatment (e..g., hydrolysis of proteins) of various plant cell wall-derived materials or waste materials, e.g. from wine or juice production, or agricultural residues such as vegetable hulls, bean hwulls, sugar beet pulp, olive pulp, potato pulp, and the like. The proteases of the invermtion can be used to modify the consistency and appearance of processed fruit or vegetables. The proteases of the invention can be used to treat plant material to facili~tate processing of plant material, including foods, facilitate purification or extraction of plant components. The proteases of the invention can be used to improve feed value, Cecrease the water binding capacity, improve the degradability in waste water plants and/or improve the conversion of plant material to ensilage, and the like.
Animal feeds and food or feed additives
The invention provides methods for treating animal feeds and foods and food or feed additives using proteases of the inventiown, animals including mammals (e.g., humans), birds, fish and the like. The invention provides animal feeds, foods, and additives comprising proteases of ®he invention. In one aspect, treating animal feeds, foods and additives using protease=s of the invention can help in the availability of nutrients, e.g., starch, in the animal feed or additive. By breaking down difficult to digest proteins or indirectly or directly uramasking starch (or other nutrients), the protease makes nutrients more accessible to other wcndogenous or exogenous enzymes. The protease can _ also simply cause the release of rezadily digestible and easily absorbed nutrients and sugars. -
Proteases of the pre=sent invention, in the modification of animal feed or a food, can process the food or feed either in vitro (by modifying components of the feed or food) or in vivo. Proteases can be added to animal feed or food compositions containing high amounts of arabinogalactans «or galactans, e.g. feed or food containing plant material from soy bean, rape seed, lupin aned the like. When added to the feed or food the protease
Lo significantly improves the in vivo Boreak-down of plant cell wall material, whereby a better utilization of the plant nutrients by— the animal (e.g., human) is achieved. In one aspect, the growth rate and/or feed conver—sion ratio (i.e. the weight of ingested feed relative to weight gain) of the animal is improved. For example a partially or indigestible galactan- comprising protein is fully or parti ally degraded by a protease of the invention, e.g. in combination with another enzyme, e.g., beta-galactosidase, to peptides and galactose and/or galactooligomers. These erazyme digestion products are more digestible by the animal. Thus, proteases of the inv—ention can contribute to the available energy of the feed or food. Also, by contributing to t_he degradation of galactan-comprising proteins, a protease of the invention can impreove the digestibility and uptake of carbohydrate and non-carbohydrate feed or food cormstituents such as protein, fat and minerals.
In another aspect, pr Totease of the invention can be supplied by expressing : the enzymes directly in transgenic feed crops (as, e.g., transgenic plants, seeds and the like), such as corn, soy bean, rape seed, lupin and the like. As discussed above, the invention provides transgenic plan _ts, plant parts and plant cells comprising a nucleic acid sequence encoding a polypeptide of the invention. In one aspect, the nucleic acid is expressed such that the protease of the invention is produced in recoverable quantities.
The protease can be recovered frorm any plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide can be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.
® ‘WO 2004/033668 ’ PCT/US2003/032819
Paper or pulp treatment
The proteases of the invention can be in papesr or pulp treatment or paper deinking. . For example, in one aspect, the invention provide-s a paper treatment process using proteases of the invention. In another aspect, paper ccamponents of recycled 6 photocopied paper during chemical and enzymatic deinking processes. In one aspect, proteases of the invention can be used in combination with cellulases, pectate lyases or other enzymes. The paper can be treated by the following tharee processes: 1) disintegration in the presence of proteases of the invention, 22) disintegration with a deinking chemical and proteases of the invention, and/or 3) disintegration after soaking with proteases of the invention. The recycled paper treated with proteases can have a higher brightness due to removal of toner particles as compa red to the paper treated with just cellulase. While the invention is not limited by any particular mechanism, the effect of proteases of the invention may be due to its behavior as s_irface-active agents in pulp : suspension,
The invention provides methods of treating paper and paper pulp using one or more proteases of the invention. The proteases of the invention can be used in any paper- or pulp-treating method, which are well known in the art, see, e.g., U.S. Patent No. 6,241,849; 6,066,233; 5,582,681. For example, in one aspect, the invention provides a : method for deinking and decolorizing a printed paper containing a dye, comprising pulping a printed paper to obtain a pulp slurry, and dislodging an ink from the pulp slurry in the presence of proteases of the invention (other enzymes «can also be added). In another aspect, the invention provides a method for enhancing the freeness of pulp, e.g., pulp made from secondary fiber, by adding an enzymatic mixture comprising proteases of : the invention (can also include other enzymes, €.g., pectate lyase, cellulase, amylase or glucoamylase enzymes) to the pulp and treating under condit ions to cause a reaction to produce an enzymatically treated pulp. The freeness of the emzymatically treated pulpis increased from the initial freeness of the secondary fiber pulp without a loss in brightness.
Waste treatment
The proteases of the invention can be used in 22 variety of other industrial applications, e.g., in waste treatment. For example, in one asppect, the invention provides a solid waste digestion process using proteases of the invention. The methods can comprise reducing the mass and volume of substantially untrezated solid waste. Solid waste can be treated with an enzymatic digestive process in the presence of an enzymatic solution (including proteases of the invention) at a controlled temperature. This results in a reaction without apprecizable bacterial fermentation from added microorganisms. The : solid waste is converted into a liquefied waste and any residual solid waste. The resulting liquefied waste can be separated from said any residual solidified waste. See e.g., US. § Patent No. 5,709,796.
In addition, the proteases of the invention can be used in the animal rendering industry, to e.g., get rid of feathers, e.g., as described by Yamamura (2002)
Biochem. Biophys. Res. Com. 294:1138-1143. Alkaline proteases can.also be used in the production of proteinaceovas fodder from waste feathers or keratin-containing materials, : e.g, as described by Guptaa (2002) Appl. Microbiol. Biotechnol. 59:15-32.
Oral care products-
The invention provides oral care product comprising proteases of the invention. Exemplary oraE care products include toothpastes, dental creams, gels or tooth powders, odontics, mouth “washes, pre- or post brushing rinse formulations, chewing gums, lozenges, or candy. See, e.g., U.S. Patent No. 6,264,925.
Brewing and ferme nting
The invention provides methods of brewing (e.g., fermenting) beer comprising proteases of thee invention. In one exemplary process, starch-containing raw materials are disintegrated and processed to form a malt. A protease of the invention is used at any point in the fermentation process. For example, proteases of the invention can be used in the processing of barley malt. The major raw material of beer brewing is barley malt. This canbea three stage process. First, the barley grain can be steeped to increase water content, e.g -, to around about 40%. Second, the grain can be germinated by incubation at 15 to 25°C for 3 to 6 days when enzyme synthesis is stimulated under the control of gibberellins. In one aspect, proteases of the invention are added at this (or any other) stage of the process.” The action of proteases results in an increase in fermentable ‘ reducing sugars. This can be expressed as the diastatic power, DP, which can rise from around 80 to 190 in 5 days at 12°C. Proteases of the invention can be used in any beer or alcoholic beverage produc ng process, as described, e.g., in U.S. Patent No. 5,762,991; - 5,536,650; 5,405,624; 5,02 1,246; 4,788,066.
Medical and resear~ch applications :
Proteases of= the invention can be used for cell isolation from tissue for cellular therapies in the sarme manner that collagenases. For example, metallo-
@® WO 2004/033- 668 PCT/US2003/032819 endoproteimases and other enzymes of the invention that can cleave collagen into smaller peptide fragayments, can be used as “liberase enzymes” for tissue d_issociation and to improve thes health of isolated cells. “Liberase enzymes” can repBace traditional collagenase=. Proteases of the invention having collagenase I, colBagenase II, clostripain 6 and/or neutral protease activity can be used for tissue dissociatiomm. In one aspect, for tissue disso- ciation, collagenase isoforms of the invention are blen_ded with each other, and, optionzally, with a neutral protease. In one aspect, the neutral protease is a neutral protease disspase and/or the neutral protease thermolysin.
Additionally, proteases of the invention can be use=d as antimicrobial agents, due to their bacteriolytic properties, as described, e.g., in Ii, S. et. al. Bacteriolytic
Activity ancl Specificity of Achromobacter b-Lytic Protease, J. Biochem. 124, 332-339 (1998).
Proteases of the invention can also be used therapeutically to cleave and destroy specific proteins. Potential targets include toxin proteins,. such as Anthrax, :
Clostridiunr: botulinum, Ricin, and essential viral or cancer cell proteins.
Proteases of the invention can also be used in disin—fectants, as described, e.g. in J. Ge=n Microbiol (1991) 137(5): 1145-1153; Science (200E.)249:2170-2172.
Additional medical uses of the proteases of the invention include lipoma removal, wound debraidment and scar prevention (collagenases), cdebriding chronic dermal ulcers and severely burned areas.
Proteases of the invention can be used to in sterile ©nzymatic debriding . composition_s, e.g., ointments, in one aspect, containing about 250 collagenase units per gram. Whites petrolatum USP can be a carrier. In one aspect, proteases of the invention can be used Zin indications similar to Santyl® Ointment (BTC, Lyn brook, NY). Proteases of the inventzion can also be used in alginate dressings, antimicrobi_al barrier dressings, burn dressings, compression bandages, diagnostic tools, gel dressirgs, hydro-selective dressings, hy~drocellular (foam) dressings, hydrocolloid Dressings, IV dressings, incise drapes, low eadherent dressings, odor absorbing dressings, paste bamndages, post operative dressings, sczar management, skin care, transparent film dressings amnd/or wound closure.
Proteases of ~the invention can be used in wound cleansing, wound bed preparation, to treat pressure= ulcers, leg ulcers, burns, diabetic foot ulcers, scars, IV fixation, surgical oo wounds and minor wounds.
Additionally, proteases of the invention can be used in proteeomics and lab work in general. Fox instance, proteases can be used in the same manner ems DNA restriction enzymes.
Other industarial applications
The i mvention also includes a method of increasing the flovw of production fluids from a subterranean formation by removing a viscous, protein-contamining, damaging fluid formmed during production operations and found within the subterranean formation which surrounds a completed well bore comprising allowing preoduction fluids to flow from the weL 1 bore; reducing the flow of production fluids from thes formation below expected flows rates; formulating an enzyme treatment by blending ®ogether an aqueous fluid and a gpolypeptide of the invention; pumping the enzyme tresatment to a desired location within the well bore; allowing the enzyme treatment to de=grade the viscous, protein-con taining, damaging fluid, whereby the fluid can be rem_oved from the subterranean format ®on to the well surface; and wherein the enzyme treatment is effective to attack protein in c=ell walls.
Prote ases of the invention can be used for peptide synthesis, in the leather industry, e.g., for hice processing, e.g., in hair removal and/or bating, for vaste management, e.g., removal of hair from drains, in the photography industr—y, e.g., for silver recovery from film, in the medical industry, e.g., as discussed above=, e.g., for treatment of burns, vwounds, carbuncles, furuncles and deep abscesses or t< dissolve blood clots by dissolving fSbrin, for silk degumming.
In otlner aspects, proteases of the invention can be used as Flavor enhancers in, for example, cheese and pet food, as described, e.g., in Pommer, K., In~vestigating the impact of enzymes on pet food palatability, Petfood Industry, May 2002, 20-11.
In ye® another embodiment of the invention, proteases of ttme invention can be used to increase sstarch yield from corn wet milling, as described, e.g., 1_n Johnston,
D.B., and Singh, V. Use of proteases to Reduce Steep Time and SO2 requirements in a corn wet-milling prc>cess, Cereal Chem. 78(4):405-411.
In otkaer aspects, proteases of the invention can be used in biodefense (e.g., destruction of sporess or bacteria). Use of proteases in biodefense applicatJons offer a significant benefit, imn that they can be very rapidly developed against any ~currently unknown biological warfare agents of the future. In addition, proteases of= the invention can be used for deco=ntamination of affected environments.
Additionally, proteases of the invention can be used in biofilam degradation, #&n biomass conversion to ethanol, and/or in the personal care amd cosmetics industry.
Proteases of the invention can also be used to enhance enanti_ oselectivity, asdescribed, eg, in Arisawa, A. et. al. Streptomyces Serine Protease (DHP—A) as a New
Biocatalyst Capable of Forming Chiral Intermediates of 1,4-Diohydropyridi me Calcium
Antagonists. Appl Environ Mircrobiol 2002 Jun; 68(6):2716-2725; Haring, ID et. al.
Semisynthetic Enzymes in Asymmetric Synthesis:Enantioselective Reduction of Racemic
Hydroperoxidles Catalyzed by Seleno-Subtilisin. J. Org. Chem. 1999, 64:83 2-835.
The invention will be further described with reference to the sollowing examples; however, it is to be understood that the invention is not limited to such examples.
EXAMPLES
EXAMPLE 1= Protease activity assays :
The following example describes exemplary protease activity assays to determine the catalytic activity of a protease. These exemplary assays can bes used to determine if a polypeptide is within the scope of the invention.
The activity assays used for proteinases (active on proteins) iraclude : 20 zymograms and liquid substrate enzyme assays. Three different types of zymograms : were used to measure activity: casein, gelatin and zein. For the liquid substrate enzyme assays, three main types were used: gel electrophoresis, O-pthaldialdehyde (OPA), and fluorescent encd point assays. For both the gel electrophoresis and OPA assays, four different substrates were used: zein, Soybean Trypsin Inhibitor (SBTI, SIGMCA-Aldrich,
T6522), wheat germ lectin and soybean lectin. The substrate for the fluorescent end point assay was gelatin, ‘The activity assays used for proteinases and peptidases (actives on peptides) used pNA linked small peptide substrates. The assays included specificity emd point assays, unit definition kinetic assays and pH assays.
The following example describes the above-mentioned exemplary protease activity assays. These exemplary assays can be used to determine if a polypeptide is wvithin the scope of the invention. -
®
Protein (proteinase activity)
Casein zymogram gel assays
Casein zymogram gels were used to assess proteinase activity (see Tables land 2). The protease activity assays were assessed using 4-16% gradient gels (Invitrogem Corp., Carlsbad, CA) containing casein conjugated to a blue= dye and embedded within the gel matrix. All zymogram gels were processed according to the manufacterer’s instructions. Briefly, each sample was mixed with an eqgual volume of 2x loading dy~e and incubated without heating for ten minutes before loadimmg. After electrophorresis, gels were incubated in a renaturing buffer to remove thes SDS and allow the proteirms to regain their native form. Gels were then transferred to a «developing ) solution ard incubated at 37°C for 4 to 24 hours. If a protease digests the casein in the gel, a cleam zone is produced against the otherwise blue background that corresponds to 7 the locatio-n of the protease in the gel. Negative controls (indicated wittm NC on gel images) were processed along with the experimental samples in each experiment and electrophorresed on the casein zymograms next to their corresponding pr-otease(s).
Unlike traditional SDS-PAGE, samples are not heat denatured prior to electrophoresis of casein zymograms. As a result, it is sometimes difficult to accurately assess the molecular weight of the proteases. For example, Subtilisin A. (Sigma, P5380, indicated with Subt.A on the gel images), which was used as a positive control in these experimerxts, is predicted to be approximately 27 kDa in size. However_, when electropho-resed through casein zymograms using the conditions describ ed, Subtilisin A barely miggrates into the gel and is visible only above 183kDa. Therefor €, the zymograms do not define the MW of the proteases indicated, but rather used as an iradicator of : activity.
Ge latin zymogram assays
Gelatin zymograms, Novex® Zymogram Gels, were performed according to manufacturer's instructions (Invitrogen Corp., Carlsbad, CA). Unlike the casein zymogram s, gelatin zymograms were post-stained following development using either a
Colloidal Blue Staining Kit or the SIMPLYBLUE™ Safestain, (both frosm Invitrogen).
Areas of protease activity appeared as clear bands against a dark backgreound.
Coan Zein assays
Corn zein was used as substrate for protease activity assays, using powder,
Z-3625 (Smgma Chemical Co. St. Louis, MO), and Aquazein, 10% solution (Freeman
® WO 2004/033668 PCT/US2003/032819
Industries, Tuckahoe, NY). When: fractionated through a SDS-PAGE gel, zein from both suppliers produced bands of 24 ard 22 kDa. The two zein bands correspond in molecular weight to those previously descritoed for alpha-zein, the most abundant subclass of zeinss, which are estimated to comprise 7 1-84% of total zein in com (see, e.g., Consoli (2001) § Electrophoresis 22:2983-2989). Results are illustrated in Table 3, above.
Lyophilized cultures supernatants containing active protease were resuspended, dialyzed, and incubaated with zein in 50 mM KPO, pH 7.5. Reactions wemre run in a 96-well microtiter format. “Substrate only” and “enzyme preparation only” controls were processed as well ass experimental samples. After 24 hours at 30°C, aliquots were removed and subjec=ted to OPA, SDS-PAGE, or Zymogram analysis. In some cases, fresh aliquots were re=moved and analyzed after 48 or 72 hours at 30°C.
Zein Zymogram: Aa quazein was added to a final concentration of 0.075% in a 10% polyacrylamide gel. Ali-quots of dialyzed protease samples were : } electrophoresed through the zein Zymogram using standard conditions. F ollowing electrophoresis, the zymogram gel was washed, incubated in a renaturing buffer, incubated overnight in a developirg buffer optimized for protease activity (contains NaC_l,
CaCl, and Brij 35, in Tris buffer goH 8), and stained with Coomassie blue stain.
SDS-PAGE: Aliquots of equal volume were removed from each sample and subjected to SDS-PAGE analysis. Following electrophoresis, proteins in the gels were stained with SYPRO Oranges (Molecular Probes) and visualized using UV transillumination.
OPA: In the preserce of Beta-mercaptoethanol (BME), OPA reacts with free amino ends to produce a fluorescent imidazole that can be detected using a standard fluorescence plate reader. In this z:ssay, aliquots of equal volume were removed from each sample and placed in a black fluorescence plate. Samples were then diluted 1:10 ir
OPA reagents. Fluorescence (Ex == 340 nm, Em = 450 nm) was determined after a 5- minute incubation. A summary of OPA data on all substrates is included in Table 3, above,
Soybean Trypsin Inhibitor assays :
Soybean Trypsin Inhibitor (SBTI, SIGMA-Aldrich, T6522) was used as =a substrate for protease activity. Lyophilized culture supernatants containing active protease were resuspended, dialyzed, and incubated. with SBTI (1 mg/m final conc.) at 37°C in 50 mM KPO,, pH 7.5. Substrate alone ancl enzyme preparation alone controls were processed along with experimental samples. ZA fter 24 hours, aliquots were removed and subjected to OPA and SDS-PAGE analysis. Re=sults are illustrated in Table 3, above.
SDS-PAGE: for SBTI, following electrophoresis, pmroteins in the gels were stained with
Coomassie blue.
Wheat Germ Lectin assays -
Wheat germ lectin (WGA, EY Laboratories, 1L-2101, Pure) was used as a substrate for protease activity. Lyophilized culture supernatants containing active protease were resuspended, dialysed, and incubated with WGA (1 mg/ml final concentration) at 37°C in 50 mM KPQ,, pH 7.5. Sweabstrate alone and enzyme preparation alone controls were processed along with experimeratal samples. After 24 hours, aliquots were removed and subjected to OPA and SDS-PAG-E analysis as. Results are illustrated in Table 3, above. SDS-PAGE: for WGA, followin sg electrophoresis, proteins in the gels were stained with Coomassie blue.
Soybean lectin assays
Soybean lectin (SBA, EY Laboratori «es, L-1300, Crude) was used as a substrate for protease activity. Lyophilized culture supernatants containing active protease were resuspended, dialysed, and incubated with SBA (1 mg/ml final concentration) at 37°C in 50 mM KPOq, pH 7.5. Substrate alone and. enzyme preparation alone controls were processed along with experimental samples. Aw fter 24 hours, aliquots were removed and subjected to OPA and SDS-PAGE analysis. Re sults are illustrated in Table 3, above.
SDS-PAGE: for SBA, following electrophoresis, p=—oteins in the gels were stained with
Coomassie blue.
Gelatin in fluorescent liquid end point assay
DQ Gelatin (Molecular Probes, flucreescein conjugate, D-12054) was used to assess the proteolytic activity of the proteases of t-he invention. DQ gelatin is a protein : that is so heavily labeled with a fluorophore that its fluorescence is quenched when the molecule is intact. Proteases that cleave the substra€e will release the fluorophores from internal quenching and fluorescence will increase in proportion to the protease activity.
DQ Gelatin was diluted to a final concentration of 225 ug/ml in 100 ul reactions containing a suitable buffer such as zymogram developing buffeer (Invitrogen) and varying amounts of protease preparations. Reactions were incubated in a 384 well, clear, flat-bottom o WO 2004/033668 PCT/US2003/032819 microtiter plate at 37°C for various timee periods from 1 hr to overnight. Fluorescence was monitored using a fluorescence plate reader after incubation at 37°C for various times.
As an example of the ressults obtained from the fluorescent liquid end point assay, see Table 5 and Figure 5, which show the activity of SEQ ID NO: 144 (encoded by
SEQID NO:143). Samples were assay~ed in duplicate and the raw data is shown in the
Table 4, below. Duplicates were averagyed and the background from the negative control was subtracted to depict the increase in fluorescence caused by SEQ ID NO: 144 activity in one hour using a bar graph (Figure 5). [0 [0 Thew Jer]
Ey Ea LL EE
ICE LC cn Ls
Table 5
Peptides (proteinase and Deptidase activi-ty)
Specificity end point assay
Synthetic small peptide sumbstrates linked to a chromophore are often used to determine the specificity and aid in biochemical characterization of proteases. To gauge the substrate specificity of the protecases of the invention, several para-nitroanalide linked synthetic peptides were obtained fi-om Sigma including Ala-Ala-Pro-Phe-pNA (AAPF), Ala-Ala-Ala-pNA (AAA), N-Bz-D,L-Arg-pNa (BAPNA), Gly-Gly-Phe-pNA,
Ile-Glu-Gly-Arg-pNA, and Pro-Phe-Arg-ppNA. When the peptide bond between the pNA group and the amino acid in the P1 substrate position is cleaved, a yellow color is produced whose absorbance can be measuered at 410nm. 25 mM stocks of small peptide substrates were prepared in DMSO. Substrates were used at a final concentration of 250 uM in 100 ul reaction volumes including v-arying amounts of protease preparations.
Reactions were run in a suitable protease beuffer such as 1X Zymogram developing buffer from Invitrogen and were incubated in a 3834 well, clear, flat-bottom microtiter plate at 37°C for various time periods from 1 hr to -overnight. This “end point” assay provides a qualitative instead of quantitative method teo assess substrate specificity. However, the process can be adapted to provide qualitative data by determining initial rates for the : various small peptide substrates,
Unit definition kinetic assay
The following assay was devel<oped to determine protease unit activity using pNA linked small peptide substrates. Thais assay allows for the direct comparison of enzymes of the invention to Subtilisin on a un-it per unit basis, Free pNA was used to create a standard curve to allow conversion of” pNA absorbance (A405nm) to moles of pNA, allowing direct quantification of the amount of PNA released by a protease (Figure ( 6.
Subtilisin A activity (initial rate) on AAPF-pNA was measured over a 160 fold concentration range of enzyme (0.1 to 10 U/m_L in assay, based on Sigma’s supplied activity). The activity of Subtilisin A was linear with enzyme concentration over this range and allowed the determination of equivalent units of enzymes of the invention over a broad activity range. A Subtilisin A standarci curve is shown in Figure 7.
DH Assay
The following assay was developed using Subtilisin A to determine the relative activity of proteases at various pH’s. Wour different buffers were identified that would permit the testing of a range of different pH’s. Protease activity was assayed using the small peptide substrate p-nitroanalide linke=d Alanine-Alanine-Proline-Phenylalanine (AAPF-pNA, Sigma, S-7388) as follows: The amount of Subtilisin A. required to obtain an initial rate using the assay conditions was determined at the desired pH (5mM AAPF-
PNA, 37°C). Reactions were performed in tripmlicate. Initial rates were determined and averaged. The percent activity at various pH’ss were determined relative to the sample with the highest activity, and percent relative activity was then plotted vs. pH. Substrate stability at the pH’s tested was verified in the mmbsence of activity. Results are illustrated in Table 6 and in Figure 8. [see next page for Table 6]
Table6
Rates (Assam X 10° min™™)
o WO 2004/033668 PCT/US2003/0332819 ’ % % Relative pH ~~ Buffer 1 2 3 Ave. StdDev Deviation Activa 5.0 [Malic Acid | 3.78] 380] 362] 3.71] 0.00 | 25 | 10.09 6.0 | Malic Acid 13.5 613.24 12.23[13.01] 069 | 532 | 3538 : | 55] MBS [510] 482[5.19] 500] 026 [ 51 | 13.61 60] MES | 11.8 I[11.53[ 11.18 ILS 032 | 275 | 31.3 65] MES [20.43 194812049(20.14 0.57 | 2.85 | 34.76 70 | MES [27.54]27.51] 27.03 27.36] 028 | 1.03 | 7441
MOPS | 19.68]19322020{ 19.73] 044 | 224 | 53.66
MOPS 29.6529.50] 0.55 | 1.37 | 80.23
MOPS 32.65/33.64] 0.86 | 255 | 91.47 8.0 | MOPS [36.76 37.19 36.37 136.77] 041 | 112 | io [8.0 | Boric Acid] 34.55] 32.97 3410133.87] 081 | 239 | 62.132 8.5 | Boric Acid] 35.39( 32.01] 35.41] 3427] 196 | 572 93.19 9.0 [Boric Acid] 34.85B3.99 33.45
A number -of embodiments of the invention have been described.
Nevertheless, it will be uraderstood that various modifications may be made withovat § departing from the spirit sand scope of the invention. Accordingly, other embodiments are within the scope of the foRlowing claims.

Claims (1)

  1. WHAT IS CLAIMED IS: :
    1. An isolated or recombinant nucleic acid comprising a nucleic acid sequence having at least 50% s-equence identity to SEQ ID NO:1 ; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID 1NO:9; SEQ ID NO: 11; SEQ ID NO:13; SEQ ID NO:15; SEQID NO:17; SEQ ID NO:1%9; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SE-Q ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQID : NO:37; SEQ ID NO:39; SEQ I'D NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQID NO:47; SEQ ID NO:49; SEQ I'D NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ I'D NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ I'D NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ I'D NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID i NO:87; SEQ ID NO:89; SEQ ITD NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ I'D 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; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEEQ ID NO:254, over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having a protease activity, and the sequence ident ities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
    2. The isolated or recombinant nucleic acid of claim 1, wherein the sequence identity is at least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63% or 64%.
    3. The isolated or recombinant nucleic acid of claim 2, wherein the sequence identity is at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 15%, 16%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94°24, 95%, 96%, 97%, 98%, 99%, or more, or is 100%.
    4, The isolated or recombinant nucleic acid of claim 1, wherein the Sequence identity is over a region of at least about 50, 75, 100, 150, 200, 250 _, 300, 350, 400, 450, 500, 55€0, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 11003, 1150 or oo more residues, or the full length of a gene or a transcript.
    5. The isolated or recombinant nucleic acid of claim 1, whherein the nucleic acid seque=nce comprises a sequence as set forth in SEQ ID NO:1; SE-&D NO:3; SEQ ID NO:5; SE=Q ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:1 3; SEQID NO:15; SEQ ID NTO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEEQ ID NO:25; SEQ ID N 0:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEEQID NO:35; SEQ ID N 0:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEEQID NO:45; SEQ ID N" 0:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEEQID } NO:55; SEQ ID N 0:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEEQID : NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEEQID NO:75; SEQ ID N«O:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SE-QID NO:85; SEQ ID N«O:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SE-QID NO:95; SEQ ID N€O:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQID NO:105; SEQ ID M0:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:1 M3; SEQ ID NO:115; SEQ ID NNO0:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO: 123; SEQ ID NO:125; SEQ ID N0:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO: 133; SEQ ID NO:135; SEQ ID NJ0:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO: 143; SEQ ID NO:145; SEQ ID NJ0:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO: 1654; SEQ ID NO:171; SEQ ID NJ0:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:19#9; SEQ ID NO:204; SEQ ID NI0:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:22.9; SEQ ID NO:234; SEQ ID NT0:241; SEQ ID NO:248 or SEQ ID NO:254. :
    6. The isolated or recombinant nucleic acid of claim 1, whesrein the nucleic acid sequence encodes a polypeptide having a sequence as set forth in SEQID NO:2; SEQ ID NO: 4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID INO: 12; SEQID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO-:22; SEQ ID NO:24; SEQ ID 1INO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQID NO:34; SEQ ID NO» :36; SEQ ID NO:3 8; 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 IDINO:72; SEQID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID INO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID INO:92; SEQID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SE» ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ» ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ» ID NO:142; SEQ ID NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ» ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ» ID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ» ID NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or the polypeptide encoded Bby SEQ ID NO: 145.
    7. The isolated or recombinant nucleic acid of cBaim 1, 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 op®&ions are set to : default.
    8. The isolated or recombinant nucleic acid of claim 1, wherein the protease activity comprises catalyzing hydrolysis of peptide bonds.
    9. The isolated or recombinant nucleic acid of cL aim 8, wherein the protease zxctivity comprises an endoprotease activity or an exoprotea se activity.
    10. The isolated or recombinant nucleic acid of cl aim 8, wherein the protease a_ctivity comprises a proteinase activity or a peptidase activi ty.
    11. The isolated or recombinant nucleic acid of claim 10, wherein the peptidase activity comprises a carboxypeptidase activity.
    12. The isolated or recombinant nucleic acid of cl=aim 10, wherein the peptidase =mctivity comprises an aminopeptidase activity.
    : o WO 2004/033668 PCT/US2003,032819 Co
    13. The isolated or recombinant nucleic acid of claim 1, wherezin the protease activity come prises a serine protease activity.
    14. The isolated or recombinant nucleic acid of claim 1, wherein the protease activity com prises a metalloprotease activity, a matrix metalloprotease activity or a collagenase activ ity. : : 15. The isolated or recombinant nucleic acid of claim 1, where=in the protease activity comprises a cysteine protease activity.
    16. The isolated or recombinant nucleic acid of claim 1, wheredn the protease activity comprises an aspartic protease activity.
    17. The isolated or recombinant nucleic acid of claim 1, wherein the protease activity comprises a chymotrypsin, a trypsin, an elastase, a kallikrein or & subtilisin activity.
    18. ‘The isolated or recombinant nucleic acid of claim 1, wherei nthe protease activity comprises a peptidase activity.
    19. “The isolated or recombinant nucleic acid of claim 18, where=in the peptidase activity comprises a dipeptidylpeptidase activity.
    20. “The isolated or recombinant nucleic acid of claim 1, whereir the protease activity is thermostable,
    21. “The isolated or recombinant nucleic acid of claim 20, where3n the polypeptide retains a protease activity under conditions comprising a temperature range of between about 37°C to about 95°C, or between about 55°C to about 85°C, or between about 70°C to about 75°C, or between about 70°C to about 95°C, or between about *90°C to about 95°C. }
    22. T he isolated or recombinant nucleic acid of claim 1, wherein _ the protease activity is therrmotolerant. :
    23. The isolated or recombinant nucleic acid Of claim 22, wherein the polypeptide retains a protease activity after exposure to a temperature in the range from greater than 37°C to about 95°C, from greater than 55°C to about: 85°C, or between about 70°C to about 75°C, or from greater than 90°C to about 95°C.
    24. Anisolated or recombinant nucleic acid, vwherein the nidleic 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 ; SEQID NO:7; SEQ ID NO:9; SEQ IDNO:!1; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:I 7; SEQ ID NO:19; SEQ ) IDNO: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; SEQID NO:39; SEQ ID NO:431; 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 I'D NO:59; SEQID NO:61; SEQ ID N0:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:#1; 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; SEQ ID NO:89; SEQ ID NO:21; SEQ ID N0O:93; SEQ ID NO:95; SEQ ID NO:97; SEQ I'D NO:99; SEQ ID NO:1 01; SEQ ID NO:103; SEQ ID NO:105; SEQ ID NO:107; S EQ ID NO:109; SEQ ID NO:L 11; SEQ ID NO:113; SEQ ID NO:115; SEQ ID NO:1 17; SEQ ID NO:119; SEQ ID NO:1 21; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127; S EQ ID NO:129; SEQ ID NO:1.31; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; S EQ ID NO:139; SEQID NO:L 41; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO: 146; SEQ ID NO:150; SEQ ID NO:L 58; SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:1 93; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:2222; SEQ ID N0O:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID N«O:254, wherein the nucleic acid encodes a polypeptide having a protease activity.
    25. The isolated or recombinant nucleic acid of claim 24, wherein the nucleic acid is at least about 50, 75, 100, 150, 200, 300, 400, S00», 600, 700, 800, 900, 1000 or more residues in length or the full length of the gene or transcript.
    o WO 2004/033668 PCT/US20034032819
    26. The isolated or recombinant nucleic acid of claim 24, wherein the stringent conditions Anclude a wash step comprising a wash in 0.2X SSC ata tempperature of about 65°C for abeout 15 minutes. :
    27. A nucleic acid probe for identifying a nucleic acid encoding a polypeptide with a protease activity, wherein the probe comprises at least 10 conseecutive bases of a sequence comprising SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5;7SEQID NO:7; SEQ ID NO:9= SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID MO0:17; SEQ IDNO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID N 0:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO: #1; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47: SEQ IID ) NO:49; SEQ ID NO:5 1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID» - NO:59; SEQ ID NO:6 1; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID» NO:69; SEQ ID NO:7 1; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:8 1; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:9 1; 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:10s; SEQ ID NO:107; SEQID NO:109; SEQ IDNO: R11; SEQ ID NO:113; SEQ ID NO:1 15; SEQ ID NO:117; SEEQ ID NO:119; SEQ ID NO: L 21; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127; SEQID NO:129; SEQ ID NO:1 31; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQID NO:139; SEQ ID NO:1 41; SEQ ID NO:143; SEQ ID NO: 145; SEQ ID NO:146; SEZQ ID NO:150; SEQ ID NO:1 58; SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SE-QID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SE QID NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SE«QID NO:248 or SEQ ID NO=254, wherein the probe identifies the nucleic acid by bindings or hybridization.
    28. THe nucleic acid probe of claim 27, wherein the probe comprisses an oligonucleotide comp rising at least about 10 to 50, about 20 to 60, about 30 to 70, 3¢ about 40 to 80, about 60 to 100, or about 50 to 150 consecutive bases,
    29. A mucleic acid probe for identifying a nucleic acid encoding a polypeptide having a protease activity, wherein the probe comprises a nucleic acid comprising at least about 10 consecutive residues of SEQ ID NO:1; SEQ ID NO:3; SEZQ
    ID "NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:1 1; 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:3 1; SEQ IDNO: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:5 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; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQID 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; SEQ ID NO:89; SEQ ID NO:91; 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; SEQIDNO: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; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO :135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO :145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID NO :171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO :204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NO :234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID NO:254, wherein the sequence idertities are determined by analysis with a sequence comparison algorithm or by visual inspoection.
    30. The nucleic acid probe of claim 29, wherein the probe comprises an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, about 60 to 100, or about 50 to 150 consecutive bases.
    31. An amplification primer pair for amplifying a nucleic acid encding a polypeptide having a protease activity, wherein the [orimer pair is capable of amplifying a nucleic acid comprising a sequence as set forth in <laim 1 or claim 24,0ra subssequence thereof.
    32. The amplification primer pair of claim 31 , wherein a member of the zmmoplification primer sequence pair comprises an oligonucleotide comprising at least abouat 10 to 50 consecutive bases of the sequence, or, about 12, 13, 14, 15, 16, 17, 18,19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive ba ses of the sequence.
    ® WO 2004/033668 } PCT/US2003/032819
    33. An amplification primer pair, 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, oo 17,18, 19,20, 21, 222, 23, 24, 25, 26, 27,28, 29, 30 or more residues -of SEQ ID NO:1; SEQIDNO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ IID NO:11; SEQ ID NO:13; SEQ ID NO: 15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NCD:21; SEQ ID NO:23; SEQ ID NO: 25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID N(D:31; SEG ID NO:33; SEQ ID NO: 35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID N(O:41; SEQ ID NO:43; SEQ ID NO: 45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID N(D:51; SEQ ID NO:53; SEQ ID NO: S5; SEQ ID NO:57; SEQ ID NO:59; SEQ IDNO:61; SEQ ID NO:63; SEQ ID NO: «65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:~75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID N(CD:81; SEQ ID } NO:83; SEQ ID NO: 35; SEQ ID NO:87; SEQ ID NO:89; SEQ ID N(:91; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID N(O:101; SEQ ID NO:103; SEQ ID NO»:105; SEQ ID NO:107; SEQ ID NO:109; SEQ IRD NO:111; SEQ ID NO:113; SEQ ID NO»:115; SEQ ID NO:117; SEQ ID NO:119; SEQ IED NO:121; SEQ ID ~ NO:123; SEQ ID NO-:125; SEQ ID NO:127; SEQ ID NO:129; SEQ IED NO:131; SEQ ID NO:133; SEQ ID NO :135; SEQ ID NO:137; SEQ ID NO:139; SEQ [ED NO:141; SEQ ID NO:143; SEQ ID NO :145; SEQ ID NO: 146; SEQ ID NO:150; SEQ IND NO:158; SEQ ID NO:164; SEQ ID NO :171; SEQ ID NO:179; SEQ ID NO:187; SEQ [ED NO:193; SEQID NO:199; SEQ ID NO :204; SEQ ID NO:210; SEQ ID NO:218; SEQ IID NO:222; SEQ ID NO:229; SEQ ID NO =234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID NO:254, and a second member havin_g a sequence as set forth by about the first (the 57°)12, 13, 14, 15, 16,17, 18, 19, 20,21, 22,23, 24,25, 26, 27, 28, 29, 30 or more residuces of the complementary strand of the first member.
    34. A protease-encoding nucleic acid generated by amplification of a polynucleotide using zan amplification primer pair as set forth in claim 33.
    35. The protease-encoding nucleic acid of claim 34, wherein the amplification is by polymerase chain reaction (PCR).
    36. The protease-encoding nucleic acid of claim 34_, wherein the nucleic acid generated: by amplification of a gene library.
    ’ 377. The protease-encoding nucleic acid of claim 34, wherein the gene library is an environmental library.
    33. Anisolated or recombinant protease encoaded by a protease- encoding nucleic= acid as set forth in claim 34.
    3%. Amethod of amplifying a nucleic acid encoding a polypeptide having a proteasee activity comprising amplification of a template nucleic acid with an amplification pri_mer sequence pair capable of amplifying a nucleic acid sequence as set forth in claim 1 ©r claim 24, or a subsequence thereof, . 4. An expression cassette comprising a nucle=i¢ acid comprising a - sequence as set f-orth in claim 1 or claim 24.
    471. A vector comprising a nucleic acid compr-ising a sequence as set forth in claim | or claim 24.
    42. A cloning vehicle comprising a nucleic ac-id comprising a sequence asset forth in claim 1 or claim 24, wherein the cloning vehicle ceomprises a viral vector, a plasmid, a phage=, a phagemid, a cosmid, a fosmid, a bacteriopha_ge or an artificial chromosome.
    43. The cloning vehicle of claim 42, wherein the viral vector comprises 26 an adenovirus ve=ctor, a retroviral vector or an adeno-associated wiral vector.
    41. The cloning vehicle of claim 42, comprisi mg a bacterial artificial chromosome (BAAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromo =some (YAC), or a mammalian artificial chromo some (MAC).
    45. A transformed cell comprising a nucleic a-cid comprising a sequence as set feorth in claim 1 or claim 24. :
    PY PCT/US2003/032819
    46. A transformed cell comprising an expression cassette as set forth in claim 40. 47 The trarzesformed cell of claim 40, wherein the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
    48. A transgzenic non-human animal comprising a sequence as set forth in claim or claim 24 _
    49. The trarx sgenic non-human animal of claim 48, wherein the animal is a mouse.
    50. A transg=enic plant comprising a sequence as set forth in claim 1 or claim 24.
    Sl. The transgenic plant of claim 50, wherein the plant is a corn plant, a sorghum plant, a potaxto plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a s oybean plant, a rice plant, a barley plant, a grass, or a tobacco plant.
    52. A transgsenic seed comprising a sequence as set forth in claim 1 or claim 24.
    53. The tran sgenic seed of claim 52, wherein the seed is a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a rice, a barley, a peanut or a tobacco plant seed.
    54. An antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a sequence as set forth in claim 1 or claim 24, or a subsequence thereof.
    55. The antisense oligonucleotide of claim 54, wherein the antisense oligonucleotide is between about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 bases in length.
    56. A metho-d of inhibiting the translation of a protease message in a cell comprising admi nistering to the cell or expressing in the cell an 173 AMENIDDED SHEET
    A\ P PCT/US2003/032819 antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under strin. gent conditions to a sequence as set forth in claim 1 or claim 24.
    57. A double-stranded inhibitory RNA (RNAi) molecule comprising a subsequence of a sequ ence as set forth in claim 1 or claim 24.
    58. The double-stranded inhibitory RNA (RNAi) molecule of claim 57, wherein the RNAI is about 15, 16,17, 18, 19,20, 21, 22,23, 24,25 or more duplex nucleotides in length.
    59. A method of inhibiting the expression of a protease ina cell comprising administering to th_e cell or expressing in the cell a double- stranded inhibitory RNA (iRNA), «wherein the RNA comprises a subsequence ofa sequence as set forth in claim 1 or claim 24.
    60. An isolated or recombinant polypeptide (i) having at least S0% sequence identity to SEQ ID NO:2; SEQ ID NO:4; SEQ 1D 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 XD 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 NO0:40; 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 RE 25 NO:64; SEQ ID NO:66; SEQ ID NO:68: SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92: SEQ ID NO:94; SEQ 1D NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO: 102; SEQ ID NO:104; SEEQ ID NO: 106; SEQ ID NO:108; SEQ ID NO:110: SEQ ID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO: 122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128: SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:1 40: SEQ ID NO:142; SEQ ID NO: 144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO: 1.88; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or the polypeptide encoded by SEQ ID NO: 145, 174 AMENDED SHEET
    PCT/US2003/032819 over a region of at least about 100 residues, where=in the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection, or, (ii) encoded by a nucleic acid havirag at least 50% sequence identity to a sequence as set forth in SEQ ID NO: E ; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEEQ 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 [D NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ [D NO:39; SEQ 1D 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 ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ 1D NO:69; SEQ ID NO:71; 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; SEQ ID NO:39; SEQ ID NO:91; 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: 1405; 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 NQO:121; SEQ ID NO:1223; SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO: 1-41; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:1 87; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID N0O:229; SEQ ID NO:234; SEQ ID NO:2-41; SEQ ID NO:248 or SEQ ID NO:254, over a region of at least about 100 residue=s, and the sequence identities are determined by analysis with a sequence compa.rison algorithm or by a visual inspection, or encoded by a nucleic acid capable of hybridizing under stringent conditions to a sequence as set forth in SEQ ID N(O:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO=z 11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID INO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ XID NO:3!; 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 INO:49; SEQ ID NO:51; SEQ [ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ XD NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NQ:73; SEQ ID NO:75; SEQ ID INO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ XD NO:87; SEQ ID NO:89; SEQ ID NO:91; 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 IID NO: 105; SEQ ID NO:107; 175 AMENDED SHEET
    PC T/US2003/032819 ® SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID IMNO: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; SEQ ID NO:129; SEQ ID NO:131; SEQ ID ™MNO0:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO: 146; SEQ ID NO:130; SEQ ID INO:158; SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID MNO0:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID NO:254.
    61. The isolated or recombinant polypeptide of claim 60, wherein the sequence identity is over a region of at least about 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%, 17%, 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 is 100% sequence identity.
    62. The isolated or recombinant polypeptide of claim 60, wherein the sequence identity is over a region of at least about 10, 15, 20, 25, 30, 35,40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more residues, or tke full length of an enzyme.
    63. The isolated or recombinant polypeptide of claim 60, wherein the polypeptide has a sequence as set forth in SEQ ID INO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID INO: 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; SEEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:<10; 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; SEEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SI=Q ID NO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO: 100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO: 106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQ ID NJO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; 176 AMENDED SHEET
    PCT/US20003/032819 SEQ ID NO:12«6; SEQ ID NO:128; SEQ ID NQO:130; SEQ ID NO:1323 SEQ ID NO:134; SEQ I' DNO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID» NO:142; SEQ ID NO:14=4; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159z SEQ ID NO:165; SEQ I'D NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID» NO:194; SEQ ID NO:20€; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219z SEQ ID NO:223; SEQ I'D NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQ ID» NO:249 or SEQ ID NO: 255, or the polypeptide encoded by SEQ ID NO: 145.
    64. The isolated or recombinant polypeptide of cRaim 60, wherein the polypeptide has a protease activity.
    65. The isolated or recombinant polypeptide of cRaim 64, wherein the pro tease activity comprises catalyzing hydrolysis of peptid e bonds.
    66. The isolated or recombinant polypeptide of cRaim 63, wherein the pro-tease activity comprises an endoprotease activity or an exoprotease activity.
    67. The isolated or recombinant polypeptide of cRaim 65, wherein the pro tease activity comprises a proteinase activity or a pepticlase activity.
    68. The isolated or recombinant polypeptide of cRaim 67, wherein the peptidase activity comprises a carboxypeptidase activity.
    69. The isolated or recombinant polypeptide of cRaim 67, wherein the peptidase activity comprises an aminopeptidase activity.
    70. The isolated or recombinant polypeptide of claim 64, wherein the pro tease activity comprises a serine proteinase activity.
    71. The isolated or recombinant polypeptide of cRaim 64, wherein the pro tease activity comprises a metalloproteinase activity, a mnatrix metalloproteina se activity or a collagenase activity.
    72. The isolated or recombinant polypeptide of cEaim 64, wherein the pro tease activity comprises a cysteine protease activity. 177 AMENDED SHEET
    PCT/US2003/032819
    73. The isolated or recombinant polypeptide of claim 64-, wherein the protease activity comprises an aspartic protease activity.
    74. The isolated or recombinant polypeptide of claim 64, wherein the protease acctivity comprises a chymotrypsin, a trypsin, an elastase,. a kallikrein or a subtilisimn activity.
    75. The isolated or recombinant polypeptide of claim 64-, wherein the protease acctivity comprises a peptidase activity.
    76. The isolated or recombinant polypeptide of claim 64-, wherein the peptidase activity comprises a dipeptidylpeptidase activity.
    77. The isolated or recombinant polypeptide of claim 64-, wherein the protease activity is thermostable.
    78. The isolated or recombinant polypeptide of claim 77, wherein the polypeptide retains a protease activity under conditions comprisin_ga temperature range of b=etween about 1°C to about 5°C, between about 5°C to atoout 15°C, between about 1 =5°C to about 25°C, between about 25°C to about 37°C, between about 37°C to about 95°C, between about 55°C to about 85°C, betwee=n about 70°C to about 95 °C, between about 70°C to about 75°C, or between about 90°C to about 95°C.
    79. The isolated or recombinant polypeptide of claim 64-, wherein the protease activity is thermotolerant.
    80. The isolated or recombinant polypeptide of claim 79 , wherein the polypeptid_e retains a protease activity after exposure to a temperature in the range from between about 1°C to about 5°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°C to about 95°C, between about 55°C to about 85°C, betwee=n about 70°C to about 75 °C, or between about 90°C to about 935°C, or more.
    81. An isolated or recombinant polypeptide comprising = polypeptide as set forth in claim 60 and lacking a signal sequence or a prepro sequence. 178 AMENDED SHEET
    PCT/US2003/03281 9 ® 82. An is olated or recombinant polypeptide comprising a polypeptide as set forth in clairm 60 and having a heterologous signal sequence or a heterologous prepro sequence.
    83. The 1ssolated or recombinant polypeptide of claim 64, wherein the protease activity comprises a specific activity at about 37°C in the range from about 100 to about 1000 units per milligram of protein, from about 500 to about 750 units per mill& gram of protein, from about 500 to about 1200 units per milligram of protein, er from about 750 to about 1000 units per milligram of protein.
    84. The isolated or recombinant polypeptide of claim 79, wherein the thermotolerance comprises retention of at least half of the specific activity of the protease at 37°C after being heated to an elevated temperature.
    85. The isolated or recombinant polypeptide of claim 79, wherein the thermotolerance comprises retention of specific activity at 37°C in thee range from about 500 to about 1200 units per milligram of protein after being heated to an elevated temperatiare.
    86. The isolated or recombinant polypeptide of claim 60, wherein the polypeptide comprises at least one glycosylation site.
    87. The isolated or recombinant polypeptide of claim 86, wherein the glycosylation is an N-linked glycosylation.
    88. The isolated or recombinant polypeptide of claim 87, wherein the polypeptide is glycosylated after being expressed in a P. pastoris or a
    S. pombe.
    89. The isolated or recombinant polypeptide of claim 64, wherein the polypeptide retains a protease activity under conditions comprising about pH 6.5, pH 6.0, pH 5.5, 5.0, pH 4.5 or 4.0.
    90. The isolated or recombinant polypeptide of claim 64, wherein the polypeptide retains a protease activity under conditions comprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5. 179 AMENDED SHEET
    PCT/US2003/032819
    91. A protein preparation comprising a polypeptide as set forth in claim 60, wherein the proteix preparation comprises a liquid, a solid ora gel.
    92. A heterodimmer comprising a polypeptide as set forth in claim 60 and a second domain.
    93. The heterodimer of claim 92, wherein the second domain is a polypeptide and the heterodimer is a fusion protein. 94, The heteroadimer of claim 92, wherein the second domain is an epitope or a tag.
    05. A homodirmer comprising a polypeptide as set forth in claim 60.
    96. An immobilized polypeptide, wherein the polypeptide comprises a sequence as set forth in claim 60, or a subsequence thereof.
    97. The immotoilized polypeptide of claim 96, wherein the polypeptide is immobilized on a cell a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic p article, a bead, a gel, a plate, an array or a capillary tube.
    98. An array comprising an immobilized polypeptide as set forth in claim 60.
    99. An array comprising an immobilized nucleic acid as set forth in claim 1 or claim 24.
    100. An isolated or recombinant antibody that specifically binds to a polypeptide as set forth in claim 60.
    101. The isolated or recombinant antibody of claim 100, wherein the antibody is a monoclonal] or a polyclonal antibody.
    102. A hybridoma comprising an antibody that specifically binds to a polypeptide as set forth in -claim 60. 1&0 AMENDED SHEET
    @® PCT/US2003/032819
    103. A method of isolati ng or identifying a polypeptide with a protease activity comprising the steps of: (a) providing an antibody as set forth in claim 100; (b) providing a sample comprising p olypeptides; 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 a protease activity.
    104. A method of makimg an anti-protease antibody comprising administering to a non-human an imal a nucleic acid as set forth in claim | or claim 24 or a subsequence thereof” in an amount sufficient to generate a humoral immune response, thereby making a n anti-protease antibody.
    105. A method of makirag an anti-protease antibody comprising administering to a non-human an imal a polypeptide as set forth in claim 60 or a subsequence thereof in an amowunt sufficient to generate a humoral immune response, thereby making an anti-pretease antibody.
    106. A method of produ cing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid operably, linked to a promoter, wherein the nucleic acid comprises a sequence as set forth in claim 1 or claim 24; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby produ <ing a recombinant polypeptide.
    107. The method of claiam 106, further comprising transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producin ga recombinant polypeptide in a transformed cell.
    108. A method for identifying a polypeptide having a protease activity comprising the following steps: (a) providing a polypeptide as set foxth in claim 64; {b) providing a protease substrate; ard (c) contacting the polypeptide with t he substrate of step (b) and detecting a decrease in the amount of substrate or an irmcrease 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 poly~peptide having a protease activity. 181 AMENDED SHEET
    ® PCT/US2003/032819
    109. A method for identifying a protease substrate comprising the following steps: (a) providing a polypeptide as set forth in claim 64; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with thme test substrate of step (b) amd detecting a decrease in the amount of substrate or aL increase in the amount o freaction product, wherein a decrease in the amount of _ the substrate or an iracrease in the amount of a reaction product identifies thee test substrate as a perotease substrate.
    110. A method of determining whe=ther a test compound specifically binds to a polypeptide comprising the followwing steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid wander conditions permissive for translation of the nucle ic acid to a polypeptide, —wherein the nucleic acid has a sequence as set forth in claim 1 or claim 24; (b) providing a test compound; (c) contacting the polypeptide with the test cornpound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide. :
    111. A method of determining whaether a test compound specifically binds to a polypeptide comprising the foll@owing steps: (a) providing a polypeptide as set forth in claim 60; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound o {step (b) specifically binds 10 the polypeptide.
    112. A method for identifying a modulator of a protease activity comprising the following steps: (a) providing a polypeptide as set forth in claim 64; (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 protease, wherein a change in the protease 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 protease activity. i 182 AMENDED SHEET
    PC I/US2003/032819 ®
    113. The method of claim 112, wherein the protease activity is measured by providing a protease substrate and detecting a d ecrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increas © in the amount of the substrate or a decrease in the amo unt of a reaction product. 114, The method of claim 113, wherein a clecrease 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 resaction product without the test compound identifies the test compound as an activator of protease activity=.
    115. The method of claim 113, wherein an. increase in the amoung 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 resaction product without the test compound identifies the test compound as an irahibitor of protease activity,
    116. A computer system comprising a processor and a data storage= device wherein said data storage device has stored thereon a polypeptide sequensce or a nucleic acid sequence, wherein the polypeptide sequence comprises sequence as set forth in claim 60, a polypeptide encoded by a nucleic acid as set forth ire claim 1 or claim 24.
    117. The computer system of claim 116, fiarther comprising a sequence comparison algorithm and a data storage device havirag at least one referenece sequence stored thereon.
    118. The computer system of claim 117, w~herein the sequenece comparison algorithm comprises a computer program. that indicates polymorphisms.
    119. The computer system of claim 117, fuarther comprising an identifier that identifies one or more features in said sequence.
    120. A computer readable medium having stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide 183 AMENDED SHEET
    PCT/US2003_/032819 ® sequence comprisses a polypeptide as set forth in claim 60; a polypeptide e=ncoded by a nucleic acid as set forth in claim | or claim 24.
    121. A method for identifying a feature in a sequences S comprising the stesps of: (a) reading the sequence using a computer program which identifIes one or more features Ln a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide as sset forth in claim 60; a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; and (b) ident®m fying one or more features in the sequence with the come puter program.
    122. A method for comparing a first sequence to a se cond sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer prograrm which compares sequences, wherein the first sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein thes polypeptide sequesnce comprises a polypeptide as set forth in claim 60 or & polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; and (b) determmining differences between the first sequence and the se cond sequence with the computer program.
    123. The method of claim 122, wherein the step of determining differences between the first sequence and the second sequermce further comprises the step of identifying polymorphisms.
    124. The method of claim 123, further comprising an. identifier that idemtifies one or more features in a sequence.
    125. The method of claim 124, comprising reading tine first sequence using a computer program and identifying one or more features in the sequence.
    126. A method for isolating or recovering a nucleic a_cid encoding a polypeptide with a protease activity from an environmental sample comprising the steps of: 184 AMENDED SHEET
    PCT/US2003/0328 19 o (a) providing ar amplification primer sequence pair as set forth in claim. 31 or claim 33; (b) isolating a reucleic acid from the environmental sample or treating th e environmental sample saich 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 with a protease activity from am environmental sample.
    127. The method of claim 126, wherein each member of the amplification primer sequence pair comprises an oligonucleotide comprising at least about 10 to 50 consecutive bases of 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; SEQ ID NO:13; SEQ IID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23z 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 IID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID NO:51z 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; SEQ ID NO:67; SEQ ID NO:69; SEQ IID NO:71; SEQ ID NO:73; SEQ ID NO: 75; SEQ ID NO:77; SEQ ID NO:79z SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO=89; SEQ ID NO:91; SEQ ID N0:93; 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:1Q7; SEQ ID NO:109; SEQ ID NO:111; SEQID NO:1133 SEQ ID NO:115; SEQ LD NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO:129; SEQID NO:131 3 SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO: 143; SEQ ID NO: 145; SEQ ID NO:146; SEQ ID NO:1503 SEQ ID NO:158; SEQ LD NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ [D NO:199; SEQ ID NO:204; SEQ ID NO:2103 SEQ ID NO:218; SEQ [LD N0:222; SEQ ID N0:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID NQO:254, or a subsequence thereof.
    128. A method for isolating or recovering a nucleic acid encoding a polypeptide -with a protease activity from an environmental sample comprising the steps of: 185 AMENDED SHEET
    PCT/US2003/032819 o (a) providing a polynucleotide probe comprising a sequence as set forth in claim 1 or claim 24, 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 isol ated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (2); and (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (2&2), thereby isolating or recovering a nucleic acid encoding a polypeptide with a protease activity from an environmental sample.
    129. The mmethod of claim 127 or claim 128, wherein the environmental sample comprises a water sample, a liquid sample, a soil sample, an air sample or a biological sample.
    130. The mmethod of claim 129, wherein the biological sample is derived from a bacterial cell , a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mamma lian cell.
    131. A method of generating a variant of a nucleic acid encoding a polypeptide with a protease activity comprising the steps of: (a) providing a template nucleic acid comprising a sequence as set forth in claim 1 or claim 24; and (b) modifying, deletin_g or adding one or more nucleotides in the template sequence, or a combination thesreof, to generate a variant of the template nucleic acid.
    132. The amethod of claim 131, further comprising expressing the variant nucleic acid to generate a variant protease polypeptide.
    133. The amethod of claim 131, wherein the modifications, additions or deletions are intro- duced by a method comprising error-prone PCR, shuffling, oligonucleotide-dire=cted mutagenesis, assembly PCR, sexual PCR mutagenesis, in Vivo mutagenessis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturatecd mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof. 186 AMENDED SHEET
    PCT/US2003/032819 o
    134. The method of claim 131, wherein the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phiosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-cleficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis. deletion mutagenesis, restriction-selection mutagenesis, restriction-purification rmutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
    135. The method of claim 131, wherein the method is iteratively repeated until a protease 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.
    136. The method of claim 135, wherein the variant protease polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature.
    137. The method of claim 135, wherein the variant protease polypeptide has increased glycosylati on as compared to the protease encoded by a template nucleic acid.
    138. The method of claim 135, wherein the variant protease polypeptide has a protease activity urader a high temperature, wherein the protease encoded by the template nucleic acid 1s not active under the high temperature.
    139. The method of claim 131, wherein the method is iteratively repeated until a protease coding sequence having an altered codon usage from that of the template nucleic acid is produced.
    140. The method of claim 131, wherein the method 1s iteratively repeated until a protease gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.
    141. A method for modifying codons in a nucleic acid encoding a polypeptide with a protease activity to increase its expression in a host cell, the method comprising the following steps: 187 AMENDED SHEET
    PCT/US2003/032819 ® (a) providing a nucleic acid encodling a polypeptide with a protease activity comprising a sequence as set forth. in claim 1 or claim 24; 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 codorm, 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-reepresented in coding sequences in genes in the host cell, thereby modifying the nuc leic acid to increase its expression in a host cell.
    142. A method for modifying codons in a nucleic acid encoding a protease polypeptide, the methe«od comprising the following steps: (a) providing a nucleic acid encodling a polypeptide with a protease activity comprising a sequence as set forth. in claim | or claim 24; and, (b) identifying a codon in the nucBeic 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 encodi ng a protease.
    143. A method for modifying codons in a nucleic acid encoding a protease polypeptide to increas-e its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid encoding a protease polypeptide comprising a sequence as set forth in claim | or claim 24; 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-rezpresented in coding sequences in genes in the host cell, thereby modifying the nucJeic acid to increase its expression in a host cell.
    144. A method for modifying a codon in a nucleic acid encoding a polypeptide having a protease activity to decrease its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid encod. ing a protease polypeptide comprising a sequence as set forth in claim | or claim 24; 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 188 AMENDED SHIEET
    PCT/US2003/032819 ® amino acid as the replaced codon, wherein a preferred codon is a codon over- represented in coding sequences in genes in a ost 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.
    145. The method of claim 144, wherein the host cell isa bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.
    146. A method for produc ing a library of nucleic acids encoding a plurality of modified protease activ-e 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 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:4!; 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 ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO: 31; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; 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 N€:109; SEQ ID NO:111; SEQ ID NO:113; SEQID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO: 121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID N©:127; SEQ ID NO: 129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO: 137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID N&O: 145; SEQ ID NO:146; SEQ ID NO: 150; SEQ ID NO:158; SEQ ID NO:164; S EQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID N€D:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; S EQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID INO:254, or a subsequence thereof, 189 AMENDED SHEET
    PCT/US2003/032819 ® and the nucleic acid encodes a protease active site or a protease 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 set of mutagenic oligonucleotides tc generate a set of active site-encoding or substrate binding site-encoding variant n ‘ucleic acids encoding a range of amino acid variations at each amino acid codon t-hat was mutagenized, thereby producing a library of nucleic acids encoding a pl urality of modified protease active sites or substrate binding sites.
    147. The method of claim 145, comperising mutagenizing the first nucleic acid of step (a) by a method comprising an opotimized directed evolution system, gene site-saturation mutagenesis (GSSMM), or a synthetic ligation reassembly (SLR).
    148. The method of claim 145, comprising mutagenizing the first nucleic acid of step (a) or variants by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembls/ PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), sym thetic ligation reassembly (SLR) and a combination thereof.
    149. The method of claim 145, comperising mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modifaed DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex m_utagenesis, point mismatch repair mutagenesis, repair-deficient host strain amutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenes-is, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer cresation and a combination thereof.
    150. A method for making a small m olecule comprising the following steps: (a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymx es comprises a protease 190 AMENDED SHEET
    PS PCT/US2003/032819 enzyme encoded by a nucleic acid comprising a sequence as set forth in claim or claim 24; (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 smal 1 molecule by a series ©f biocatalytic reactions.
    151. A method for modifying a small molecules comprising the follo wing steps: (a) providing a protease enzyme, wherein the enzyme comporises a polypeptide as set forth in claim 64, or a polypeptide encoded by a mucleic acid comprising a nucleic acid sequence as set forth in claim 1 or claim 24; (b) providing a small molecule; and (c) reacting the enzyme of step (a) with the small molecules of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the protease enzyme, thereby modifying a small molecule by a protease enzymatic reaction.
    152. The method of claim 151, comprising 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 catalyze d by the protease enzyme.
    153. The method of claim 151, further comprising 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.
    154. The method of claim 153, further comprising the step of testing the library to determine if a particular modified small molec ule which exhibits a desired activity is present within the library.
    155. The method of claim 154, wherein the step of testing the library further comprises 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 biocatal ytic reaction that procduces the particular modified small molecule of desired act@ vity. 191 AMENDED SHEET
    PCT/US2003/032819 @
    156. A method for determining a functional fragment o=fa : protease enzym € comprising the steps of: (a) pro=iding a protease enzyme, wherein the enzyme comprises a polypeptide as sset forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in clairm | or claim 24; and (b) deleting a plurality of amino acid residues from the sequence of step (a) and testing t he remaining subsequence for a protease activity, thereby determining a feanctional fragment of a protease enzyme.
    157. The method of claim 156, wherein the protease activity 1s measured by providing a protease substrate and detecting a decrease in th_e amount of the substrate or an increase in the amount of a reaction product.
    158. A method for whole cell engineering of new or me=odified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps = (a) malxing a modified cell by modifying the genetic composition o=fa cell, wherein th e genetic composition is modified by addition to the cell of &a nucleic acid cormnprising a sequence as set forth in claim 1 or claim 24; (b) cul€uring the modified cell to generate a plurality of modified ceells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) ana_lyzing the data of step (c) to determine if the measured parammneter differs from a c omparable measurement in an unmodified cell under similar conditions, ther-eby identifying an engineered phenotype in the cell using re-al- time metabolic flux analysis.
    159. The method of claim 158, wherein the genetic composition of the cell is modified by a method comprising deletion of a sequence or momdification of a sequence in the cell, or, knocking out the expression of a gene.
    160. The method of claim 158, further comprising sele=cting a cell comprising a newly engineered phenotype. 192 AMENDED SHEET
    PY PCT/US2003/032819
    161. The method of claim 160, further comprising culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.
    162. An isolated or recombinant signal sequence consisting of (i) a sequence as set forth in residues 1 to 17,1 to 18, 1to 19,1 t0 20, 1 to 21, 1 t022,1t023,1t024, 1 to 25,1t026,[t027,1t028,11t028, 1t030,1t031,1 t0 32,1 t033,11034, 1 to 35,1t036,1t037,1to0380r! to390of 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:2 4; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID INO: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:5 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; SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:8 0; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO: 100; SEQ ID NO:102; SEQ ID NO: 104; SEQ ID NO: 106; SEQ IID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO: 124; SEQ IID NO: 126; SEQ ID NO: 128; SEQ ID NO: 130; SEQ ID NO:132; SEQ ID NO: 134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO: 142; SEQ IID NO: 144; SEQ ID NO: 147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO: 188; SEQ ID NO: 194; SEQ IID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NQO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or the polypeptide encoded by SEQ ID NO: 145, or, (ii) a signal sequence consisting of a sequence as set forth in Table 4.
    163. A chimeric polypeptide comprising at least a first domain comprising signal peptide (SP) having a sequence as set forth in claim 162, and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP).
    164. The chimeric polypeptide of claim 163, wherein the heterologous polypeptide or peptide is not a protease. 193 AMENDED SHEET
    PCT/US2003/032819 o
    165. The chimeric polypeptide of claim 163, wherein the heterologous polypeptide or peptide is amino terminal to, carboxy terminal to or on both ends of the signal peptide € SP) or a protease catalytic domain (CD).
    166. An isolated or recombinant nucleic acid encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises at least a first domain comprising signal peptide (SP) having a sequence as set forth in claim 162 and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide CSP).
    167. A method of increasing thermotolerance or thermostability of a protease polypmeptide, the method comprising glycosylating a protease, wherein the polypeptide «comprises at least thirty contiguous amino acids of a polypeptide as set forth in clai m 60, or a polypeptide encoded by a nucleic acid as set forth in claim I or claim 24, thereby increasing the thermotolerance or thermostability of the protease.
    168. A method for overexpressing a recombinant protease in a cell comprising expressing a vector comprising a nucleic acid sequence as set forth in claim 1 or claim 24, wheresin overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.
    169. A method of making a transgenic plant comprising the following steps: (a) introducing a heterologzous nucleic acid sequence into the cell, wherein the heterologous nucleic s-equence comprises a sequence as set forth in claim 1 or claim 24, thereby produ cing a transformed plant cell; (b) producing a transgenics plant from the transformed cell.
    170. The method as set forth in claim 169, wherein the step (a) further comprises introducing the heterologous nucleic acid sequence by electroporation or microinjection cf plant cell protoplasts.
    171. The metkod as set forth in claim 169, wherein the step (a) comprises introducing the heter-ologous nucleic acid sequence directly to plant 194 AMENIDED SHEET
    PCT/US2003/032819 ® tissue by DNA particle bombardment or by using an Agrobacterium tumefaciens host.
    172. A method of expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps: (a) transforming the plant cell w-ith a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a sequence as set forth in clairm | or claim 24; (b) growing the plant under conditions wherein the heterologous nucleic acids sequence is expressed in the plant cell.
    173. A method for hh ydrolyzing, breaking up or disrupting a protein-comprising composition compris ing the following steps: . (a) providing a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; (b) providing a composition comprising a protein; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the protease hydrolyzes, breaks up or disrupts the protein-comprising composition.
    174. The method as set forth in claim 173, wherein the composition comprises a plant cell, a bacterial cell, a yeast cell, an insect cell, or an animal cell.
    175. A method for liquefying or removing a protein from a composition comprising the following steps: (a) providing a polypeptide havimg a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; (b) providing a composition comprising a protein; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the protease rexmoves or liquefies the protein.
    176. A detergent cormposition comprising a polypeptide as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim [ or claim 24, wherein the polypeptide has a protease activity.
    177. The detergent composition of claim 176, wherein the . protease is a nonsurface-active protease or a surface-active protease. 195 AMENDED SHEET
    PCT/US2003/032819 _
    178. The detergent composi tion of claim 176, wherein the protease is formulated in a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel form, a paste or a slurry form.
    179. A method for washing an object comprising the following steps: (a) providing a composition comprising a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; (b) providing an object; and (c) contacting the polypeptide of step (a2) and the object of step (b) under conditions wherein the composition can wash the object. 18 180. A textile or fabric comprising a polypeptide as set forth in claim 64, or a polypeptide encoded by a nucledc acid as set forth in claim 1 or claim 24.
    181. The textile or fabric of claim 180, wherein the textile or fabric comprises a cellulose-containing fiber.
    182. A method for removing protein stains from a composition comprising the following steps: (a) providing a composition comprising a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; (b) providing a composition having a protein stain; and (c) contacting the polypeptide of step (a)» and the composition of step (b) under conditions wherein the protease can remov-e the stain.
    183. A method for improving the finish of a fabric comprising the following steps: (a) providing a composition comprising a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; (b) providing a fabric; and 196 AMENDED SHEET
    PCT/US2003/032819 (c) contacting the polypeptide of step (a) and the fabric of step (b) under co nditions wherein the polypeptide can treat the fabric ther eby improving the firaish of the fabric.
    184. The method as set forth in claim 183, wherein the fabric is a wool or a silk.
    185. A feed or a food comprising a polypeptide as set forth in claaim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24.
    186. A method for hydrolyzing proteirs in a feed or a food prior to consumption by an animal comprising the followin g steps: (a) obtaining a feed material comprising a protease, wherein the protease has a sequence as set forth in claim 64, or is encoded by a rucleic acid as set forth in claim | or claim 24; and (b) adding the polypeptide of step (a) to the feed oT food material in an ammount sufficient for a sufficient time period to cause hydrolysis of the protein ax d formation of a treated food or feed, thereby hydrolyzing the proteins in the fo «od or the feed prior to consumption by the animal.
    187. The method as set forth in claim 186, wherein the food or the feed is corn.
    188. A method for improving texture and flavor of a dairy product comprising the following steps: (a) providing a polypeptide having a protease activity, wherein the protease has a sequence as set forth in claim 64, or is encoded by a nucleic acid as set forth in claim | or claim 24; (b) providing a dairy product; and {c) contacting the polypeptide of step (a) and the dL airy product of step (b) umder conditions wherein the protease can improve the texture or flavor of the da_iry product.
    189. The method as set forth in claim 188, wherein the dairy product comprises a cheese or a yogurt. 197 AMENDED SHEET
    PCT/US22003/032819 ® 190. A dairy product comprising a protease having a sequen <e as set forth in claim 64, or is encoded by a nucleic acid as s-et forth in claim L or claim 24.
    191. A method for tenderizing a meat or a fish c-omprising the foll owing steps: (a) providing a polypeptide having a protcase activity, wherein the proteasse has a sequence as set forth in claim 64, or is encoded by a nwmicleic acid as set fort hin claim 1 or claim 24; (b) providing a composition comprising meat or fish; and (c) contacting the polypeptide of step (a) and the compositio=n of step (b) under conditions wherein the polypeptide can tenderize the meat or tke fish.
    192. A method improving the extraction of oil frmom an oil- rich plant material comprising the following steps: (a) providing a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; {(b) providing an oil-rich plant material; and (c) contacting the polypeptide of step (a) with the oil-rich plant material under conditions wherein the polypeptide having a protease activity can catalyze the hydrolysis of a peptide bond.
    193. The method of claim 192, wherein the oil-rach plant material comprises an oil-rich seed.
    194. The method of claim 193, wherein the oil iss a soybean oil, an olive oil, a rapeseed (canola) oil or a sunflower oil.
    195. A method for preparing a fruit or vegetable juice, syrup, puree o=r extract comprising the following steps: (a) providing a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; (b) providing a composition or a liquid comprising a fruit or vegetable ‘material; and (c) contacting the polypeptide of step (a) and the compositiom, thereby preparimg the fruit or vegetable juice, syrup, puree or extract. 198 AMENDED SHEET
    PCT/US2003/0328 19
    196. A paper or paper product or paper pulp comprising a protease as set forth in claim 64, or a polypeptide encoded by a nucleic acid as s-et forth in claim 1 or claim 24.
    197. A method for treating a paper or a paper or wood pulp comprising the following steps: (a) providing a polypeptide having a protease activity as set forth in claam 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 243; (b) providing a composition comprising a paper or a paper or wood pulp; and (c) contacting the polypeptide of step (a) and the composition of step (be) under conditiorzs wherein the protease can treat the paper or paper or wood pulp .
    198. A pharmaceutical composition comprising a polypeptide as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth im claim 1 or claim 24.
    199. The pharmaceutical composition of claim 198, whereim the pharmaceutical composition acts as a digestive aid or as a topical skin care.
    200. Use of a polypeptide as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24 in the manufacture of” a preparation for treating an imbalance of desquamation.
    201. A method of preventing an imbalance of desquamatiomn comprising topical application of the composition of claim 199.
    202. An oral care product comprising a polypeptide as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in clairm 1 or claim 24.
    203. The oral care product of claim 202, wherein the product comprises a toothpaste, a dental cream, a gel or a tooth powder, an odontic, a mouth wash, a pre- or post brushing rinse formulation, a chewing gum, a lozengze or a candy. 199 AMENDED SHEET
    PCT/US2003/03281%2 ® 204. A contact lens cleaning composition comprising a polypeptide as seat forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24.
    205. A method for treating solid or liquid animal waste products compri sing the following steps: (a) providing a polypeptide as set forth in claim 64, or a polypeptide encoded by a nu <leic acid as set forth in claim 1 or claim 24; (b) prov=iding a solid or a liquid animal waste; and (c) cont acting the polypeptide of step (a) and the solid or liquid waste of step (b) under conditions wherein the protease can treat the waste.
    206. A processed waste product comprising a polypeptide having a proteas- e activity, wherein the polypeptide comprises a sequence as set forth in claim 648, or a polypeptide encoded by a nucleic acid as set forth in claim or claim 24.
    207. Use of a polypeptide having a protease activity, whercirm the polypeptide «comprises a sequence as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24 in the manufacture off a preparation for use as a hairball remedy.
    208. A hairball prevention composition comprising a polypeptide having a protease activity, wherein the polypeptide comprises a sequence as set Xorth in claim 59, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24.
    209. A blood or organic spot remover comprising a : polypeptide hav-ng a protease activity, wherein the polypeptide comprises a sequence as set Forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24.
    210. A method for disinfecting a solid or a liquid comprising the following steps: (a) providing a composition comprising a polypeptide having a protease activity as set fo-rth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24; (b) providing a solid or a liquid to be disinfected; and 200 AMENDED SHEET
    PCT/US2003/032819 (c) contacting the composition of step (a) and the solid or liquid of step (b) under conditions wherein the protease can disinfect the solid or liquid. 21 1. The method of claim 210, wherein the composition of step (a) is formulated as a spray or a liquid.
    212. An antimicrobial, anti-viral or anti-spore agent comprising a polypeptide having a protease activity, wherein the polypeptide comprises a sequence as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24.
    213. A disinfectant comprising a polypeptide having a protease activity, wherein the polypeptide comprises a sequence as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24.
    214. A method for tissue dissociation comprising the following steps: (a) providing a composition comprising a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24; and (b) contacting the composition of step (a) and with a tissue to be dissociated.
    215. Use ofapolypeptide having a protease activity as set forth in claim 64, ox a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24 in the manufacture of a preparation for the treatment of a wound by tissue dissociation. 21 6. Use of claim 215, wherein said preparation is used for wound cleansing, wound bed preparation, to treat pressure ulcers, leg ulcers, burns, diabetic foot ulcers, scars, [V fixation, surgical wounds or minor wounds.
    217. A medical dressing comprising a polypeptide having a : 35 protease activity, wherein the polypeptide comprises a sequence as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24, 201 AMENDED SHEET
    PCT/US2003/032819 ® 218. Use of an antisense oligonucleotide comprising a nucleic acid sequence complemen tary to or capable of hybridizing under stringent conditions to a sequence as set for-th in claim 1 or claim 24 in the manufacture of a preparation for inhibiting the translation of a protease message in a cell.
    219. Use of & double-stranded inhibitory RNA (RNA), wherein the RNA comprises a subbsequence of a sequence as set forth in claim 1 or claim 24 in the manufacture of a preparation for inhibiting the expression of a protease in a cell.
    220. Use of a nucleic acid as set forth in claim | or claim 24 or a subsequence thereof in the m anufacture of a preparation for generating a humoral immune response, theret>y making an anti-protease antibody.
    221. Use of a polypeptide as set forth in claim 60 or a subsequence thereof in the manufacture of a preparation for generating a humoral immune response, thereby makings an anti-protease antibody.
    222. Use of aa polypeptide as set forth in claim 64, or a peptide encoded by a nucleic acid as set forth in claim 1 or claim 24 in the manufacture of a preparation for preventing an imbalance of desquamation.
    223. Use of aa polypeptide having a protease activity, wherein the polypeptide comprises a sequence as set forth in claim 59, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24 in the manufacture of a preparation for hairball preventaon.
    224. Use of a polypeptide having a protease activity as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24 in the manufacture «of a preparation for tissue dissociation
    225. A subst ance or composition for use in a method of inhibiting the translation of a pro&ease message in a cell, said substance or composition comprising an antise=nse oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a sequence as set forth in claim | or claim 24, and said method comprising administering said substance or composition to the cell. 202 AMENDED SHEET
    PCT/US2003/032819 ®
    226. A substance ox composition for use in a method of inhibiting the expression of a protease ix a cell, said substance or composition comprising a double-stranded inhibitory” RNA (iRNA), wherein the RNA comprises a subsequence of a sequence as set forth in claim | or claim 24, and said method comprising administering said substance or composition to the cell.
    227. A substance o r composition for use in a method of generating a humoral immune response and thereby making an anti-protease antibody, said substance or compositiora comprising a nucleic acid as set forth in claim 1 or claim 24 or a subsequence th ereof, and said method comprising administering said substance or compossition to a non-human animal.
    228. A substance oer composition for use in a method of generating a humoral immune response and thereby making an anti-protease antibody, said substance or compositiora comprising a polypeptide as set forth in claim 60 or a subsequence thereof, and said method comprising administering said substance or composition to a non—human animal.
    229. A substance or composition for use in a method of treating an imbalance or desquamation said substance or composition comprising a polypeptide as set forth in claim 64, oer a polypeptide encoded by a nucleic acid as set forth in claim 1 or claims 24, ancE said method comprising topical application of said substance or composition.
    230. A substance or composition for use in a method of treatment or prevention of claim 229, wherein the treatment is prophylactic.
    231. A substance Or composition for use in a method of treatment as a hairball remedy, said sulostance or composition comprising a polypeptide having a protease activity, wherein the polypeptide comprises a sequence as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set forth in claim | or claim 24, and sai d method comprising administering said substance or composition.
    232. A substance or composition for use in a method of prevention of a hairball, said substances or composition comprising a polypeptide having a protease activity, wherein the polypeptide comprises a sequence as set forth in claim 59, or a polypeptide encoded by a nucleic acid as set forth in claim 203 AMENDED» SHEET
    PCT/US2003/032819 1 or claim 24, and said method comprising administering said substance or composition.
    233. A substance or composi. tion for use in a method for tissue dissociation, said substance or composition comprising a polypeptide having a protease activity as set forth in claim 64,. or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 24, amd said method comprising contacting said substance or composition with a tassue to be dissociated.
    234. A substance or composi tion for use in a method of treatment or prevention of claim 233, wherein the= tissue is wound tissue.
    23s. A substance or compostLtion for use in 2 method of treatment or prevention of claim 233 wherein the contacting of said substance or composition with said tissue is used for wound cleansing, wound bed preparation, to treat pressure ulcers, leg ulcers, burns, diabetic foot ulcers, scars, [V fixation, surgical wounds or minor wounds.
    236. A nucleic acid according to any one of claims 1, or 24, or 34, or 166, substantially as herein described amd illustrated.
    237. A probe according to cl aim 27, or claim 29, substantially as herein described and illustrated.
    238. A primer pair according to claim 31, or claim 33, substantially as herein described and illustrated.
    239. A protease according tom claim 38, substantially as herein described and illustrated.
    240. A method according to any one of claims 39, or 103, or 106, 0r 108 to 112, 0r 121, 0r 122, 0r 126, 0r 128 , or 131, or 141, or 142 to 144, or 146, or 150, or 151, or 156, 0r 158,0r 167 to 1 69, or 172, or 175, 0r 179, or 182, or 183, or 186, or 188, or 191, or 192, or 195, or 197, or 205, or 210, substantially as herein described and illustrated.
    241. A cassette according to claim 40, substantially as herein described and illustrated. 204 AMENDED SHEET
    PC TI/US2003/032819
    242. A vector according to claim 41, substantially as herein described and illustrated.
    243. A vehicle according to claim 42, subs-tantially as herein described and illustrated. 244, A cell according to claim 45, or claim 46, substantially as herein described and illustrated.
    245. An animal according to claim 48, substantially as herein described and illustrated.
    246. A plant according to claim 50, substamtially as herein described and illustrated.
    247. A seed according to claim 52, substartially as herein described and illustrated.
    248. An oligonucleotide according to clairm 54, substantially as herein described and illustrated.
    249. A method according to any one of claims 56, or 59, or 104, or 105, or 201, or 214, substantially as herein described ard illustrated.
    250. An RNA according to claim 57, substantially as herein described and illustrated.
    251. A polypeptide according to any one Of claims 60, or 81, or 82, or 96, or 163, substantially as herein described and illust=rated.
    252. A preparation according to claim 91, substantially as herein described and illustrated.
    253. A heterodimer according to claim 92 , substantially as herein described and illustrated. 205 AMENDED SHEET
    C PCT/US2003/032819
    254. A homodimer according to claim 95, substantizally as herein described and illustrated.
    255. An array according to claim 98, or claim 99, substantially as herein described and illustrated.
    256. An antibody according to claim 100, substanti ally as hereim described and illustrated.
    257. A hybridoma according to claim 102, substan tially as herein described and illustrated.
    758. A system according to claim 116, substantial ly as herein described and illustrated.
    259. A medium according to claim 120, substantially as herein described and illustrated.
    260. A signal sequence according to claim 162, s-ubstantially as herein described and illustrated.
    261. A composition according to any one of claims 176, 0r 19 8, or 204, or 208, substantially as herein described and illustrated.
    262. A textile or fabric according to claim 180, substantially as herein described and illustrated.
    263. A feed or a food according to claim 185, substantially as herein described and illustrated.
    264. A product according to any one of claims 190, or 196, or 202, or 206, substantially as herein described and illustrated.
    265. Use according to any one of claims 200, <r 207, or 215, or 218 to 224, substantially as herein described and illustrated.
    266. A spot remover according to claim 209, substantially as Therein described and illustrated. 206 AMENDED SHEET
    PCT/US2003/032819 @
    267. An agent according to claim 212, substantially as herein described and illustrated.
    268. A disinfectant according to claim 213, substantially as herein described and illustrated.
    269. A dressing according to claim 217, substantially as herein described and illustrated.
    270. A substance or composition for use in a method of treatment or prevention according to any one of claims 225 to 233, substantially as herein described and illustrated.
    271. A new nucleic acid; a new probe; a new primer pair; a new protease; a new method of amplifying a nucleic acid; a new method of isolating or identifying a polypeptide; a new method of producing a polypeptide; a new method of identifying a polypeptide; a new method of identifying a substrate; a new method of determining binding of a polypeptide; a new method of identifying a modulator; a new method of identifying a feature; a new method of comparing sequences; a new method of isolating or recovering a nucleic acid; a new method of generating a variant; a new method of modifying codons; a new method for producing a library; a new method of making a small molecule; a new method for modifying a small molecule; a new method of determining a functional fragment; a new method for whole cell engineering; a new method of increasing thermotolerance or thermostability; a new method for overexpressing a protease; a new method for making a transgenic plant; a new method of expressing a nucleic acid sequence; a new method of hydrolyzing, breaking up or disrupting a composition; a new method for liquefying or removing a protein; a new method for washing an object; a new method for removing stains; a new method for impro ving the finish of a fabric; a new method for hydrolyzing proteins; a new method for improving texture and flavour of a product; a new oN method of tenderi zing meat or fish; a new method of improving the extraction of h oil; a new method of preparing a juice, syrup, puree or extract; a new method of treating a pulp; a new method of treating a waste product; a new method of disinfecting a solid or a liquid; a new cassette; a new vector; a new vehicle; a new cell; a new non-th erapeutic method of treatment; a new RNA; a new transgenic animal; a new transgenic plant; a new seed; a new oligonucleotide; a new 207 AMENDED SHEET
    PCT/US2003/032819 (J polypeptide; a new preparation; a rew heterodimer; a new homodimer; a new array; a new antibody; a new hybridoma; a new system; a new medium; a new signal sequence; a new compositio=n; a new textile or fabric; a new feed or food; a new product; a new use of a polypeptide as set forth in claim 64, or a polypeptide encoded by a nucleic acid as set fo-rth in claim 1 or claim 24; a new use of an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a sequence as set forth in claim 1 or claim 24; a new use of &a double-stranded inhibitory RNA (1IRNA) comprising a subsequence or a seq uence as set forth in claim 1 or claim 24; a new use of a nucleic acid as set forth in_ claim 1 or claim 24 or a subsequence thereof; 2 new use of a polypeptide as set for—th in claim 60 or a subsequence thereof; a new use of a polypeptide comprising a sequence as set forth in claim 59, or a polypeptide encoded by a nucleic =acid as set forth in claim 1 or claim 24; a new spot remover; a new agent; a new «disinfectant; a new dressing; or a substance or composition for a new use in 2a me thod of treatment or prevention, substantially as herein described. : 208 AMENIDED SHEET
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GB201001791D0 (en) * 2010-02-03 2010-03-24 Ucb Pharma Sa Process for obtaining antibodies
WO2013026796A1 (en) * 2011-08-19 2013-02-28 Novozymes A/S Polypeptides having protease activity
CN104745613B (en) * 2013-12-27 2016-01-20 江苏众红生物工程创药研究院有限公司 A kind of Human kallikrein and encoding gene thereof and application preparation method
CN107475228B (en) * 2015-03-18 2020-08-04 江南大学 Keratinase mutant with improved substrate specificity and preparation method thereof
CN106434607B (en) * 2016-10-18 2019-05-17 江南大学 A kind of keratinase mutant and its application of thermal stability and specific enzyme activity raising
US20200248159A1 (en) * 2017-10-04 2020-08-06 Novozymes A/S Polypeptides having protease activity and polynucleotides encoding same
CN107828764B (en) * 2017-12-13 2020-11-06 中国科学院理化技术研究所 Heat-resistant cysteine protease and coding gene and application thereof
CN110333341A (en) * 2019-07-04 2019-10-15 浙江理工大学 A method of silk fabric cultural relics sample is identified based on protein biochip technology
CN115725552A (en) * 2022-08-15 2023-03-03 西南大学 Method for recombinant expression of p37k protease with gelatinase activity and method for improving p37k protease performance
CN117169519B (en) * 2023-10-26 2024-01-30 艾康生物技术(杭州)有限公司 Dissociation agent and kit for detecting TT3 and/or TT4 in sample

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