System for expressing hyperthermostable protein

专利摘要:
The amino acid sequence shown in SEQ ID NO: 1 of the sequence table and the amino acid sequence deleted or substituted, inserted or added in order to provide a protease of Bacillus Bacillus which is advantageous for industrial use by genetic engineering techniques. Disclosed are a heat resistant protease, a heat resistant protease gene encoding the same, and a method for producing the protease.
公开号:KR20010013540A
申请号:KR1019997011547
申请日:1998-06-04
公开日:2001-02-26
发明作者:히까루 다까꾸라；미오 모리시따；도모꼬 시모조；기요조 아사다；이꾸노신 가또
申请人:오미야 히사시；다까라 슈조 가부시키가이샤；
IPC主号:

专利说明:

System for expressing hyperthermostable protein
SEQUENCE LISTING
APPLICANT NAME: TAKARA SHUZO CO., LTD.
TITLE OF :: SYSTEM FOR EXPRESSING HYPERTHERMOSTABLE PROTEIN
REFERENCE / DOCKET NUMBER: 660782
CURRENT APPLICATION NUMBER:
CURRENT FILING DATE:
PRIOR APPLICATION NUMBER: 151969/1997
PRIOR FILING DATE: 10-JUN-1997
NUMBER OF SEQUENCES: 29
INFORMATION FOR SEQ ID NO: 1:
LENGTH: 412
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION:
Ala Glu Leu Glu Gly Leu Asp Glu Ser Ala Ala Gln Val Met Ala
5 10 15
Thr Tyr Val Trp Asn Leu Gly Tyr Asp Gly Ser Gly Ile Thr Ile
20 25 30
Gly Ile Ile Asp Thr Gly Ile Asp Ala Ser His Pro Asp Leu Gln
35 40 45
Gly Lys Val Ile Gly Trp Val Asp Phe Val Asn Gly Arg Ser Tyr
50 55 60
Pro Tyr Asp Asp His Gly His Gly Thr His Val Ala Ser Ile Ala
65 70 75
Ala Gly Thr Gly Ala Ala Ser Asn Gly Lys Tyr Lys Gly Met Ala
80 85 90
Pro Gly Ala Lys Leu Ala Gly Ile Lys Val Leu Gly Ala Asp Gly
95 100 105
Ser Gly Ser Ile Ser Thr Ile Ile Lys Gly Val Glu Trp Ala Val
110 115 120
Asp Asn Lys Asp Lys Tyr Gly Ile Lys Val Ile Asn Leu Ser Leu
125 130 135
Gly Ser Ser Gln Ser Ser Asp Gly Thr Asp Ala Leu Ser Gln Ala
140 145 150
Val Asn Ala Ala Trp Asp Ala Gly Leu Val Val Val Val Ala Ala
155 160 165
Gly Asn Ser Gly Pro Asn Lys Tyr Thr Ile Gly Ser Pro Ala Ala
170 175 180
Ala Ser Lys Val Ile Thr Val Gly Ala Val Asp Lys Tyr Asp Val
185 190 195
Ile Thr Ser Phe Ser Ser Arg Gly Pro Thr Ala Asp Gly Arg Leu
200 205 210
Lys Pro Glu Val Val Ala Pro Gly Asn Trp Ile Ile Ala Ala Arg
215 220 225
Ala Ser Gly Thr Ser Met Gly Gln Pro Ile Asn Asp Tyr Tyr Thr
230 235 240
Ala Ala Pro Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ile
245 250 255
Ala Ala Leu Leu Leu Gln Ala His Pro Ser Trp Thr Pro Asp Lys
260 265 270
Val Lys Thr Ala Leu Ile Glu Thr Ala Asp Ile Val Lys Pro Asp
275 280 285
Glu Ile Ala Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Ala Tyr
290 295 300
Lys Ala Ile Asn Tyr Asp Asn Tyr Ala Lys Leu Val Phe Thr Gly
305 310 315
Tyr Val Ala Asn Lys Gly Ser Gln Thr His Gln Phe Val Ile Ser
320 325 330
Gly Ala Ser Phe Val Thr Ala Thr Leu Tyr Trp Asp Asn Ala Asn
335 340 345
Ser Asp Leu Asp Leu Tyr Leu Tyr Asp Pro Asn Gly Asn Gln Val
350 355 360
Asp Tyr Ser Tyr Thr Ala Tyr Tyr Gly Phe Glu Lys Val Gly Tyr
365 370 375
Tyr Asn Pro Thr Asp Gly Thr Trp Thr Ile Lys Val Val Ser Tyr
380 385 390
Ser Gly Ser Ala Asn Tyr Gln Val Asp Val Val Ser Asp Gly Ser
395 400 405
Leu Ser Gln Pro Gly Ser Ser
410
INFORMATION FOR SEQ ID NO: 2:
LENGTH: 1236
TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: Genomic DNA
ORIGINAL SOURCE:
ORGANISM: Pyrococcus furiosus
STRAIN: DSM3638
SEQUENCE DESCRIPTION:
GCAGAATTAG AAGGACTGGA TGAGTCTGCA GCTCAAGTTA TGGCAACTTA CGTTTGGAAC 60
TTGGGATATG ATGGTTCTGG AATCACAATA GGAATAATTG ACACTGGAAT TGACGCTTCT 120
CATCCAGATC TCCAAGGAAA AGTAATTGGG TGGGTAGATT TTGTCAATGG TAGGAGTTAT 180
CCATACGATG ACCATGGACA TGGAACTCAT GTAGCTTCAA TAGCAGCTGG TACTGGAGCA 240
GCAAGTAATG GCAAGTACAA GGGAATGGCT CCAGGAGCTA AGCTGGCGGG AATTAAGGTT 300
CTAGGTGCCG ATGGTTCTGG AAGCATATCT ACTATAATTA AGGGAGTTGA GTGGGCCGTT 360
GATAACAAAG ATAAGTACGG AATTAAGGTC ATTAATCTTT CTCTTGGTTC AAGCCAGAGC 420
TCAGATGGTA CTGACGCTCT AAGTCAGGCT GTTAATGCAG CGTGGGATGC TGGATTAGTT 480
GTTGTGGTTG CCGCTGGAAA CAGTGGACCT AACAAGTATA CAATCGGTTC TCCAGCAGCT 540
GCAAGCAAAG TTATTACAGT TGGAGCCGTT GACAAGTATG ATGTTATAAC AAGCTTCTCA 600
AGCAGAGGGC CAACTGCAGA CGGCAGGCTT AAGCCTGAGG TTGTTGCTCC AGGAAACTGG 660
ATAATTGCTG CCAGAGCAAG TGGAACTAGC ATGGGTCAAC CAATTAATGA CTATTACACA 720
GCAGCTCCTG GGACATCAAT GGCAACTCCT CACGTAGCTG GTATTGCAGC CCTCTTGCTC 780
CAAGCACACC CGAGCTGGAC TCCAGACAAA GTAAAAACAG CCCTCATAGA AACTGCTGAT 840
ATCGTAAAGC CAGATGAAAT AGCCGATATA GCCTACGGTG CAGGTAGGGT TAATGCATAC 900
AAGGCTATAA ACTACGATAA CTATGCAAAG CTAGTGTTCA CTGGATATGT TGCCAACAAA 960
GGCAGCCAAA CTCACCAGTT CGTTATTAGC GGAGCTTCGT TCGTAACTGC CACATTATAC 1020
TGGGACAATG CCAATAGCGA CCTTGATCTT TACCTCTACG ATCCCAATGG AAACCAGGTT 1080
GACTACTCTT ACACCGCCTA CTATGGATTC GAAAAGGTTG GTTATTACAA CCCAACTGAT 1140
GGAACATGGA CAATTAAGGT TGTAAGCTAC AGCGGAAGTG CAAACTATCA AGTAGATGTG 1200
GTAAGTGATG GTTCCCTTTC ACAGCCTGGA AGTTCA 1236
INFORMATION FOR SEQ ID NO: 3:
LENGTH: 29
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION:
Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr
5 10 15
Leu Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala
20 25
INFORMATION FOR SEQ ID NO: 4:
LENGTH: 522
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: peptide
FEATURES:
OTHER INFORMATION: Xaa at position 428 is Gly or Val
SEQUENCE DESCRIPTION:
Ala Glu Leu Glu Gly Leu Asp Glu Ser Ala Ala Gln Val Met Ala
5 10 15
Thr Tyr Val Trp Asn Leu Gly Tyr Asp Gly Ser Gly Ile Thr Ile
20 25 30
Gly Ile Ile Asp Thr Gly Ile Asp Ala Ser His Pro Asp Leu Gln
35 40 45
Gly Lys Val Ile Gly Trp Val Asp Phe Val Asn Gly Arg Ser Tyr
50 55 60
Pro Tyr Asp Asp His Gly His Gly Thr His Val Ala Ser Ile Ala
65 70 75
Ala Gly Thr Gly Ala Ala Ser Asn Gly Lys Tyr Lys Gly Met Ala
80 85 90
Pro Gly Ala Lys Leu Ala Gly Ile Lys Val Leu Gly Ala Asp Gly
95 100 105
Ser Gly Ser Ile Ser Thr Ile Ile Lys Gly Val Glu Trp Ala Val
110 115 120
Asp Asn Lys Asp Lys Tyr Gly Ile Lys Val Ile Asn Leu Ser Leu
125 130 135
Gly Ser Ser Gln Ser Ser Asp Gly Thr Asp Ala Leu Ser Gln Ala
140 145 150
Val Asn Ala Ala Trp Asp Ala Gly Leu Val Val Val Val Ala Ala
155 160 165
Gly Asn Ser Gly Pro Asn Lys Tyr Thr Ile Gly Ser Pro Ala Ala
170 175 180
Ala Ser Lys Val Ile Thr Val Gly Ala Val Asp Lys Tyr Asp Val
185 190 195
Ile Thr Ser Phe Ser Ser Arg Gly Pro Thr Ala Asp Gly Arg Leu
200 205 210
Lys Pro Glu Val Val Ala Pro Gly Asn Trp Ile Ile Ala Ala Arg
215 220 225
Ala Ser Gly Thr Ser Met Gly Gln Pro Ile Asn Asp Tyr Tyr Thr
230 235 240
Ala Ala Pro Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ile
245 250 255
Ala Ala Leu Leu Leu Gln Ala His Pro Ser Trp Thr Pro Asp Lys
260 265 270
Val Lys Thr Ala Leu Ile Glu Thr Ala Asp Ile Val Lys Pro Asp
275 280 285
Glu Ile Ala Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Ala Tyr
290 295 300
Lys Ala Ile Asn Tyr Asp Asn Tyr Ala Lys Leu Val Phe Thr Gly
305 310 315
Tyr Val Ala Asn Lys Gly Ser Gln Thr His Gln Phe Val Ile Ser
320 325 330
Gly Ala Ser Phe Val Thr Ala Thr Leu Tyr Trp Asp Asn Ala Asn
335 340 345
Ser Asp Leu Asp Leu Tyr Leu Tyr Asp Pro Asn Gly Asn Gln Val
350 355 360
Asp Tyr Ser Tyr Thr Ala Tyr Tyr Gly Phe Glu Lys Val Gly Tyr
365 370 375
Tyr Asn Pro Thr Asp Gly Thr Trp Thr Ile Lys Val Val Ser Tyr
380 385 390
Ser Gly Ser Ala Asn Tyr Gln Val Asp Val Val Ser Asp Gly Ser
395 400 405
Leu Ser Gln Pro Gly Ser Ser Pro Ser Pro Gln Pro Glu Pro Thr
410 415 420
Val Asp Ala Lys Thr Phe Gln Xaa Ser Asp His Tyr Tyr Tyr Asp
425 430 435
Arg Ser Asp Thr Phe Thr Met Thr Val Asn Ser Gly Ala Thr Lys
440 445 450
Ile Thr Gly Asp Leu Val Phe Asp Thr Ser Tyr His Asp Leu Asp
455 460 465
Leu Tyr Leu Tyr Asp Pro Asn Gln Lys Leu Val Asp Arg Ser Glu
470 475 480
Ser Pro Asn Ser Tyr Glu His Val Glu Tyr Leu Thr Pro Ala Pro
485 490 495
Gly Thr Trp Tyr Phe Leu Val Tyr Ala Tyr Tyr Thr Tyr Gly Trp
500 505 510
Ala Tyr Tyr Glu Leu Thr Ala Lys Val Tyr Tyr Gly
515 520
INFORMATION FOR SEQ ID NO: 5:
LENGTH: 4765
TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: Genomic DNA
ORIGINAL SOURCE:
ORGANISM: Pyrococcus furiosus
STRAIN: DSM3638
SEQUENCE DESCRIPTION:
TTTAAATTAT AAGATATAAT CACTCCGAGT GATGAGTAAG ATACATCATT ACAGTCCCAA 60
AATGTTTATA ATTGGAACGC AGTGAATATA CAAAATGAAT ATAACCTCGG AGGTGACTGT 120
AGAATGAATA AGAAGGGACT TACTGTGCTA TTTATAGCGA TAATGCTCCT TTCAGTAGTT 180
CCAGTGCACT TTGTGTCCGC AGAAACACCA CCGGTTAGTT CAGAAAATTC AACAACTTCT 240
ATACTCCCTA ACCAACAAGT TGTGACAAAA GAAGTTTCAC AAGCGGCGCT TAATGCTATA 300
ATGAAAGGAC AACCCAACAT GGTTCTTATA ATCAAGACTA AGGAAGGCAA ACTTGAAGAG 360
GCAAAAACCG AGCTTGAAAA GCTAGGTGCA GAGATTCTTG ACGAAAATAG AGTTCTTAAC 420
ATGTTGCTAG TTAAGATTAA GCCTGAGAAA GTTAAAGAGC TCAACTATAT CTCATCTCTT 480
GAAAAAGCCT GGCTTAACAG AGAAGTTAAG CTTTCCCCTC CAATTGTCGA AAAGGACGTC 540
AAGACTAAGG AGCCCTCCCT AGAACCAAAA ATGTATAACA GCACCTGGGT AATTAATGCT 600
CTCCAGTTCA TCCAGGAATT TGGATATGAT GGTAGTGGTG TTGTTGTTGC AGTACTTGAC 660
ACGGGAGTTG ATCCGAACCA TCCTTTCTTG AGCATAACTC CAGATGGACG CAGGAAAATT 720
ATAGAATGGA AGGATTTTAC AGACGAGGGA TTCGTGGATA CATCATTCAG CTTTAGCAAG 780
GTTGTAAATG GGACTCTTAT AATTAACACA ACATTCCAAG TGGCCTCAGG TCTCACGCTG 840
AATGAATCGA CAGGACTTAT GGAATACGTT GTTAAGACTG TTTACGTGAG CAATGTGACC 900
ATTGGAAATA TCACTTCTGC TAATGGCATC TATCACTTCG GCCTGCTCCC AGAAAGATAC 960
TTCGACTTAA ACTTCGATGG TGATCAAGAG GACTTCTATC CTGTCTTATT AGTTAACTCC 1020
ACTGGCAATG GTTATGACAT TGCATATGTG GATACTGACC TTGACTACGA CTTCACCGAC 1080
GAAGTTCCAC TTGGCCAGTA CAACGTTACT TATGATGTTG CTGTTTTTAG CTACTACTAC 1140
GGTCCTCTCA ACTACGTGCT TGCAGAAATA GATCCTAACG GAGAATATGC AGTATTTGGG 1200
TGGGATGGTC ACGGTCACGG AACTCACGTA GCTGGAACTG TTGCTGGTTA CGACAGCAAC 1260
AATGATGCTT GGGATTGGCT CAGTATGTAC TCTGGTGAAT GGGAAGTGTT CTCAAGACTC 1320
TATGGTTGGG ATTATACGAA CGTTACCACA GACACCGTGC AGGGTGTTGC TCCAGGTGCC 1380
CAAATAATGG CAATAAGAGT TCTTAGGAGT GATGGACGGG GTAGCATGTG GGATATTATA 1440
GAAGGTATGA CATACGCAGC AACCCATGGT GCAGACGTTA TAAGCATGAG TCTCGGTGGA 1500
AATGCTCCAT ACTTAGATGG TACTGATCCA GAAAGCGTTG CTGTGGATGA GCTTACCGAA 1560
AAGTACGGTG TTGTATTCGT AATAGCTGCA GGAAATGAAG GTCCTGGCAT TAACATCGTT 1620
GGAAGTCCTG GTGTTGCAAC AAAGGCAATA ACTGTTGGAG CTGCTGCAGT GCCCATTAAC 1680
GTTGGAGTTT ATGTTTCCCA AGCACTTGGA TATCCTGATT ACTATGGATT CTATTACTTC 1740
CCCGCCTACA CAAACGTTAG AATAGCATTC TTCTCAAGCA GAGGGCCGAG AATAGATGGT 1800
GAAATAAAAC CCAATGTAGT GGCTCCAGGT TACGGAATTT ACTCATCCCT GCCGATGTGG 1860
ATTGGCGGAG CTGACTTCAT GTCTGGAACT TCGATGGCTA CTCCACATGT CAGCGGTGTC 1920
GTTGCACTCC TCATAAGCGG GGCAAAGGCC GAGGGAATAT ACTACAATCC AGATATAATT 1980
AAGAAGGTTC TTGAGAGCGG TGCAACCTGG CTTGAGGGAG ATCCATATAC TGGGCAGAAG 2040
TACACTGAGC TTGACCAAGG TCATGGTCTT GTTAACGTTA CCAAGTCCTG GGAAATCCTT 2100
AAGGCTATAA ACGGCACCAC TCTCCCAATT GTTGATCACT GGGCAGACAA GTCCTACAGC 2160
GACTTTGCGG AGTACTTGGG TGTGGACGTT ATAAGAGGTC TCTACGCAAG GAACTCTATA 2220
CCTGACATTG TCGAGTGGCA CATTAAGTAC GTAGGGGACA CGGAGTACAG AACTTTTGAG 2280
ATCTATGCAA CTGAGCCATG GATTAAGCCT TTTGTCAGTG GAAGTGTAAT TCTAGAGAAC 2340
AATACCGAGT TTGTCCTTAG GGTGAAATAT GATGTAGAGG GTCTTGAGCC AGGTCTCTAT 2400
GTTGGAAGGA TAATCATTGA TGATCCAACA ACGCCAGTTA TTGAAGACGA GATCTTGAAC 2460
ACAATTGTTA TTCCCGAGAA GTTCACTCCT GAGAACAATT ACACCCTCAC CTGGTATGAT 2520
ATTAATGGTC CAGAAATGGT GACTCACCAC TTCTTCACTG TGCCTGAGGG AGTGGACGTT 2580
CTCTACGCGA TGACCACATA CTGGGACTAC GGTCTGTACA GACCAGATGG AATGTTTGTG 2640
TTCCCATACC AGCTAGATTA TCTTCCCGCT GCAGTCTCAA ATCCAATGCC TGGAAACTGG 2700
GAGCTAGTAT GGACTGGATT TAACTTTGCA CCCCTCTATG AGTCGGGCTT CCTTGTAAGG 2760
ATTTACGGAG TAGAGATAAC TCCAAGCGTT TGGTACATTA ACAGGACATA CCTTGACACT 2820
AACACTGAAT TCTCAATTGA ATTCAATATT ACTAACATCT ATGCCCCAAT TAATGCAACT 2880
CTAATCCCCA TTGGCCTTGG AACCTACAAT GCGAGCGTTG AAAGCGTTGG TGATGGAGAG 2940
TTCTTCATAA AGGGCATTGA AGTTCCTGAA GGCACCGCAG AGTTGAAGAT TAGGATAGGC 3000
AACCCAAGTG TTCCGAATTC AGATCTAGAC TTGTACCTTT ATGACAGTAA AGGCAATTTA 3060
GTGGCCTTAG ATGGAAACCC AACAGCAGAA GAAGAGGTTG TAGTTGAGTA TCCTAAGCCT 3120
GGAGTTTATT CAATAGTAGT ACATGGTTAC AGCGTCAGGG ACGAAAATGG TAATCCAACG 3180
ACAACCACCT TTGACTTAGT TGTTCAAATG ACCCTTGATA ATGGAAACAT AAAGCTTGAC 3240
AAAGACTCGA TTATTCTTGG AAGCAATGAA AGCGTAGTTG TAACTGCAAA CATAACAATT 3300
GATAGAGATC ATCCTACAGG AGTATACTCT GGTATCATAG AGATTAGAGA TAATGAGGTC 3360
TACCAGGATA CAAATACTTC AATTGCGAAA ATACCCATAA CTTTGGTAAT TGACAAGGCG 3420
GACTTTGCCG TTGGTCTCAC ACCAGCAGAG GGAGTACTTG GAGAGGCTAG AAATTACACT 3480
CTAATTGTAA AGCATGCCCT AACACTAGAG CCTGTGCCAA ATGCTACAGT GATTATAGGA 3540
AACTACACCT ACCTCACAGA CGAAAACGGT ACAGTGACAT TCACGTATGC TCCAACTAAG 3600
TTAGGCAGTG ATGAAATCAC AGTCATAGTT AAGAAAGAGA ACTTCAACAC ATTAGAGAAG 3660
ACCTTCCAAA TCACAGTATC AGAGCCTGAA ATAACTGAAG AGGACATAAA TGAGCCCAAG 3720
CTTGCAATGT CATCACCAGA AGCAAATGCT ACCATAGTAT CAGTTGAGAT GGAGAGTGAG 3780
GGTGGCGTTA AAAAGACAGT GACAGTGGAA ATAACTATAA ACGGAACCGC TAATGAGACT 3840
GCAACAATAG TGGTTCCTGT TCCTAAGAAG GCCGAAAACA TCGAGGTAAG TGGAGACCAC 3900
GTAATTTCCT ATAGTATAGA GGAAGGAGAG TACGCCAAGT ACGTTATAAT TACAGTGAAG 3960
TTTGCATCAC CTGTAACAGT AACTGTTACT TACACTATCT ATGCTGGCCC AAGAGTCTCA 4020
ATCTTGACAC TTAACTTCCT TGGCTACTCA TGGTACAGAC TATATTCACA GAAGTTTGAC 4080
GAATTGTACC AAAAGGCCCT TGAATTGGGA GTGGACAACG AGACATTAGC TTTAGCCCTC 4140
AGCTACCATG AAAAAGCCAA AGAGTACTAC GAAAAGGCCC TTGAGCTTAG CGAGGGTAAC 4200
ATAATCCAAT ACCTTGGAGA CATAAGACTA TTACCTCCAT TAAGACAGGC ATACATCAAT 4260
GAAATGAAGG CAGTTAAGAT ACTGGAAAAG GCCATAGAAG AATTAGAGGG TGAAGAGTAA 4320
TCTCCAATTT TTCCCACTTT TTCTTTTATA ACATTCCAAG CCTTTTCTTA GCTTCTTCGC 4380
TCATTCTATC AGGAGTCCAT GGAGGATCAA AGGTAAGTTC AACCTCCACA TCTCTTACTC 4440
CTGGGATTTC GAGTACTTTC TCCTCTACAG CTCTAAGAAG CCAGAGAGTT AAAGGACACC 4500
CAGGAGTTGT CATTGTCATC TTTATATATA CCGTTTTGTC AGGATTAATCTTTAGCTCAT 4560
AAATTAATCC AAGGTTTACA ACATCCATCC CAATTTCTGG GTCGATAACC TCCTTTAGCT 4620
TTTCCAGAAT CATTTCTTCA GTAATTTCAA GGTTCTCATC TTTGGTTTCT CTCACAAACC 4680
CAATTTCAAC CTGCCTGATA CCTTCTAACT CCCTAAGCTT GTTATATATC TCCAAAAGAG 4740
TGGCATCATC AATTTTCTCT TTAAA 4765
INFORMATION FOR SEQ ID NO: 6:
LENGTH: 1398
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION:
Met Asn Lys Lys Gly Leu Thr Val Leu Phe Ile Ala Ile Met Leu
5 10 15
Leu Ser Val Val Pro Val His Phe Val Ser Ala Glu Thr Pro Pro
20 25 30
Val Ser Ser Glu Asn Ser Thr Thr Ser Ile Leu Pro Asn Gln Gln
35 40 45
Val Val Thr Lys Glu Val Ser Gln Ala Ala Leu Asn Ala Ile Met
50 55 60
Lys Gly Gln Pro Asn Met Val Leu Ile Iles Lys Thr Lys Glu Gly
65 70 75
Lys Leu Glu Glu Ala Lys Thr Glu Leu Glu Lys Leu Gly Ala Glu
80 85 90
Ile Leu Asp Glu Asn Arg Val Leu Asn Met Leu Leu Val Lys Ile
95 100 105
Lys Pro Glu Lys Val Lys Glu Leu Asn Tyr Ile Ser Ser Leu Glu
110 115 120
Lys Ala Trp Leu Asn Arg Glu Val Lys Leu Ser Pro Pro Ile Val
125 130 135
Glu Lys Asp Val Lys Thr Lys Glu Pro Ser Leu Glu Pro Lys Met
140 145 150
Tyr Asn Ser Thr Trp Val Ile Asn Ala Leu Gln Phe Ile Gln Glu
155 160 165
Phe Gly Tyr Asp Gly Ser Gly Val Val Val Ala Val Leu Asp Thr
170 175 180
Gly Val Asp Pro Asn His Pro Phe Leu Ser Ile Thr Pro Asp Gly
185 190 195
Arg Arg Lys Ile Ile Glu Trp Lys Asp Phe Thr Asp Glu Gly Phe
200 205 210
Val Asp Thr Ser Phe Ser Phe Ser Lys Val Val Asn Gly Thr Leu
215 220 225
Ile Ile Asn Thr Thr Phe Gln Val Ala Ser Gly Leu Thr Leu Asn
230 235 240
Glu Ser Thr Gly Leu Met Glu Tyr Val Val Lys Thr Val Tyr Val
245 250 255
Ser Asn Val Thr Ile Gly Asn Ile Thr Ser Ala Asn Gly Ile Tyr
260 265 270
His Phe Gly Leu Leu Pro Glu Arg Tyr Phe Asp Leu Asn Phe Asp
275 280 285
Gly Asp Gln Glu Asp Phe Tyr Pro Val Leu Leu Val Asn Ser Thr
290 295 300
Gly Asn Gly Tyr Asp Ile Ala Tyr Val Asp Thr Asp Leu Asp Tyr
305 310 315
Asp Phe Thr Asp Glu Val Pro Leu Gly Gln Tyr Asn Val Thr Tyr
320 325 330
Asp Val Ala Val Phe Ser Tyr Tyr Tyr Gly Pro Leu Asn Tyr Val
335 340 345
Leu Ala Glu Ile Asp Pro Asn Gly Glu Tyr Ala Val Phe Gly Trp
350 355 360
Asp Gly His Gly His Gly Thr His Val Ala Gly Thr Val Ala Gly
365 370 375
Tyr Asp Ser Asn Asn Asp Ala Trp Asp Trp Leu Ser Met Tyr Ser
380 385 390
Gly Glu Trp Glu Val Phe Ser Arg Leu Tyr Gly Trp Asp Tyr Thr
395 400 405
Asn Val Thr Thr Asp Thr Val Gln Gly Val Ala Pro Gly Ala Gln
410 415 420
Ile Met Ala Ile Arg Val Leu Arg Ser Asp Gly Arg Gly Ser Met
425 430 435
Trp Asp Ile Ile Glu Gly Met Thr Tyr Ala Ala Thr His Gly Ala
440 445 450
Asp Val Ile Ser Met Ser Leu Gly Gly Asn Ala Pro Tyr Leu Asp
455 460 465
Gly Thr Asp Pro Glu Ser Val Ala Val Asp Glu Leu Thr Glu Lys
470 475 480
Tyr Gly Val Val Phe Val Ile Ala Ala Gly Asn Glu Gly Pro Gly
485 490 495
Ile Asn Ile Val Gly Ser Pro Gly Val Ala Thr Lys Ala Ile Thr
500 505 510
Val Gly Ala Ala Ala Val Pro Ile Asn Val Gly Val Tyr Val Ser
515 520 525
Gln Ala Leu Gly Tyr Pro Asp Tyr Tyr Gly Phe Tyr Tyr Phe Pro
530 535 540
Ala Tyr Thr Asn Val Arg Ile Ala Phe Phe Ser Ser Arg Gly Pro
545 550 555
Arg Ile Asp Gly Glu Ile Lys Pro Asn Val Val Ala Pro Gly Tyr
560 565 570
Gly Ile Tyr Ser Ser Leu Pro Met Trp Ile Gly Gly Ala Asp Phe
575 580 585
Met Ser Gly Thr Ser Met Ala Thr Pro His Val Ser Gly Val Val
590 595 600
Ala Leu Leu Ile Ser Gly Ala Lys Ala Glu Gly Ile Tyr Tyr Asn
605 610 615
Pro Asp Ile Ile Lys Lys Val Leu Glu Ser Gly Ala Thr Trp Leu
620 625 630
Glu Gly Asp Pro Tyr Thr Gly Gln Lys Tyr Thr Glu Leu Asp Gln
635 640 645
Gly His Gly Leu Val Asn Val Thr Lys Ser Trp Glu Ile Leu Lys
650 655 660
Ala Ile Asn Gly Thr Thr Leu Pro Ile Val Asp His Trp Ala Asp
665 670 675
Lys Ser Tyr Ser Asp Phe Ala Glu Tyr Leu Gly Val Asp Val Ile
680 685 690
Arg Gly Leu Tyr Ala Arg Asn Ser Ile Pro Asp Ile Val Glu Trp
695 700 705
His Ile Lys Tyr Val Gly Asp Thr Glu Tyr Arg Thr Phe Glu Ile
710 715 720
Tyr Ala Thr Glu Pro Trp Ile Lys Pro Phe Val Ser Gly Ser Val
725 730 735
Ile Leu Glu Asn Asn Thr Glu Phe Val Leu Arg Val Lys Tyr Asp
740 745 750
Val Glu Gly Leu Glu Pro Gly Leu Tyr Val Gly Arg Ile Ile Ile
755 760 765
Asp Asp Pro Thr Thr Pro Val Ile Glu Asp Glu Ile Leu Asn Thr
770 775 780
Ile Val Ile Pro Glu Lys Phe Thr Pro Glu Asn Asn Tyr Thr Leu
785 790 795
Thr Trp Tyr Asp Ile Asn Gly Pro Glu Met Val Thr His His Phe
800 805 810
Phe Thr Val Pro Glu Gly Val Asp Val Leu Tyr Ala Met Thr Thr
815 820 825
Tyr Trp Asp Tyr Gly Leu Tyr Arg Pro Asp Gly Met Phe Val Phe
830 835 840
Pro Tyr Gln Leu Asp Tyr Leu Pro Ala Ala Val Ser Asn Pro Met
845 850 855
Pro Gly Asn Trp Glu Leu Val Trp Thr Gly Phe Asn Phe Ala Pro
860 865 870
Leu Tyr Glu Ser Gly Phe Leu Val Arg Ile Tyr Gly Val Glu Ile
875 880 885
Thr Pro Ser Val Trp Tyr Ile Asn Arg Thr Tyr Leu Asp Thr Asn
890 895 900
Thr Glu Phe Ser Ile Glu Phe Asn Ile Thr Asn Ile Tyr Ala Pro
905 910 915
Ile Asn Ala Thr Leu Ile Pro Ile Gly Leu Gly Thr Tyr Asn Ala
920 925 930
Ser Val Glu Ser Val Gly Asp Gly Glu Phe Phe Ile Lys Gly Ile
935 940 945
Glu Val Pro Glu Gly Thr Ala Glu Leu Lys Ile Arg Ile Gly Asn
950 955 960
Pro Ser Val Pro Asn Ser Asp Leu Asp Leu Tyr Leu Tyr Asp Ser
965 970 975
Lys Gly Asn Leu Val Ala Leu Asp Gly Asn Pro Thr Ala Glu Glu
980 985 990
Glu Val Val Val Glu Tyr Pro Lys Pro Gly Val Tyr Ser Ile Val
995 1000 1005
Val His Gly Tyr Ser Val Arg Asp Glu Asn Gly Asn Pro Thr Thr
1010 1015 1020
Thr Thr Phe Asp Leu Val Val Gln Met Thr Leu Asp Asn Gly Asn
1025 1030 1035
Ile Lys Leu Asp Lys Asp Ser Ile Ile Leu Gly Ser Asn Glu Ser
1040 1045 1050
Val Val Val Thr Ala Asn Ile Thr Ile Asp Arg Asp His Pro Thr
1055 1060 1065
Gly Val Tyr Ser Gly Ile Ile Glu Ile Arg Asp Asn Glu Val Tyr
1070 1075 1080
Gln Asp Thr Asn Thr Ser Ile Ala Lys Ile Pro Ile Thr Leu Val
1085 1090 1095
Ile Asp Lys Ala Asp Phe Ala Val Gly Leu Thr Pro Ala Glu Gly
1100 1105 1110
Val Leu Gly Glu Ala Arg Asn Tyr Thr Leu Ile Val Lys His Ala
1115 1120 1125
Leu Thr Leu Glu Pro Val Pro Asn Ala Thr Val Ile Ile Gly Asn
1130 1135 1140
Tyr Thr Tyr Leu Thr Asp Glu Asn Gly Thr Val Thr Phe Thr Tyr
1145 1150 1155
Ala Pro Thr Lys Leu Gly Ser Asp Glu Ile Thr Val Ile Val Lys
1160 1165 1170
Lys Glu Asn Phe Asn Thr Leu Glu Lys Thr Phe Gln Ile Thr Val
1175 1180 1185
Ser Glu Pro Glu Ile Thr Glu Glu Asp Ile Asn Glu Pro Lys Leu
1190 1195 1200
Ala Met Ser Ser Pro Glu Ala Asn Ala Thr Ile Val Ser Val Glu
1205 1210 1215
Met Glu Ser Glu Gly Gly Val Lys Lys Thr Val Thr Val Glu Ile
1220 1225 1230
Thr Ile Asn Gly Thr Ala Asn Glu Thr Ala Thr Ile Val Val Pro
1235 1240 1245
Val Pro Lys Lys Ala Glu Asn Ile Glu Val Ser Gly Asp His Val
1250 1255 1260
Ile Ser Tyr Ser Ile Glu Glu Gly Glu Tyr Ala Lys Tyr Val Ile
1265 1270 1275
Ile Thr Val Lys Phe Ala Ser Pro Val Thr Val Thr Val Thr Tyr
1280 1285 1290
Thr Ile Tyr Ala Gly Pro Arg Val Ser Ile Leu Thr Leu Asn Phe
1295 1300 1305
Leu Gly Tyr Ser Trp Tyr Arg Leu Tyr Ser Gln Lys Phe Asp Glu
1310 1315 1320
Leu Tyr Gln Lys Ala Leu Glu Leu Gly Val Asp Asn Glu Thr Leu
1325 1330 1335
Ala Leu Ala Leu Ser Tyr His Glu Lys Ala Lys Glu Tyr Tyr Glu
1340 1345 1350
Lys Ala Leu Glu Leu Ser Glu Gly Asn Ile Ile Gln Tyr Leu Gly
1355 1360 1365
Asp Ile Arg Leu Leu Pro Pro Leu Arg Gln Ala Tyr Ile Asn Glu
1370 1375 1380
Met Lys Ala Val Lys Ile Leu Glu Lys Ala Ile Glu Glu Leu Glu
1385 1390 1395
Gly Glu Glu
INFORMATION FOR SEQ ID NO: 7:
LENGTH: 35
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
GGWWSDRRTG TTRRHGTHGC DGTDMTYGAC ACBGG 35
INFORMATION FOR SEQ ID NO: 8:
LENGTH: 32
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
KSTCACGGAA CTCACGTDGC BGGHACDGTT GC 32
INFORMATION FOR SEQ ID NO: 9:
LENGTH: 33
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
ASCMGCAACH GTKCCVGCHA CGTGAGTTCC GTG 33
INFORMATION FOR SEQ ID NO: 10:
LENGTH: 34
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
CHCCGSYVAC RTGBGGAGWD GCCATBGAVG TDCC 34
INFORMATION FOR SEQ ID NO: 11:
LENGTH: 1977
TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: Genomic DNA
ORIGINAL SOURCE:
ORGANISM: Thermococcus celer
STRAIN: DSM2476
SEQUENCE DESCRIPTION:
ATGAAGAGGT TAGGTGCTGT GGTGCTGGCA CTGGTGCTCG TGGGTCTTCT GGCCGGAACG 60
GCCCTTGCGG CACCCGTAAA ACCGGTTGTC AGGAACAACG CGGTTCAGCA GAAGAACTAC 120
GGACTGCTGA CCCCGGGACT GTTCAAGAAA GTCCAGAGGA TGAACTGGAA CCAGGAAGTG 180
GACACCGTCA TAATGTTCGG GAGCTACGGA GACAGGGACA GGGCGGTTAA GGTACTGAGG 240
CTCATGGGCG CCCAGGTCAA GTACTCCTAC AAGATAATCC CTGCTGTCGC GGTTAAAATA 300
AAGGCCAGGG ACCTTCTGCT GATCGCGGGC ATGATAGACA CGGGTTACTT CGGTAACACA 360
AGGGTCTCGG GCATAAAGTT CATACAGGAG GATTACAAGG TTCAGGTTGA CGACGCCACT 420
TCCGTCTCCC AGATAGGGGC CGATACCGTC TGGAACTCCC TCGGCTACGA CGGAAGCGGT 480
GTGGTGGTTG CCATCGTCGA TACGGGTATA GACGCGAACC ACCCCGATCT GAAGGGCAAG 540
GTCATAGGCT GGTACGACGC CGTCAACGGC AGGTCGACCC CCTACGATGA CCAGGGACAC 600
GGAACCCACG TTGCGGGTAT CGTTGCCGGA ACCGGCAGCG TTAACTCCCA GTACATAGGC 660
GTCGCCCCCG GCGCGAAGCT CGTCGGCGTC AAGGTTCTCG GTGCCGACGG TTCGGGAAGC 720
GTCTCCACCA TCATCGCGGG TGTTGACTGG GTCGTCCAGA ACAAGGACAA GTACGGGATA 780
AGGGTCATCA ACCTCTCCCT CGGCTCCTCC CAGAGCTCCG ACGGAACCGA CTCCCTCAGT 840
CAGGCCGTCA ACAACGCCTG GGACGCCGGT ATAGTAGTCT GCGTCGCCGC CGGCAACAGC 900
GGGCCGAACA CCTACACCGT CGGCTCACCC GCCGCCGCGA GCAAGGTCAT AACCGTCGGT 960
GCAGTTGACA GCAACGACAA CATCGCCAGC TTCTCCAGCA GGGGACCGAC CGCGGACGGA 1020
AGGCTCAAGC CGGAAGTCGT CGCCCCCGGC GTTGACATCA TAGCCCCGCG CGCCAGCGGA 1080
ACCAGCATGG GCACCCCGAT AAACGACTAC TACACCAAGG CCTCTGGAAC CAGCATGGCC 1140
ACCCCGCACG TTTCGGGCGT TGGCGCGCTC ATCCTCCAGG CCCACCCGAG CTGGACCCCG 1200
GACAAGGTGA AGACCGCCCT CATCGAGACC GCCGACATAG TCGCCCCCAA GGAGATAGCG 1260
GACATCGCCT ACGGTGCGGG TAGGGTGAAC GTCTACAAGG CCATCAAGTA CGACGACTAC 1320
GCCAAGCTCA CCTTCACCGG CTCCGTCGCC GACAAGGGAA GCGCCACCCA CACCTTCGAC 1380
GTCAGCGGCG CCACCTTCGT GACCGCCACC CTCTACTGGG ACACGGGCTC GAGCGACATC 1440
GACCTCTACC TCTACGACCC CAACGGGAAC GAGGTTGACT ACTCCTACAC CGCCTACTAC 1500
GGCTTCGAGA AGGTCGGCTA CTACAACCCG ACCGCCGGAA CCTGGACGGT CAAGGTCGTC 1560
AGCTACAAGG GCGCGGCGAA CTACCAGGTC GACGTCGTCA GCGACGGGAG CCTCAGCCAG 1620
TCCGGCGGCG GCAACCCGAA TCCAAACCCC AACCCGAACC CAACCCCGAC CACCGACACC 1680
CAGACCTTCA CCGGTTCCGT TAACGACTAC TGGGACACCA GCGACACCTT CACCATGAAC 1740
GTCAACAGCG GTGCCACCAA GATAACCGGT GACCTGACCT TCGATACTTC CTACAACGAC 1800
CTCGACCTCT ACCTCTACGA CCCCAACGGC AACCTCGTTG ACAGGTCCAC GTCGAGCAAC 1860
AGCTACGAGC ACGTCGAGTA CGCCAACCCC GCCCCGGGAA CCTGGACGTT CCTCGTCTAC 1920
GCCTACAGCA CCTACGGCTG GGCGGACTAC CAGCTCAAGG CCGTCGTCTA CTACGGG 1977
INFORMATION FOR SEQ ID NO: 12:
LENGTH: 659
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION:
Met Lys Arg Leu Gly Ala Val Val Leu Ala Leu Val Leu Val Gly
5 10 15
Leu Leu Ala Gly Thr Ala Leu Ala Ala Pro Val Lys Pro Val Val
20 25 30
Arg Asn Asn Ala Val Gln Gln Lys Asn Tyr Gly Leu Leu Thr Pro
35 40 45
Gly Leu Phe Lys Lys Val Gln Arg Met Asn Trp Asn Gln Glu Val
50 55 60
Asp Thr Val Ile Met Phe Gly Ser Tyr Gly Asp Arg Asp Arg Ala
65 70 75
Val Lys Val Leu Arg Leu Met Gly Ala Gln Val Lys Tyr Ser Tyr
80 85 90
Lys Ile Ile Pro Ala Val Ala Val Lys Ile Lys Ala Arg Asp Leu
95 100 105
Leu Leu Ile Ala Gly Met Ile Asp Thr Gly Tyr Phe Gly Asn Thr
110 115 120
Arg Val Ser Gly Ile Lys Phe Ile Gln Glu Asp Tyr Lys Val Gln
125 130 135
Val Asp Asp Ala Thr Ser Val Ser Gln Ile Gly Ala Asp Thr Val
140 145 150
Trp Asn Ser Leu Gly Tyr Asp Gly Ser Gly Val Val Val Ala Ile
155 160 165
Val Asp Thr Gly Ile Asp Ala Asn His Pro Asp Leu Lys Gly Lys
170 175 180
Val Ile Gly Trp Tyr Asp Ala Val Asn Gly Arg Ser Thr Pro Tyr
185 190 195
Asp Asp Gln Gly His Gly Thr His Val Ala Gly Ile Val Ala Gly
200 205 210
Thr Gly Ser Val Asn Ser Gln Tyr Ile Gly Val Ala Pro Gly Ala
215 220 225
Lys Leu Val Gly Val Lys Val Leu Gly Ala Asp Gly Ser Gly Ser
230 235 240
Val Ser Thr Ile Ile Ala Gly Val Asp Trp Val Val Gln Asn Lys
245 250 255
Asp Lys Tyr Gly Ile Arg Val Ile Asn Leu Ser Leu Gly Ser Ser
260 265 270
Gln Ser Ser Asp Gly Thr Asp Ser Leu Ser Gln Ala Val Asn Asn
275 280 285
Ala Trp Asp Ala Gly Ile Val Val Cys Val Ala Ala Gly Asn Ser
290 295 300
Gly Pro Asn Thr Tyr Thr Val Gly Ser Pro Ala Ala Ala Ser Lys
305 310 315
Val Ile Thr Val Gly Ala Val Asp Ser Asn Asp Asn Ile Ala Ser
320 325 330
Phe Ser Ser Arg Gly Pro Thr Ala Asp Gly Arg Leu Lys Pro Glu
335 340 345
Val Val Ala Pro Gly Val Asp Ile Ile Ala Pro Arg Ala Ser Gly
350 355 360
Thr Ser Met Gly Thr Pro Ile Asn Asp Tyr Tyr Thr Lys Ala Ser
365 370 375
Gly Thr Ser Met Ala Thr Pro His Val Ser Gly Val Gly Ala Leu
380 385 390
Ile Leu Gln Ala His Pro Ser Trp Thr Pro Asp Lys Val Lys Thr
395 400 405
Ala Leu Ile Glu Thr Ala Asp Ile Val Ala Pro Lys Glu Ile Ala
410 415 420
Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Val Tyr Lys Ala Ile
425 430 435
Lys Tyr Asp Asp Tyr Ala Lys Leu Thr Phe Thr Gly Ser Val Ala
440 445 450
Asp Lys Gly Ser Ala Thr His Thr Phe Asp Val Ser Gly Ala Thr
455 460 465
Phe Val Thr Ala Thr Leu Tyr Trp Asp Thr Gly Ser Ser Asp Ile
470 475 480
Asp Leu Tyr Leu Tyr Asp Pro Asn Gly Asn Glu Val Asp Tyr Ser
485 490 495
Tyr Thr Ala Tyr Tyr Gly Phe Glu Lys Val Gly Tyr Tyr Asn Pro
500 505 510
Thr Ala Gly Thr Trp Thr Val Lys Val Val Ser Tyr Lys Gly Ala
515 520 525
Ala Asn Tyr Gln Val Asp Val Val Ser Asp Gly Ser Leu Ser Gln
530 535 540
Ser Gly Gly Gly Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro Thr
545 550 555
Pro Thr Thr Asp Thr Gln Thr Phe Thr Gly Ser Val Asn Asp Tyr
560 565 570
Trp Asp Thr Ser Asp Thr Phe Thr Met Asn Val Asn Ser Gly Ala
575 580 585
Thr Lys Ile Thr Gly Asp Leu Thr Phe Asp Thr Ser Tyr Asn Asp
590 595 600
Leu Asp Leu Tyr Leu Tyr Asp Pro Asn Gly Asn Leu Val Asp Arg
605 610 615
Ser Thr Ser Ser Asn Ser Tyr Glu His Val Glu Tyr Ala Asn Pro
620 625 630
Ala Pro Gly Thr Trp Thr Phe Leu Val Tyr Ala Tyr Ser Thr Tyr
635 640 645
Gly Trp Ala Asp Tyr Gln Leu Lys Ala Val Val Tyr Tyr Gly
650 655
INFORMATION FOR SEQ ID NO: 13:
LENGTH: 28
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
AGAGGGATCC ATGAAGGGGC TGAAAGCT 28
INFORMATION FOR SEQ ID NO: 14:
LENGTH: 28
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
AGAGGCATGC GCTCTAGACT CTGGGAGAGT 28
INFORMATION FOR SEQ ID NO: 15:
LENGTH: 1962
TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: Genomic DNA
ORIGINAL SOURCE:
ORGANISM: Pyrococcus furiosus
STRAIN: DSM3638
SEQUENCE DESCRIPTION:
ATGAAGGGGC TGAAAGCTCT CATATTAGTG ATTTTAGTTC TAGGTTTGGT AGTAGGGAGC 60
GTAGCGGCAG CTCCAGAGAA GAAAGTTGAA CAAGTAAGAA ATGTTGAGAA GAACTATGGT 120
CTGCTAACGC CAGGACTGTT CAGAAAAATT CAAAAATTGA ATCCTAACGA GGAAATCAGC 180
ACAGTAATTG TATTTGAAAA CCATAGGGAA AAAGAAATTG CAGTAAGAGT TCTTGAGTTA 240
ATGGGTGCAA AAGTTAGGTA TGTGTACCAT ATTATACCCG CAATAGCTGC CGATCTTAAG 300
GTTAGAGACT TACTAGTCAT CTCAGGTTTA ACAGGGGGTA AAGCTAAGCT TTCAGGTGTT 360
AGGTTTATCC AGGAAGACTA CAAAGTTACA GTTTCAGCAG AATTAGAAGG ACTGGATGAG 420
TCTGCAGCTC AAGTTATGGC AACTTACGTT TGGAACTTGG GATATGATGG TTCTGGAATC 480
ACAATAGGAA TAATTGACAC TGGAATTGAC GCTTCTCATC CAGATCTCCA AGGAAAAGTA 540
ATTGGGTGGG TAGATTTTGT CAATGGTAGG AGTTATCCAT ACGATGACCA TGGACATGGA 600
ACTCATGTAG CTTCAATAGC AGCTGGTACT GGAGCAGCAA GTAATGGCAA GTACAAGGGA 660
ATGGCTCCAG GAGCTAAGCT GGCGGGAATT AAGGTTCTAG GTGCCGATGG TTCTGGAAGC 720
ATATCTACTA TAATTAAGGG AGTTGAGTGG GCCGTTGATA ACAAAGATAA GTACGGAATT 780
AAGGTCATTA ATCTTTCTCT TGGTTCAAGC CAGAGCTCAG ATGGTACTGA CGCTCTAAGT 840
CAGGCTGTTA ATGCAGCGTG GGATGCTGGA TTAGTTGTTG TGGTTGCCGC TGGAAACAGT 900
GGACCTAACA AGTATACAAT CGGTTCTCCA GCAGCTGCAA GCAAAGTTAT TACAGTTGGA 960
GCCGTTGACA AGTATGATGT TATAACAAGC TTCTCAAGCA GAGGGCCAAC TGCAGACGGC 1020
AGGCTTAAGC CTGAGGTTGT TGCTCCAGGA AACTGGATAA TTGCTGCCAG AGCAAGTGGA 1080
ACTAGCATGG GTCAACCAAT TAATGACTAT TACACAGCAG CTCCTGGGAC ATCAATGGCA 1140
ACTCCTCACG TAGCTGGTAT TGCAGCCCTC TTGCTCCAAG CACACCCGAG CTGGACTCCA 1200
GACAAAGTAA AAACAGCCCT CATAGAAACT GCTGATATCG TAAAGCCAGA TGAAATAGCC 1260
GATATAGCCT ACGGTGCAGG TAGGGTTAAT GCATACAAGG CTATAAACTA CGATAACTAT 1320
GCAAAGCTAG TGTTCACTGG ATATGTTGCC AACAAAGGCA GCCAAACTCA CCAGTTCGTT 1380
ATTAGCGGAG CTTCGTTCGT AACTGCCACA TTATACTGGG ACAATGCCAA TAGCGACCTT 1440
GATCTTTACC TCTACGATCC CAATGGAAAC CAGGTTGACT ACTCTTACAC CGCCTACTAT 1500
GGATTCGAAA AGGTTGGTTA TTACAACCCA ACTGATGGAA CATGGACAAT TAAGGTTGTA 1560
AGCTACAGCG GAAGTGCAAA CTATCAAGTA GATGTGGTAA GTGATGGTTC CCTTTCACAG 1620
CCTGGAAGTT CACCATCTCC ACAACCAGAA CCAACAGTAG ACGCAAAGAC GTTCCAAGGA 1680
TCCGATCACT ACTACTATGA CAGGAGCGAC ACCTTTACAA TGACCGTTAA CTCTGGGGCT 1740
ACAAAGATTA CTGGAGACCT AGTGTTTGAC ACAAGCTACC ATGATCTTGA CCTTTACCTC 1800
TACGATCCTA ACCAGAAGCT TGTAGATAGA TCGGAGAGTC CCAACAGCTA CGAACACGTA 1860
GAATACTTAA CCCCCGCCCC AGGAACCTGG TACTTCCTAG TATATGCCTA CTACACTTAC 1920
GGTTGGGCTT ACTACGAGCT GACGGCTAAA GTTTATTATG GC 1962
INFORMATION FOR SEQ ID NO: 16:
LENGTH: 654
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION:
Met Lys Gly Leu Lys Ala Leu Ile Leu Val Ile Leu Val Leu Gly
5 10 15
Leu Val Val Gly Ser Val Ala Ala Ala Pro Glu Lys Lys Val Glu
20 25 30
Gln Val Arg Asn Val Glu Lys Asn Tyr Gly Leu Leu Thr Pro Gly
35 40 45
Leu Phe Arg Lys Ile Gln Lys Leu Asn Pro Asn Glu Glu Ile Ser
50 55 60
Thr Val Ile Val Phe Glu Asn His Arg Glu Lys Glu Ile Ala Val
65 70 75
Arg Val Leu Glu Leu Met Gly Ala Lys Val Arg Tyr Val Tyr His
80 85 90
Ile Ile Pro Ala Ile Ala Ala Asp Leu Lys Val Arg Asp Leu Leu
95 100 105
Val Ile Ser Gly Leu Thr Gly Gly Lys Ala Lys Leu Ser Gly Val
110 115 120
Arg Phe Ile Gln Glu Asp Tyr Lys Val Thr Val Ser Ala Glu Leu
125 130 135
Glu Gly Leu Asp Glu Ser Ala Ala Gln Val Met Ala Thr Tyr Val
140 145 150
Trp Asn Leu Gly Tyr Asp Gly Ser Gly Ile Thr Ile Gly Ile Ile
155 160 165
Asp Thr Gly Ile Asp Ala Ser His Pro Asp Leu Gln Gly Lys Val
170 175 180
Ile Gly Trp Val Asp Phe Val Asn Gly Arg Ser Tyr Pro Tyr Asp
185 190 195
Asp His Gly His Gly Thr His Val Ala Ser Ile Ala Ala Gly Thr
200 205 210
Gly Ala Ala Ser Asn Gly Lys Tyr Lys Gly Met Ala Pro Gly Ala
215 220 225
Lys Leu Ala Gly Ile Lys Val Leu Gly Ala Asp Gly Ser Gly Ser
230 235 240
Ile Ser Thr Ile Ile Lys Gly Val Glu Trp Ala Val Asp Asn Lys
245 250 255
Asp Lys Tyr Gly Ile Lys Val Ile Asn Leu Ser Leu Gly Ser Ser
260 265 270
Gln Ser Ser Asp Gly Thr Asp Ala Leu Ser Gln Ala Val Asn Ala
275 280 285
Ala Trp Asp Ala Gly Leu Val Val Val Val Ala Ala Gly Asn Ser
290 295 300
Gly Pro Asn Lys Tyr Thr Ile Gly Ser Pro Ala Ala Ala Ser Lys
305 310 315
Val Ile Thr Val Gly Ala Val Asp Lys Tyr Asp Val Ile Thr Ser
320 325 330
Phe Ser Ser Arg Gly Pro Thr Ala Asp Gly Arg Leu Lys Pro Glu
335 340 345
Val Val Ala Pro Gly Asn Trp Ile Ile Ala Ala Arg Ala Ser Gly
350 355 360
Thr Ser Met Gly Gln Pro Ile Asn Asp Tyr Tyr Thr Ala Ala Pro
365 370 375
Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ile Ala Ala Leu
380 385 390
Leu Leu Gln Ala His Pro Ser Trp Thr Pro Asp Lys Val Lys Thr
395 400 405
Ala Leu Ile Glu Thr Ala Asp Ile Val Lys Pro Asp Glu Ile Ala
410 415 420
Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Ala Tyr Lys Ala Ile
425 430 435
Asn Tyr Asp Asn Tyr Ala Lys Leu Val Phe Thr Gly Tyr Val Ala
440 445 450
Asn Lys Gly Ser Gln Thr His Gln Phe Val Ile Ser Gly Ala Ser
455 460 465
Phe Val Thr Ala Thr Leu Tyr Trp Asp Asn Ala Asn Ser Asp Leu
470 475 480
Asp Leu Tyr Leu Tyr Asp Pro Asn Gly Asn Gln Val Asp Tyr Ser
485 490 495
Tyr Thr Ala Tyr Tyr Gly Phe Glu Lys Val Gly Tyr Tyr Asn Pro
500 505 510
Thr Asp Gly Thr Trp Thr Ile Lys Val Val Ser Tyr Ser Gly Ser
515 520 525
Ala Asn Tyr Gln Val Asp Val Val Ser Asp Gly Ser Leu Ser Gln
530 535 540
Pro Gly Ser Ser Pro Ser Pro Gln Pro Glu Pro Thr Val Asp Ala
545 550 555
Lys Thr Phe Gln Gly Ser Asp His Tyr Tyr Tyr Asp Arg Ser Asp
560 565 570
Thr Phe Thr Met Thr Val Asn Ser Gly Ala Thr Lys Ile Thr Gly
575 580 585
Asp Leu Val Phe Asp Thr Ser Tyr His Asp Leu Asp Leu Tyr Leu 590 595 600
Tyr Asp Pro Asn Gln Lys Leu Val Asp Arg Ser Glu Ser Pro Asn
605 610 615
Ser Tyr Glu His Val Glu Tyr Leu Thr Pro Ala Pro Gly Thr Trp
620 625 630
Tyr Phe Leu Val Tyr Ala Tyr Tyr Thr Tyr Gly Trp Ala Tyr Tyr
635 640 645
Glu Leu Thr Ala Lys Val Tyr Tyr Gly
650
INFORMATION FOR SEQ ID NO: 17:
LENGTH: 25
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
TCTGAATTCG TTCTTTTCTG TATGG 25
INFORMATION FOR SEQ ID NO: 18:
LENGTH: 20
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
TGTACTGCTG GATCCGGCAG 20
INFORMATION FOR SEQ ID NO: 19:
LENGTH: 25
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
AGAGGCATGC GTATCCATCA GATTTTTGAG 30
INFORMATION FOR SEQ ID NO: 20:
LENGTH: 20
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
AGTGAACGGA TACTTGGAAC 20
INFORMATION FOR SEQ ID NO: 21:
LENGTH: 20
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
GTTCCAAGTA TCCGTTCACT 20
INFORMATION FOR SEQ ID NO: 22:
LENGTH: 12
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION:
Ala Glu Leu Glu Gly Leu Asp Glu Ser Ala Ala Gln
5 10
INFORMATION FOR SEQ ID NO: 23:
LENGTH: 24
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
TCATGGATCC ACCCTCTCCT TTTA 24
INFORMATION FOR SEQ ID NO: 24:
LENGTH: 26
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
GTCTGCGCAG GCTGCCGGAN NNNNNATGAA GGGGCTGAAA GCTCTC 26
INFORMATION FOR SEQ ID NO: 25:
LENGTH: 29
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
GAGAGCTTTC AGCCCCTTCA TNNNNNNTCC GGCAGCCTGC GCAGACATG 29
INFORMATION FOR SEQ ID NO: 26:
LENGTH: 27
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
AGAGGGGGAT CCGTGAGAAG CAAAAAA 27
INFORMATION FOR SEQ ID NO: 27:
LENGTH: 20
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
GATGACTAGT AAGTCTCTAA 20
INFORMATION FOR SEQ ID NO: 28:
LENGTH: 20
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
AAGCCTGAGG TTGTTGCTCC 20
INFORMATION FOR SEQ ID NO: 29:
LENGTH: 29
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: Other nucleic acid (synthetic DNA)
SEQUENCE DESCRIPTION:
GGGCATGCTC ATGAACTTCC AGGCTGTGA 29
Proteases are enzymes that cleave peptide bonds in proteins, and numerous enzymes have been found in animals, plants and microorganisms. In addition to research reagents and medicines, their use is very important in the industrial fields such as the addition of detergents, processing of foods, and chemical synthesis using reverse reactions. Proteases used in industrial fields require high stability physically and chemically, and particularly heat resistant enzymes are used. Protease produced by Bacillus genus bacteria has a relatively high heat resistance and thus occupies the mainstream of the current protease. However, better enzymes are desired, and attempts have been made to acquire enzymes from microorganisms that grow at high temperatures, such as thermophilic Bacillus bacteria or streptococci.
For example, it is known that Pyrococcus furiosus, one of the Bacillus thermophiles, produces a protease [Appl. Environ. Microbiol. 56, pp. 1992-1998 (1990); FEMS Microbiol. Letters, vol. 71, pp. 17-20 (1990); J. Gen. Microbiol. Vol. 137, pp. 1193-1199 (1991).
In addition, Pyrococcus spp., Strain KOD1 (Pyrococcus sp. Strain KOD1) belonging to the genus Pyrococcus has been reported to produce a thiol protease (cysteine protease) [Appl. Environ. Microbiol., Vol. 60, pp. 4559-4566 (1994). Similarly, the production of proteases is known for bacteria in the genus Thermococcus, Staphylothermus, and Thermobacteroides (Appl. Microbiol. Biotechnol.), Vol. 34, 715. P. 719 (1991).
As described above, the protease derived from S. aureus has high thermal stability and is expected to be used instead of the heat-resistant protease currently used or in fields where the use of the protease has not been considered until now.
However, most of the microorganisms producing these enzymes grow only at high temperature conditions. For example, in the case of pyrococcus puriosus, culture at 90 to 100 ° C is required. This high temperature culture operation is disadvantageous in terms of energy cost. In addition, the protease productivity of these streptococci is lower than that of microbial proteases that have been used so far. As described above, the industrial production method of protease derived from S. aureus has a problem.
On the other hand, it is now widely used to isolate genes of the desired enzymes, introduce them into host microorganisms that are easy to culture, and produce genetically engineered enzymes. However, the gene of the enzyme introduced into the host is not necessarily expressed at the same efficiency as intended. It is thought that this is mainly due to the GC content of the gene to be introduced and the frequency of use of codons being different from those of the host gene. Therefore, in order to obtain the enzyme productivity suitable for the purpose of use, it is necessary to optimize the expression method for each transgene and host.
SUMMARY OF THE INVENTION An object of the present invention is to provide a protease of chorionic bacillus, which is advantageous for industrial use, to isolate the gene encoding the protease of the genus thermophilus, and to provide a superheat resistant protease gene using the gene. It is to provide an engineering manufacturing method.
Some of the proteases produced by Bacillus thermophiles can be classified into alkaline proteases called subtilisin-type from the homology of the amino acid sequence. When these genes are introduced into, for example, Bacillus subtilis, which is commonly used for genetic engineering production, the productivity of the enzyme is less than the protein originally produced by Bacillus subtilis.
As a result of intensive research, the present inventors have located a gene encoding a signal peptide (signal sequence) derived from subtilisin upstream of the genophil bacterium-derived protease gene to be expressed and further modified the amino acid sequence around the break point. It was found that the gene of interest was expressed with high efficiency in Bacillus subtilis. In addition, the present inventors have found that the amount of enzyme expression is increased even by deleting a portion of the protease-derived protease-derived protease gene that is not essential for the expression of enzymatic activity, thereby completing the present invention.
Briefly describing the present invention, the first invention of the present invention is a heat-resistant protease having an amino acid sequence shown in SEQ ID NO: 1 in the sequence table, and one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 of the sequence table, It is a protease which has a substituted, inserted or added amino acid sequence and has heat resistant protease activity.
Moreover, the 2nd invention of this invention is the gene which codes the heat resistant protease of 1st invention, and the heat resistant protease gene hybridizing to the gene.
The third invention of the present invention is a gene used to genetically prepare a heat resistant protease derived from Bacillus thermophilus, which is represented by Formula I:
SIG-Ala-Gly-Gly-Asn-PRO [Ⅰ]
The amino acid sequence represented by [wherein, SIG represents the amino acid sequence of the signal peptide derived from subtilisin and PRO represents the amino acid sequence of the protein to express, respectively] is characterized by the above-mentioned. SIG is preferably the amino acid sequence shown in SEQ ID NO: 3 of the sequence table, and PRO is preferably the amino acid sequence of a heat resistant protease derived from Bacillus thermophilus, particularly preferably a protease derived from Pyrococcus furiosus.
The fourth invention of the present invention relates to a method for producing a genetically engineered protein, comprising the step of culturing Bacillus bacteria into which the gene of the third invention is introduced and collecting a desired protein from the culture. It is done.
The fifth invention of the present invention is characterized in that the gene of the third invention is inserted into a plasmid used for genetically preparing a protein.
In addition to the polymorphisms and mutations of the gene encoding the protease disclosed in the present invention, and naturally occurring protein, one or several amino acid sequences in the amino acid sequence may be modified by in vivo or purification of the protein after production. Mutations such as deletion, insertion, addition, substitution of amino acids can occur, but nevertheless mutant proteins are known to exhibit substantially equivalent physiological and biological activities to proteins without mutations. The above-mentioned mutations are artificially introduced into the amino acid sequence of the protein, and in this case, it is possible to manufacture a variety of mutants. For example, polypeptides in which certain cysteine residues in the amino acid sequence of human interleukin 2 (IL-2) have been substituted with serine are known to maintain interleukin 2 activity (Science, Vol. 224, page 1431 (1984)). As described above, a protease having an amino acid sequence in which one or several amino acids are deleted, inserted, added, substituted, or the like in the amino acid sequence disclosed in the present invention, and which has the same activity as the protease of the present invention is within the scope of the present invention.
In this specification, "a gene to hybridize with a specific gene" is a gene having a nucleotide sequence similar to that of a specific gene. A gene having a nucleotide sequence similar to a specific gene is likely to have a similar amino acid sequence of the protein encoded therein or a function of the protein. The similarity of nucleotide sequences of genes can be examined by whether or not both genes or parts thereof hybridize (hybridize) under constriction conditions, and use them to identify proteins having the same function as the proteins encoded by a particular gene. The gene to be encoded can be obtained. That is, a gene having a nucleotide sequence similar to that of the gene of the present invention can be obtained by performing hybridization by a known method using a gene obtained by the present invention or a part thereof as a probe. Hybridization can be performed, for example, in 1989 by the Cold Spring Harbor Laboratory, by T. Maniatis et al., And by the method described in Molecular Cloning: A Laboratory Manua1 2nd ed. More specifically, for example, hybridization can be performed under the following conditions. That is, 6 × SSC (1 × SSC containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylphyllolidon, 0.1% picol 400, 0.01% denatured salmon sperm DNA) 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0)), incubated at 50 ° C for 12-20 hours simultaneously with the probe. After incubation, the DNA was fixed by changing the temperature at 37 ° C in the 2 × SSC containing 0.5% SDS, lowering the SSC concentration to 0.1 × and raising the temperature to 50 ° C. The membrane is cleaned until the signal of origin can be distinguished from the background.
Alternatively, instead of hybridization, a gene amplification method such as a PCR method using a part of the nucleotide sequence of the gene obtained by the present invention as a primer may be used. Whether or not the gene thus obtained encodes a protein having a desired function can be known by examining the activity of the protein obtained by expressing the obtained gene using an appropriate host and expression system.
The present invention relates to a super heat resistant protease useful as an industrial enzyme, a gene encoding the same, and a method for producing genetic engineering of the enzyme.
1 is a restriction map of plasmid pSTC3.
FIG. 2 is a diagram comparing the amino acid sequences of protease PFUS, protease TCES and subtilisin.
FIG. 3 is a comparison of the amino acid sequences of protease PFUS, protease TCES and subtilisin.
4 is a diagram comparing the amino acid sequences of protease PFUS, protease TCES and subtilisin.
FIG. 5 is a comparison of the amino acid sequences of protease PFUS, protease TCES and subtilisin. FIG.
6 is a restriction map of plasmid pSNP1.
7 is a restriction map of plasmid pPS1.
8 is a restriction map of plasmid pNAPS1.
As the superheat resistant protease of the present invention, various proteases derived from S. aureus bacteria can be mentioned. For example, WO 95/34645 discloses proteases from Thermococcus furiosus and Thermococus celer.
A protease gene from Pyrococcus puriosus DSM3638 was isolated from the strain's genomic DNA library as an indicator of heat resistant protease activity. The plasmid containing the gene is named plasmid pTPR12, and the E. coli JM109 transformed with the plasmid is named and labeled E. coli JM109 / pTPR12, and the Japanese country under the Butafest Treaty on May 24, 1994 It has been deposited as Accession No. FERM BP-5103 at the Institute of Biotechnology Industrial Technology, Tongsan Institute of Industrial Technology, 3, 1-cho, Higashi 1-chome, Tsukuba-shi, Ibaraki.
This protease is hereinafter referred to as protease PFUL. Protease PFUL is a protease with high thermal stability and shows protease activity even at 95 ° C.
The base sequence of the DNA fragment derived from pyrococcus puriosus inserted in the plasmid pTPR12 has already been determined. The nucleotide sequence of about 4.8 kb inserted in two DraI sites in the inserted DNA fragment of plasmid pTPR12 is shown in SEQ ID NO: 5 of the sequence table. The amino acid sequence of the gene product estimated from the nucleotide sequence is shown in SEQ ID NO: 6 in the sequence table. That is, the amino acid sequence shown in SEQ ID NO: 6 in the sequence table is the amino acid sequence of the above protease PFUL. As shown in the sequence, protease PFUL is a high molecular weight protease consisting of 1398 amino acid residues and having a molecular weight of more than 150,000.
A group of alkaline serine proteases represented by subtilisin and the amino acid sequence of the first half of the protease PFUL by comparing the amino acid sequence of the above-described protease PFUL shown in SEQ ID NO: 6 in the sequence table with the known amino acid sequence of the protease derived from the microorganism [Protein] Engineering, Vol. 4, pp. 719 to 737 (1991), has shown that there is a very high homology around four amino acid residues, which are particularly important for the catalytic activity of proteases. .
Thus, it is clear that the region existing in common with the protease derived from the room temperature bacteria is preserved in the amino acid sequence of the protease PFUL produced by S. aureus pyrococcus puriosus. It can be expected that this region exists in the protease of.
For example, the genes of the superheat resistant protease are oligonucleotides PRO-1F, PRO-2F, which are synthesized based on the nucleotide sequence of the protease PFUL gene encoding a region showing high homology with subtilisin in the amino acid sequence of the protease PFUL. Screening can be performed by using a combination of PRO-2R and PRO-4R as primers, and performing PCR using chromosomal DNA of various streptococci as a template.
The base sequences of the oligonucleotides PRO-1F, PRO-2F, PRO-2R, and PRO-4R are shown in SEQ ID NOs: 7, 8, 9, and 10 in the sequence table, respectively.
As the thermophilic bacterium derived from the protease of the present invention, bacteria belonging to the genus Pyrococcus, Thermococcus, Staphyllus asus, Thermobacteroides and the like can be used. As the bacteria belonging to the genus Thermococcus, for example, Thermococcus Thermococcus celer DSM2476 can be used, and the strain is a strain available from Deutsche Zamlong von Microorganismann Unttrourchuren GmbH. DNA fragments that are specific when PCR is performed using a combination of the oligonucleotides PRO-1F and PRO-2R or a combination of PRO-2F and PRO-4R as a primer using the chromosomal DNA of the thermococcus seller DSM2476 as a template. Amplification of is confirmed to know the presence of the protease gene. In addition, after producing a recombinant plasmid in which these DNA fragments are inserted into a suitable plasmid vector, the amino acid sequence encoded by the fragment can be estimated by examining the base sequence of the inserted DNA fragment by the dideoxy method. As a result, this DNA fragment coded an amino acid sequence homologous to the amino acid sequence of protease PFUL and alkaline serine protease derived from various microorganisms, and the PCR amplified DNA fragment was amplified using the protease gene as a template. Done
Next, a superheat resistant protease gene, for example, a gene of a superheat resistant protease produced by a thermococcus cellar, uses the oligonucleotide or the amplified DNA fragment obtained by the above PCR as a probe to generate a gene library from S. aureus bacteria. It can be obtained by screening.
For example, phage clones containing the gene of interest can be obtained by performing a hybridization to a library using the amplified DNA fragment obtained by the above PCR as a probe.
Such a library contains a DNA fragment obtained by partially digesting the chromosomal DNA of the thermococcus cell DSM2476 with restriction enzyme Sau3AI, and a lambda phage particle by in vitro packaging method after ligating the lambda GEM-11 vector (promega). It is obtained by packing in the middle.
Analysis of the DNA fragments contained in the phage clones thus obtained revealed that the protease gene was present in the SacI fragment of about 1.9 kb, and by examining the nucleotide sequence, the fragment lacked the 5 'region of the protease gene. Turned out to be.
This 5 'side region can be obtained using a cassette and PCR using a cassette primer (Takarashuzo Gene Engineering Product Guide, 1994-1995, pages 250-251). In this way, a DNA fragment covering the 5 'side region of the superheat resistant protease gene lacking the plasmid pTCS6 can be obtained, and the nucleotide sequence of the full length of the superheat resistant protease gene derived from the thermococcus cellar is determined from the nucleotide sequences of these two DNA fragments. Can be.
The base sequence of the open reading frame existing in the determined base sequence is shown in SEQ ID NO: 11 of the sequence table, and the amino acid sequence estimated from the base sequence is shown in SEQ ID NO: 12 of the sequence table, respectively.
The base sequence and amino acid sequence of the gene encoding the superheat resistant protease from the thermococcus cellar were determined. This protease was named Protease TCES.
By combining the two DNA fragments, an expression plasmid obtained by reconstructing the full length of the protease TCES gene can be constructed. However, when E. coli was used as a host, a transformant into which the desired expression plasmid was introduced was not obtained. This is expected to be caused by the production of the expression product of the gene in the bacteria, or harmful to E. coli. In such a case, for example, it may be considered to confirm the activity by secreting and expressing the protease outside of cells using Bacillus subtilis as a host.
Bacillus subtilis DB104 can be used as the Bacillus subtilis, and this strain is a known strain described in Gene 83, 215-233 (1989). As the cloning vector, plasmid pUB18-P43 can be used, and the plasmid is a plasmid obtained from Dr. Sui-Lam Wong, Calgary University. The plasmid also contains a kanamycin resistance gene as a selection marker.
The recombinant plasmid in which the protease TCES gene was inserted downstream of the P43 promoter of plasmid vector pUB18-P43 was named plasmid pSTC3 and Bacillus subtilis DB104 transformed with the plasmid was designated and labeled Bacillus subtilis DB104 / pSTC3 in 1995. On December 1 (original deposit date) under the Butafest Treaty, it has been deposited as accession number FERM BP-5635 to the Institute of Biotechnology Industrial Technology, Tianjin Institute of Industrial Technology, 3-1, Higashi 1-chome, Tsukuba City, Ibaraki, Japan.
1 shows a restriction map of the plasmid pSTC3. In the figure, the thick solid line shows the inserted DNA fragment into the plasmid vector pUB18-P43.
When the Bacillus subtilis DB104 / pSTC3 was cultivated and the protease activity was examined for the culture supernatant and the cell extract, heat resistance of the protease can be confirmed in all.
The main properties of the coenzyme standard of the protease obtained from the transformant culture are as follows.
(1) Action:
Casein, gelatin are degraded to produce single chain polypeptides.
Hydrolyzing Succinyl-L-leucine-L-leucine-L-valyl-L-tyrosine-4-methylcoumalin-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) Amino-4-methylcoumalin).
Hydrolyze succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-p-NA) to give a yellow substance (p- Nitroaniline).
(2) proper temperature:
Enzyme activity is shown at 37-95 degreeC, and the appropriate temperature is 70-80 degreeC.
(3) titration pH:
Enzyme activity is shown between pH 5.5-9, and the appropriate pH is pH7-8.
(4) thermal stability:
The enzyme activity was maintained at 90% or higher even after treatment at 80 ° C. for 3 hours.
Protease PFUL, protease TCES and subtilisin [subtilisin BPN ', Nucl. Acids Res. Vol. 11, pp. 7911 to 7825 (1983), lists the homologous regions as shown in Figs. 2 to 5 so that the homologous regions of the protease PFUL are not only homologous to the C-terminal side. It can be seen that there is an unidentified sequence in protease TCES and subtilisin even between these regions. From these results, in addition to protease PFUL, there may be protease such as protease TCES or subtilisin having a lower molecular weight than protease PFUL.
Therefore, when Southern hybridization was carried out using a DNA probe derived from the region having the above homology and targeting chromosomal DNA prepared from Pyrococcus puriosus, a signal was observed in addition to the gene of the protease PFUL. The presence of the protease gene became clear.
This novel protease gene can be isolated by the following operations.
For example, a DNA fragment containing a gene encoding a new protease is obtained by digesting the chromosomal DNA of Pyrococcus puriosus with an appropriate restriction enzyme, and then targeting it by Southern hybridization as described above. . The base sequence of the DNA fragment is examined to confirm whether or not this base sequence encodes an amino acid sequence having homology with the protease. If the DNA fragment does not contain the full length of the gene of interest, the lacking portion is additionally obtained by reverse PCR.
For example, when pyrococcus puriosus chromosomal DNA is digested with restriction enzymes SacI and SpeI (manufactured by Takara Shuzo) and Southern hybridization is used, a signal having a size of about 0.6 kb is observed. E. coli JM109 is transformed with a recombinant plasmid obtained by inserting a DNA fragment of this size and inserting it between the SpeI-SacI sites of the plasmid vector pBluescriptSK (-) (produced by Stratagene). From this transformant, a clone containing the desired fragment can be obtained by colony hybridization using the same probe as that used for the above-mentioned Southern hybridization. Whether the plasmid contained in the obtained clone has a sequence encoding a protease can be confirmed by examining the nucleotide sequence of the inserted DNA fragment of the plasmid. The plasmid in which the presence of the protease gene was confirmed was named plasmid pSS3.
The amino acid sequence estimated from the nucleotide sequence of the DNA fragment inserted into the plasmid pSS3 was found to be homologous to sequences such as subtilisin, protease PFUL, protease TCES, and the like. The product of a protease gene other than the protease PFUL gene newly obtained in part from Pyrococcus puriosus is named protease PFUS. The region encoding the N terminal side and the region encoding the C terminal side of the protease can be obtained by reverse PCR.
Primers used for reverse PCR can be prepared based on the nucleotide sequence of the inserted DNA fragment contained in the plasmid pSS3. Next, after digesting the chromosomal DNA of Pyrococcus puriosus with an appropriate restriction enzyme, an intramolecular ligation reaction is performed on the generated DNA fragment. Using this reaction solution as a template, PCR using the above primers can be carried out to obtain DNA fragments of regions adjacent to fragments of the protease gene contained in the plasmid pSS3. By analyzing the nucleotide sequence of the DNA fragment thus obtained, the amino acid sequence of the enzyme protein encoded in the region is estimated. Moreover, the primer which can amplify the full length of the template protease PFUS gene using the chromosomal DNA of a pyrococcus puriosus can be produced.
Primer NPF-4 which has a nucleotide sequence immediately before the start codon of protease PFUS and can introduce a BamHI site on its 5 'side, and a SphI site on its 5' side having a sequence complementary to the 3 'side of protease PFUS gene Primer NPR-4 can be set up.
The base sequences of the primers NPF-4 and NPR-4 are shown in SEQ ID NOs: 13 and 14 in the Sequence Listing. These two primers can be used to amplify the full-length of the template protease PFUS gene by chromosomal DNA of pyrococcus puriosus.
Protease PFUS can be expressed using Bacillus subtilis as a host as in the case of protease TCES. Plasmids for expressing protease PFUS can be constructed based on the expression plasmid pSTC3 of protease TCES. That is, a plasmid for expressing protease PFUS can be constructed by substituting the DNA fragment containing the full length of the protease PFUS gene amplified by PCR using the above primer with the protease TCES gene on the plasmid pSTC3. The expression plasmid thus constructed was named plasmid pSNPl and Bacillus subtilis DB104 transformed with this plasmid was named and labeled Bacillus subtilis DB104 / pSNP1, and was dated Dec. 1, 1995. Under the treaty of the Butafest, it was deposited as Accession No. FERM BP-5634 to the Institute of Biotechnology Industrial Technology, Tianjin Institute of Industrial Technology, 3, 1-cho, Higashi 1-chome, Tsukuba-shi, Japan. 6 shows a restriction map of the plasmid pSNP1.
The base sequence of the open reading frame portion of the gene encoding the protease PFUS is shown in SEQ ID NO: 15 of the Sequence Listing, and the amino acid sequence of the protease PFUS estimated from the base sequence is shown in SEQ ID NO: 16 of the Sequence Listing.
When the protease activity of the Bacillus subtilis DB104 / pSNP1 was cultured and the culture supernatant and the bacterial extracts were examined, heat-resistant protease activity was confirmed in all of them. That is, part of the expressed protease PFUS is secreted in the culture supernatant.
The main properties of the protease obtained from the culture of the transformant are as follows.
(1) Action:
Casein, gelatin are degraded to produce single chain polypeptides.
Hydrolyze succinyl-L-leucine-L-leucine-L-valyl-L-tyrosine-4-methylcoumarin-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) to hydrolyze the phosphor (7-amino 4-methylcoumarin).
Hydrolyze succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-p-NA) to give a yellow substance (p- Nitroaniline).
(2) proper temperature:
Enzyme activity is shown at 40-110 degreeC, and a suitable temperature is 80-95 degreeC.
(3) titration pH:
Enzyme activity is shown between pH 5-10, and the suitable pH is pH 6-8.
(4) thermal stability:
The enzyme activity was maintained at 90% or higher even after treatment at 95 ° C for 8 hours.
(5) pH stability:
The activity was maintained at 95% or more even after treatment at pH 5-11, 95 ° C and 60 minutes.
(6) molecular weight:
A molecular weight of about 45 kDa on SDS-PAGE.
Protease genes homologous to these genes can be obtained from pyrococcus bacteria other than pyrococcus puriosus and thermococcal cells by the same method as for the protease TCES gene and the protease PFUS gene.
A DNA fragment of about 1 kb encoding the sequence from the 323rd residue to the 650th residue of the amino acid sequence of protease PFUL shown in SEQ ID NO: 6 in the sequence table was prepared, and this was used as a probe to Staphylothermus marinus DSM3639 and Southern hybridization of the genome can be performed using the chromosomal DNA of Thermobacteroides proteoliticus DSM5265. As a result, when mutant DNA was used as Staphyll digested with PstI (product of Takara Shuzo), it was about 4.8 kb, and about chromosomal DNA of Thermobacteroides proteoliticus digested with XbaI. The signals are identified at 3.5 kb.
From these results, it became clear that the sequences homologous to genes such as protease PFUL, protease PFUS, and protease TCES also exist in the chromosomal DNA of Staphylocus marinus and Thermobacteroides proteoliticus. By using the same method as when isolated and identified genes encoding the protease TCES and the protease PFUS from the DNA fragments thus detected, the superheat resistance present in Staphyr Mariners and Thermobacteroides proteoliticus Genes encoding proteases can be isolated and identified.
In general, in the case of preparing a large amount of protein by genetic engineering techniques, it is considered to be advantageous to use a promoter that works effectively in the host rather than a promoter owned by the gene encoding the desired protein. In this respect, the P43 promoter used in constructing the expression system of the protease TCES and PFUS was a Bacillus subtilis-derived promoter, but did not show a sufficient effect on the expression of these two proteases.
Therefore, in order to improve the expression level, genes highly expressed by Bacillus subtilis, in particular, genes of secreted proteins, can be used. As such genes, α-amylase, genes of various extracellular proteases, and the like can be used. For example, it is expected to increase the expression level of protease PFUS by using a promoter of subtilisin and a signal peptide-coding region.
That is, the protease PFUS as a fusion protein under the control of promoter 1 of the subtilisin gene by linking the full length of the protease PFUS gene behind the region encoding the signal peptide of the subtilisin gene including the promoter region so that the translation frame of both genes is consistent. Can be expressed.
As the subtilisin gene used in the present invention, for example, a gene encoding subtilisin E can be used. J. Bacteriol. The promoter and signal peptide-coding region of subtilisin E inserted in plasmid pKWZ described in Vol. 171, pages 2657-2665 (1989) can be used. The base sequence of the gene is described in the above document for the 5 'upstream region containing the promoter sequence, and for J. Bacteriol. Vol. 158, pp. 411-418 (1984), respectively.
Based on these sequences, primers SUB4 for introducing the EcoRI site upstream of the promoter sequence of the gene and primers BmR1 for introducing the BamHI site behind the region encoding the signal peptide of subtilisin E are synthesized, respectively. SEQ ID NOs: 17 and 18 in the Sequence Listing show the nucleotide sequences of the primers SUB4 and BmR1, respectively. The primers SUB4 and BmR1 can be used to amplify a DNA fragment of about 0.3 kb that includes a region encoding the promoter and signal peptide of the subtilisin E gene by PCR using the plasmid pKWZ as a template.
The protease PFUS gene located downstream of the DNA fragment can be obtained from chromosomal DNA of Pyrococcus puriosus by PCR. As the primer hybridizing to the 5 'side of the gene, the primer NPF-4 described above can be used. In addition, a primer NPM-1 designed from a nucleotide sequence downstream from the stop codon of the gene and having a SphI site can be used as a primer hybridizing to the 3 'side of the gene. SEQ ID NO: 19 of the Sequence Listing shows the sequence of primer NPM-1.
On the other hand, when the protease PFUS gene is linked to the 0.3 kb DNA fragment by using the BamHI site, one of the BamHI sites present in the gene interferes. Primers mutRR and mutFR for removing this BamHI site by PCR-mutagenesis can be prepared based on the nucleotide sequence of the protease PFUS gene shown in SEQ ID NO: 15 of the Sequence Listing.
The base sequences of the primers mutRR and mutFR are shown in SEQ ID NOs: 20 and 21 in the sequence table, respectively. When the BamHI site was removed using these primers, the amino acid residue encoded by this site due to the base substitution introduced into the site, that is, the 560th amino acid sequence of the protease PFUS shown in SEQ ID NO: 16 of the sequence table. Glycine present in is substituted with valine.
By using these primers, it is possible to obtain a protease PFUS gene for linking to the region encoding the promoter and signal peptide of the subtilisin E gene. That is, two types of PCR are performed using two primer pairs, primer mutRR and NPF-4, and primer mutFR and NPM-1, using the pyrococcus furiosus chromosomal DNA as a template. In addition, a heteroduplex formed by mixing DNA fragments amplified by each PCR is used as a template, and a second PCR is performed using primers NPF-4 and NPM-1. In this way, the full length of the protease PFUS gene of about 2.4 kb that does not contain the BamHI site can be amplified.
The DNA fragment of about 2.4 kb obtained by digesting the PCR amplified DNA fragment with BamHI and SphI is isolated and replaced with the BamHI-SphI fragment containing the protease PFUS gene in the plasmid pSNP1. The expression plasmid thus constructed was named plasmid pPS1 and Bacillus subtilis DB104 transformed with the plasmid was named Bacillus subtilis DB104 / pPS1. When the transformant was cultured, the same protease activity was found as in the case of using plasmid pSNP1 in both the culture supernatant and the cell extract, confirming that the amino acid substitution did not affect the enzyme activity. 7 shows a restriction map of the plasmid pPS1.
The DNA fragment of about 0.3 kb that contains the promoter and signal peptide encoding region of the subtilisin E gene is digested with EcoRI and BamHI, and the EcoRI-BamHI fragment comprising the P43 promoter and ribosomal binding site sequence in the plasmid pPS1. Replace with The expression plasmid thus constructed was named pNAPS1 and Bacillus subtilis DB104 transformed with the plasmid was named Bacillus subtilis DB104 / pNAPS1. When the transformant was cultured and the protease activity was examined for the culture supernatant and the bacterial extract, heat-resistant protease activity was confirmed in all, and the expression amount thereof was increased compared to Bacillus subtilis DB104 / pSNP1. 8 shows a restriction map of the plasmid pNAPS1.
The protease expressed from this transformant exhibits the same enzymatic properties as those of the protease expressed by Bacillus subtilis DB104 / pSNP1. Further, the protease expressed by the transformant was purified and the N-terminal amino acid sequence was analyzed to obtain the amino acid sequence shown in SEQ ID NO: 22 in the Sequence Listing. The sequence coincides with the 133th to 144th sequences of the amino acid sequence of protease PFUS shown in SEQ ID NO: 15 of the sequence table, indicating that the mature protease PFUS is an enzyme consisting of the polypeptide after this portion. The amino acid sequence of the mature protease PFUS guessed from this result is shown by SEQ ID NO: 4 of a sequence table.
The amount of protease produced by Bacillus subtilis DB104 / pNAPS1 is increasing compared to Bacillus subtilis DB104 / pSNP1 (FERM BP-5634), but it is desirable to increase the yield. Although pNAPS1 codes for a signal peptide of subtilisin and a fusion peptide of protease PFUS, it is expected that the expression level of protease will be increased by modifying this linking portion so that the removal of signal peptide occurs efficiently. In the plasmid pNAPS1, three amino acids of Ala-Gly-Ser between the C-terminal amino acid (Ala) of the signal peptide of subtilisin E shown in SEQ ID NO: 3 of the sequence table and the N-terminal amino acid (Met) of the protease PFUS Peptides consisting of residues are inserted. A transformant having an increased protease expression level can be obtained by examining the protease production capacity of a transformant having a mutant plasmid obtained by introducing a mutation into the DNA of the portion encoding the peptide on the plasmid pNAPS1.
First, a mutant plasmid is prepared in which a portion encoding Ser in the above 3-amino acid peptide is converted into a nucleotide sequence encoding any 2 amino acids with respect to a gene encoding a fusion protein of subtilisin E-protease PFUS on plasmid pNAPS1. . Such mutant plasmids can be produced using PCR. For example, primers SPOF0 and SPOR0 having a sequence in which the codon (TCC) coding for Ser is replaced with any 6 bases (the nucleotide sequences of the primers SPOF0 and SPOR0 are shown in SEQ ID NOs: 24 and 25, respectively) of the sequence table; To encode the above Ser by performing PCR using primers SUB3 and NPR-10 (the nucleotide sequences of the primers SUB3 and NPR-10 are shown in SEQ ID NOs: 26 and 27, respectively, respectively) prepared from the base sequences of the front and rear regions. DNA fragments having the desired mutations introduced into the codon (TCC) moiety can be obtained. By replacing this fragment with the corresponding region on plasmid pNAPS1, a mutant plasmid containing the protease gene into which the mutation is introduced can be obtained.
Subsequently, the transformant obtained by introducing the mutant plasmid obtained in this way into a suitable host, for example, Bacillus subtilis DB104, by examining the amount of protease expression can be obtained. Confirmation of the amount of protease expression can be performed by culturing the isolated transformants individually and investigating the activity in the culture, but alternatively, by using agar plates containing substrates for the cultivation, it is easy to transformants with increased expression levels. It is also possible to choose.
That is, the transformants into which the above mutant plasmids have been introduced are grown on an agar plate containing skim milk, and then the plate is warmed at a temperature at which protease PFUS exhibits its activity, for example, 70 ° C. As a transformant expressing a protease, the skim milk surrounding the colony is decomposed and made transparent. The amount of expression of protease can be inferred from the size of the clear region.
One of the transformants expressing high protease activity compared to Bacillus subtilis DB104 / pNAPS1 thus obtained is named Bacillus subtilis DB104 / pSPO124. When the plasmid contained in the transformant (this plasmid is named pSPO124) was prepared and the nucleotide sequence was analyzed, the portion coding for Ser was mutated to the nucleotide sequence GGGAAT, so that Ser was Gly-. It became clear that the mutated protein was encoded with Asn.
In this way, a peptide consisting of four amino acids, Ala-Gly-Gly-Asn, is placed behind the signal peptide of subtilisin, and then fused to the N-terminus of the protein of interest to express the expression of the bacillus bacteria as a host. It became clear that it was possible to increase. Subtilisin produced by Bacillus bacteria include subtilisin BPN 'derived from Bacillus amyloliquefacience in addition to the subtilisin E (from Bacillus subtilis) used in the present invention. (Nucl. Acids Res., 11, 791-7925 (1983)), and subtilisin Carlsberg from Bacillus licheniformis (Nucl. Acids Res., Vol. 13, 8913-8926 (1985)), etc. Although there are some differences in these amino acid sequences, these signal peptides can also be suitably used in the present invention. In the present invention, a promoter derived from subtilisin E was used as a promoter for controlling expression, but it is possible to use various promoters that act as Bacillus bacteria instead.
In addition, there is no restriction on the protein to be expressed, and if the gene of the protein is available, it is possible to express it high genetically by applying the present invention. Applicability of the expression of proteins derived from organisms other than the host of the present invention is also apparent from the fact that Bacillus bacteria and proteins derived from Pyrococcus puriosus differing in taxonomic position are expressed at high levels. The present invention is particularly suitable for the genetic engineering of protease PFUL, protease TCES or Staphyrmouth Mariners, which are structurally similar to the above protease PFUS, and proteases derived from Summerbacteroides proteoliticus.
Based on such homology with subtilisin, the protease PFUS is thought to be expressed as a precursor protein having a signal peptide and propeptide, which is then processed to become a mature enzyme. Further, from the results of the N-terminal amino acid sequence analysis of the protease PFUS mature enzyme, the mature enzyme is expected to be an enzyme consisting of the amino acid sequence shown in SEQ ID NO: 4 of the sequence table. However, the molecular weight of the purified mature protease PFUS is about 45 kDa, which is smaller than that calculated from this amino acid sequence. This suggests that the protease PFUS expressed as a precursor is also processed at its C-terminal peptide to be converted into a mature protease.
If the C-terminal peptide removed by this processing is not essential for enzymatic activity and folding of the enzyme protein into the correct structure, the expression level of the protease PFUS may also be expressed by deleting and expressing the region encoding this portion of the gene. An increase is expected.
About the mature protease PFUS obtained from said Bacillus subtilis DB104 / pNAPS1, the molecular weight can be measured precisely using a mass spectrometer, for example. From this molecular weight and the N-terminal amino acid sequence of the mature protease PFUS identified above, it can be seen that the protease is a polypeptide corresponding to Thr from 133th Ala to amino acid sequence shown in SEQ ID NO: 15 of the sequence table. In addition, by introducing a stop codon in the vicinity of the 552th Thr-coding region of the protease PFUS gene included in the plasmid pNAPS1, a plasmid expressing protease PFUS having a polypeptide unnecessary for enzymatic activity can be constructed.
That is, the DNA fragment having the base sequence into which the desired stop codon was introduced introduces a stop codon (TGA) into the C-terminal side of the codon encoding the 544th amino acid (Ser) from the start codon of the protease PFUS gene on the plasmid pNAPS1. The primer NPR544 (the base sequence of primer NPR544 is shown in SEQ ID NO: 28 of the sequence table) and the primer NPFE81 (the base sequence of the primer NPFE81 are shown in SEQ ID NO: 29 of the sequence table) having the base sequence of the upstream region of the NspV region on the gene. Can be obtained by PCR using the same method. This fragment can then be replaced with the corresponding region on plasmid pNAPS1 to obtain a mutant plasmid containing the protease gene into which the desired mutation has been introduced. This plasmid was named plasmid pNAPSΔC, and Bacillus subtilis DB104 transformed with this plasmid was named Bacillus subtilis DB104 / pNAPSΔC. This transformant expresses protease activity with properties equivalent to protease PFUS, and its expression is higher than that of Bacillus DB104 / pNAPS1.
In this way, it became clear that the protease PFUS gene contained in the plasmid pNAPSΔC has a sufficient region for active expression of the enzyme. The base sequence of the region encoding the protease PFUS present in this plasmid is shown in SEQ ID NO: 2 in the sequence table. In addition, the amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table.
In addition, the same mutation as that of plasmid pNAPSΔC may be introduced into the protease PFUS gene of the plasmid pSPO124 to express the protease PFUS in which the C-terminal peptide is deleted.
That is, the desired plasmid can be constructed by ligation by mixing the plasmid pNAPSΔC digested with NspV and SphI with a plasmid pSPO124 previously digested with NspV and SphI to perform ligation. This plasmid was named plasmid pSPO124ΔCfh, and Bacillus subtilis DB104 transformed with this plasmid was named and labeled Bacillus subtilis DB104 / pSPO124ΔC, butabest, May 16, 1997 Under the treaty, it has been deposited with the accession number FERM BP-6294 at the Institute of Biotechnology Industrial Technology, Tongsan Institute of Industrial Technology, Ibaraki-cho, Higashi 1-chome, Tsukubashi, Japan. The transformant has an improved expression level of protease compared to Bacillus DB104 / pNAPS1.
The enzymatic and physicochemical properties of the protease produced by the transformant, Bacillus subtilis DB104 / pNAPSΔC and Bacillus subtilis DB104 / pSPO124ΔC, are those of the protease produced by Bacillus subtilis DB104 / pSNP1. No difference is seen. That is, the main properties of the protease obtained from these two transformant cultures are as follows.
(1) Action:
Casein, gelatin are degraded to produce single chain polypeptides.
Hydrolyze succinyl-L-leucine-L-leucine-L-valyl-L-tyrosine-4-methylcoumarin-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) to hydrolyze the phosphor (7-amino -4-methylcoumalin).
Hydrolyze succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-p-NA) to give a yellow substance (p- Nitroaniline).
(2) proper temperature:
Enzyme activity is shown at 40-110 degreeC, and the titration temperature is 80-95 degreeC.
(3) titration pH:
Enzyme activity is shown between pH 5-10, and the suitable pH is pH 6-8.
(4) thermal stability:
The enzyme activity was maintained at 90% or higher even after treatment at 95 ° C for 8 hours.
(5) pH stability:
The activity was maintained at 95% or more even after treatment at pH 5-11, 95 ° C and 60 minutes.
(6) molecular weight:
A molecular weight of about 45 kDa on SDS-PAGE.
Thus, proteases and genes thereof having high thermostability are provided. Also disclosed herein is a novel protein expression system that allows for the mass expression of the protease. The expression system is useful for the genetic engineering of various proteins in addition to those of the protease of the present invention.
Although an Example is given to the following and this invention is demonstrated in more detail, this invention is not limited to these.
Example 1
(1) Preparation of chromosomal DNA of pyrococcus puriosus
Cultivation of pyrococcus puriosus DSM3638 was performed as follows.
1% Tryptone, 0.5% Yeast Extract, 1% Soluble Starch, 3.5% Jamarin S Solid (Jamarine Laboratory), 0.5% Jamarin S Liquid (Jamarine Laboratory), 0.003% MgSO ₄ , O.001% NaCl, 0.0001% FeSO ₄ · 7H ₂ O, 0.0001% CuSO ₄ , 0.0001% CaCl ₂ · 7H ₂ O, 0.0001% ZnSO ₄ , 0.1 ppm CuSO ₄ · 5H ₂ O, 0.1 ppm H ₃ BO ₃ , 0.1 ppm KAl (SO ₄ ) ₂ , 0.1 ppm Na ₂ MoO ₄ · 2H ₂ O, 0.25 ppm NiCl ₂ · H ₂ O The composition medium is placed in a 2 liter medium bottle, sterilized at 120 ° C. for 20 minutes, and nitrogen gas is blown into the dissolved oxygen. After removing the strain, the strain was inoculated and incubated at 95 ° C. for 16 hours. Cells were collected by centrifugation after the end of the culture.
The obtained cells were then suspended in 4 ml of 50 mM Tris-HCl (pH 8.0) containing 25% scruce, and 2 ml of 0.2 M EDTA and 0.8 ml of lysozyme (5 mg / ml) were added to the suspension to give the mixture. After 1 hour warming at 20 ° C., 24 ml of SET solution (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0), 4 ml of 5% SDS, 400 μl proteinase K (10 mg / ml ) Was added, and the mixture was further kept warm at 37 ° C for 1 hour. Phenolic-chloroform extraction was performed to stop the reaction, followed by ethanol precipitation to obtain about 3.2 mg of chromosomal DNA.
Example 2
(1) Synthesis of primers for the construction of plasmid pNSP1
In order to synthesize a primer for use in amplifying the full length of the protease PFUS gene, the plasmid pSNP1 containing the full length of the gene was isolated from Bacillus subtilis DB104 / pSNP1 (FERM BP-5634) to determine the base sequence of the required region. Based on this sequence, a primer NPF-4 for introducing a BamHI site immediately before the start codon of the protease PFUS gene, and a primer NPM-1 hybridized to the 3 'side of the gene and containing a recognition sequence of SphI are synthesized. It was. The base numbers of the primers NPF-4 and NPM-1 are shown in SEQ ID NOs: 13 and 19 in the sequence table, respectively.
In addition, primers mutRR and mutFR were synthesized to remove the BamHI site existing about 1.7 kb downstream from the start codon of the protease PFUS gene. The base numbers of the primers mutRR and mutFR are shown in SEQ ID NOs: 20 and 21 in the sequence table, respectively.
(2) Preparation of Plasmid pPS1
Two sets of LA-PCR reaction mixtures each comprising a chromosomal DNA of Pyrococcus puriosus as a template and a combination of the primers NPF-4 and mutRR as a primer or a combination of the primers mutFR and NPM-1 were prepared, respectively. The reaction was carried out for 30 cycles in which 94 ° C, 30 seconds-55 ° C, 1 minute-68 ° C, and 3 minutes were used as one cycle. LA PCR Kit Ver. 2 (manufactured by Takara Shuzo Co., Ltd.) was used for the preparation of the LA-PCR reaction mixture. When agarose electrophoresis was performed using a part of the reaction solution, DNA fragments of about 1.8 kb were amplified when primers NPF-4 and mutRR were used, and about 0.6 kb when primers mutFR and NPM-1 were used.
Amplified DNA fragments were prepared by removing primers from the two PCR reaction solutions using SUPREC-02 (manufactured by Takara Shuzo Co., Ltd.). A LA-PCR reaction solution containing these two amplified DNA fragments and not containing a primer or LA Taq was prepared, subjected to thermal denaturation at 94 ° C for 10 minutes, cooled to 30 ° C over 30 minutes, and then at 30 ° C. Incubate for 15 minutes to form a hetero duplex. Subsequently, LA Taq (manufactured by Takara Shujo Co., Ltd.) was added to the reaction solution and reacted at 72 ° C. for 30 minutes. Further, primers NPF-4 and NPM-1 were further added to 94 ° C., 30 seconds to 55 ° C., 1 minute to 68 minutes. 25 cycles of reaction were performed at 3 degreeC and 1 minute as 1 degreeC. In this reaction solution, amplification of a DNA fragment of about 2.4 kb was observed.
The 2.4 kb DNA fragment was digested with BamHI, SphI (all from Takara Shuzo), and the fragment was digested with BamHI, SphI in advance, mixed with plasmid pSNP1 from which the full length of the protease PFUS gene was removed, followed by ligation. Introduced in Subtilis DB104. A plasmid was prepared from the resulting kanamycin-resistant transformant, and the plasmid containing only one molecule of the above-mentioned 2.4 kb fragment was selected and named plasmid pPS1. In addition, Bacillus subtilis DB104 transformed with this plasmid ppS1 was named Bacillus subtilis DB104 / pPS1.
7 shows a restriction map of the plasmid pPS1.
(3) Amplification of DNA fragments of promoter-signal peptide coding region of subtilisin E gene
Primers were synthesized to obtain the promoter-signal peptide coding region of the subtilisin E gene. First of all, J. Bacterio1. Primer SUB4 (SEQ ID NO: SEQ ID NO: 159), referring to the nucleotide sequence of the promoter region portion of the subtilisin E gene described in pages 2657 to 2665 (1989), containing the EcoRI site upstream of the region. The base sequence of primer SUB4 is shown at No. 17). Then J. Bacteriol. With reference to the nucleotide sequence of the subtilisin E gene described in volume 158, pages 411 to 418 (1984), primer BmR1 (SEQ ID NO: 18 of SEQ ID NO: 18) can be introduced immediately downstream of the signal peptide coding region. The base sequence of primer BmRI is shown).
J. Bacteriol. A PCR reaction mixture containing the primers SUB4 and BmR1 was prepared using a plasmid pKWZ containing the subtilisin E gene described in Vol. 171, pages 2657 to 2665 (1989), 94 ° C., 30 sec. 30 cycles of reaction which made 1 degreeC, 1 minute-68 degreeC, and 2 minutes into 1 cycle were performed. When agarose electrophoresis was performed using a part of the reaction solution, amplification of a DNA fragment of about 0.3 kb was confirmed.
(4) Construction of Protease Expression Plasmid pNAPS1
The above-described 0.3 kb DNA fragment was digested with EcoRI (Takara Shuzo Co., Ltd.) and BamHI, and then ligated by mixing with plasmid pPS1 described in Example 3, previously digested with EcoR I and BamHI, followed by Bacillus subs. Introduced to Tilly's DB104. A plasmid was prepared from the obtained kanamycin resistant transformant, and the plasmid into which only one molecule of about 0.3 kb fragment was inserted was selected and named as plasmid pNAPS1. In addition, Bacillus subtilis DB104 transformed from the plasmid pNAPS1 was named Bacillus subtilis DB104 / pNAPS1.
8 shows a restriction map of the plasmid pNAPS1.
(5) Construction of plasmid pSNP2
Primer SUB17R (the base sequence of primer SUB17R is shown in SEQ ID NO: 23 of SEQ ID NO: 23) for introducing a BamHI site upstream of the region encoding the signal peptide of subtilisin E present in the plasmid pNAPS1 was synthesized. As a template, a PCR reaction solution containing plasmid pNAPS1 and primers SUB17R and SUB4 was prepared and subjected to 25 cycles of reaction at 94 ° C for 30 seconds to 55 ° C for 1 minute to 72 ° C for 1 minute. The amplified DNA fragment of about 0.21 kb was digested with EcoRI and BamHI to obtain a DNA fragment of about 0.2 kb that contained the promoter and SD sequence of subtilisin E. This fragment was transformed into Bacillus subtilis DB104 using a reaction mixture ligated with plasmid pNAPS1 previously digested with EcoRI and BamHI. A plasmid was prepared from the obtained kanamycin resistant transformant, and the plasmid into which the above DNA fragment of about 0.2 kb was inserted was selected and named plasmid pSNP2.
(6) Construction of mutant plasmids expressing high levels of protease
Primers SPOF0 and SPOR0 (SEQ ID NO: 24 of SEQ ID NO: 24 in SEQ ID NO: 24) for replacing Ser (base sequence TCC) of the amino acid at the binding site of the start codon of the protease PFUS with the signal peptide of subtilisin E in the plasmid pNAPS1 with any two amino acids. And 25 show the base sequences of the primers SPOF0 and SPOR0, respectively). Further, primer NPR-10 (SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 1) containing primer SUB3 for introducing the BamHI site immediately before the region encoding the signal peptide of subtilisin E in the plasmid pNAPS1, and the SpeI site in the region encoding the protease PFUS. 26 and 27 show the nucleotide sequences of primers SUB3 and primers NPR-10).
Using the plasmid pNAPS1 as a template, respectively, a PCR reaction solution containing the primers SPOF0 and NPR-10 and a PCR reaction solution containing the primers SUB3 and SPOR0 were prepared. 94 ° C, 30 seconds-50 ° C, 1 minute-72 ° C. , 20 cycles of 1 minute in 1 cycle were carried out. About 0.13 kb and about 0.35 kb of DNA fragments amplified in each of the two reaction mixtures were mixed and thermally denatured at 94 DEG C for 10 minutes, and then slowly cooled to 37 DEG C to form a hetero duplex, and further, Taq polymerase Two-stranded DNA was produced from this hetero duplex by irradiation product). Thus prepared double-stranded DNA was used as a template to prepare a PCR reaction mixture containing the primers SUB3 and primers NPR-10 and 25 cycles of 94 ° C, 30 seconds-50 ° C, 1 minute-72 ° C, and 1 minute in 1 cycle. The reaction of the cycle was performed. DNA fragments obtained by digesting the amplified DNA fragments of about 0.43 kb with BamHI and SpeI (manufactured by Takara Shuzo) were mixed with the plasmid pSNP2, previously digested with BamHI and SpeI, and ligated. Bacillus subtilis DB104 was transformed.
The obtained kanamycin-resistant transformant was inoculated onto skim milk plates (10 g / ml kanamycin, LB-high temperature agar culture medium containing 1% skim milk) to form colonies, and then the plates were kept at 70 ° C. The protease activity expressed by each transformant was examined from the digested state of skim milk surrounding colonies. As a result, one clone in which particularly high activity was detected was isolated and a plasmid was prepared from this clone, which was named plasmid pSPO124. In addition, Bacillus subtilis DB104 transformed with this plasmid was named Bacillus subtilis DB104 / pSPO124. Analysis of the nucleotide sequence of the plasmid pSPO124 showed that the nucleotide sequence of the Ser-encoding region in the plasmid pNAPS1 was substituted with GGGAAT, that is, a protein in which Ser was mutated to a 2-amino acid of Gly-Asn was encoded. . In addition, at the same time as the mutation, it was found that the codon corresponding to the 25th present Pro (CCA) from the initiation codon of protease PFUS was changed to encoding Leu (CTA).
(7) Construction of protease expression plasmid pNAPSΔC
A terminal codon was introduced into the C-terminal side of the 544th amino acid from the start codon of the protease PFUS gene in the above plasmid pNAPS1 to construct a plasmid expressing the protease from which the downstream of this site was deleted. A terminal codon (base sequence TGA) is introduced at the C-terminal side of the codon encoding the 544th amino acid on the gene, and primer NPR544 having a SphI site (SEQ ID NO: 28 in the sequence table shows the base sequence of primer NPR544). Was synthesized. Further, primer NPFE81 (the nucleotide sequence of primer NPFE81 is shown in SEQ ID NO: 29 of the Sequence Listing) was synthesized based on the nucleotide sequence upstream of the NspV region on the gene.
A PCR reaction solution containing the primers NPFES1 and NPR544 as a template using plasmid pNAPS1 was prepared, and 20 cycles of reaction at 94 ° C, 30 seconds-50 ° C, 1 minute-72 ° C, and 1 minute in 1 cycle were carried out. The amplified DNA fragment of about 0.61 kb was digested with NspV (manufactured by Takara Shuzo Co., Ltd.) and SphI to obtain a DNA fragment of about 0.13 kb including the stop codon. This DNA fragment was ligated with the plasmid pNAPS1 previously digested with restriction enzymes NspV and SphI, and transformed to Bacillus subtilis DB104 using the reaction solution. A plasmid was prepared from the obtained kanamycin resistant transformant, and the plasmid into which the above DNA fragment of about 0.13 kb was inserted was selected and named plasmid pNAPSΔC. Bacillus subtilis DB104 transformed with this plasmid pNAPSΔC was named Bacillus subtilis DB104 / pNAPSΔC.
(8) Construction of protease expression plasmid pSPO124ΔC
About 1.3 kb of DNA fragment obtained by digesting the plasmid pNAPSΔC with NspV and SphI was isolated, and this was mixed with plasmid pSPO124 previously digested with NspV and SphI to perform ligation. The reaction solution was used to transform Bacillus subtilis DB104. A plasmid was prepared from the obtained kanamycin resistant transformant, and the plasmid into which the DNA fragment of about 1.3 kb was inserted was selected and named as plasmid pSPO124ΔC. The Bacillus subtilis DB104 transformed with this plasmid pSPOl24ΔC was named Bacillus subtilis DB104 / pSPO124ΔC.
Example 3
(1) Culture of Bacillus subtilis transformed with plasmid containing protease PFUS gene and preparation of coenzyme solution
LB medium containing Bacillus subtilis DB104 / pNAPS1, which is Bacillus subtilis DB104, into which plasmid pNAPS1 containing the protease PFUS gene was introduced as described in Example 2, containing 10 µg / ml kanamycin (10 g of tryptone) / Liter, yeast extract 5g / liter, NaCl 5g / liter, pH 7.2) 2ml incubated for 24 hours at 37 ℃. The culture solution was centrifuged to obtain culture supernatant (Sample 1-S) and cells.
The obtained cells were suspended in 100 µl of 50 mM Tris-HCl, pH 7.5, 2 mg of lysozyme (Sigma) was added, and then digested at 37 ° C for 45 minutes. The digested sample was further heated at 95 ° C. for 10 minutes, and then centrifuged to collect the supernatant to obtain a cell-free extract (Sample 1-L).
Similarly, Bacillus subtilis DB104 / pSPO124 containing the plasmid pSPO124, Bacillus subtilis DB104 / pNAPSΔC containing the plasmid pNAPSΔC, or Bacillus subtilis DB104 / pSPO124 containing pSPO124ΔC. Culture supernatants and cell-free extracts were prepared from ΔC in the same manner as described above. The culture supernatant of Bacillus subtilis DB104 / pSPO124 was named 124-S and the cell-free extract was designated 124-L. The culture supernatant of Bacillus subtilis DB104 / pNAPSΔC was designated as ΔC-S and the cell-free extract was designated as ΔC-L. The culture supernatant of Bacillus subtilis DB104 / pSPO124ΔC was named 124ΔC-S and the cell-free extract was named 124ΔC-L. These samples were used to measure protease activity and to examine the protease concentrations contained in each label.
(2) Comparison of Protease Production Capacity
Determination of the activity of the protease PFUS was performed by spectroscopically measuring p-nitroaniline produced by hydrolysis reaction by enzymes using Suc-Ala-Ala-Pro-Phe-p-NA (manufactured by Sigma) as a substrate. It was. That is, the enzyme standard to measure the enzyme activity is appropriately diluted, and 50 µl of the 1mM Suc-Ala-Ala-Pro-Phe-p-NA solution (100 mM phosphate buffer, pH 7.0) is added to 50 µl of the sample solution. The reaction was carried out for 30 minutes at. After stopping the reaction under ice-cooling, the absorbance at 405 nm was measured to determine the amount of p-nitroaniline produced. One unit of enzyme was made into the amount of enzyme which produces 1 micromole of p-nitroaniline in 95 degreeC for 1 minute. Based on the enzyme activity obtained, the enzyme protein mass expressed in the culture supernatant and cells was calculated as the specific activity of the protease PFUS per protein weight of 9.5 units / mg.
The protease activity of each enzyme standard prepared in Example 3 (1) was measured, and the amount of protease PFUS production per liter of each of the transformant cultures calculated from the measurement results is shown in Table 1.
In Bacillus subtilis DB104 / pSPO124, the production of protease PFUS in cells was 3.6-fold higher than in Bacillus subtilis DB104 / pNAPS1. In addition, Bacillus subtilis DB104 / pNAPSΔC showed an increase in protease PFUS production of 2.4 times in culture supernatant and 2.2 times in cells. In Bacillus subtilis DB104 / pSPO124ΔC, protease P FUS production increased by 2 fold in culture supernatant and 2.4 fold in cells. In addition, the yield per cell increased.
Comparing the sum of the protease PFUS production in the culture supernatant and cells with the values in Bacillus subtilis DB104 / pNAPS1, 2.1 times in Bacillus subtilis DB104 / pSPO124, 2.1 times in Bacillus subtilis DB104 / pNAPSΔC, In Bacillus subtilis DB104 / pSPO124ΔC increased 2.2 times.
Table 1 Production of Protease PFUS (per liter of culture, unit mg)
Transformant (plasmid) Culture supernatant Cell Culture solution + bacteria pNAPS1 15.1 12.5 27.6 pSPO124 13.1 45.4 58.5 pNAPS △ C 35.5 28.1 63.6 pSPO124 △ C 30.5 30.1 60.6
Example 4
(1) Preparation of purified enzyme standard of mature protease PFUS
Bacillus subtilis DB104 / pNAPS1 and Bacillus subtilis DB104 / pSPO124ΔC, respectively, Bacillus subtilis DB104 into which the superheat resistant protease gene of the present invention, introduced as described in Example 2, were each 10 µg / It was inoculated into a test tube containing 5 ml of LB medium containing ml of kanamycin and incubated at 37 ° C for 7 hours. TM medium containing 10 μg / ml of kanamycin (5 g / liter of soy flour, 10 g / liter of polypeptone, 5 g / liter of meat extract, 2 g / liter of yeast extract, 10 g / liter of glucose, FeSO ₄ · 7H ₂ O 10 mg /) L, 500 ml of MnSO ₄ · 4H ₂ O 10 mg / liter, ZnSO ₄ · 7H ₂ O 1 mg / liter, pH 7.0) was placed in a 5 liter Ellenmeyer flask and the culture was inoculated with 5 ml at 30 ° C. Shake culture was carried out for 3 days. The obtained culture solution was sonicated and then heated at 95 ° C. for 30 minutes, followed by centrifugation, and the supernatant was recovered. After adding ammonium sulfate so that the supernatant was saturated at 25%, the supernatant obtained by centrifugation was equilibrated with 25 mM Tris-HCl buffer (pH 7.6) containing 25% saturated ammonium sulfate (Micro-Prep Methyl). HIC, Biorad Co.) column. After washing the gel with the same buffer, the adsorbed protease PFUS on the column was eluted by stepwise elution with 25 mM Tris-HCl buffer (pH 7.6) containing 40% ethanol. The fraction containing the protease PFUS thus obtained was subjected to gel filtration using a NAP-25 column (from Pharmacia) equilibrated with 0.05% trifluoroacetic acid containing 20% acetonitrile to denature the protease PFUS and simultaneously desalting it. Was carried out to obtain the purified protease PFUS standard. In addition, the product obtained from Bacillus subtilis DB104 / pNAPS1 was named NAPS-1 and the product obtained from Bacillus subtilis DB104 / pSPO124ΔC, respectively, as SPO-124ΔC.
Electrophoresis of the above two purified enzyme samples using a 0.1% SDS-10% polyacrylamide gel, followed by staining with Coomassie Brilliant Blue R-250, resulted in both purified enzyme samples NAPS-1 and SPO-l24ΔC. A single band was shown and the estimated molecular weights were all about 45 kDa.
(2) N-terminal amino acid sequence analysis of mature protease PFUS
N-terminal amino acid sequence analysis of the purified enzyme standards NAPS-1 and SPO-124ΔC was performed by an automatic Edman method using a GOOOOA protein sequencer (manufactured by Hewlett-Packard). The N-terminal amino acid sequences of these two purified enzyme products are shown in SEQ ID NO: 22 in the sequence table. This sequence coincides with the 133 th to 144 th sequences of the amino acid sequence of the protease PFUS shown in SEQ ID NO: 15 in the sequence table, meaning that both NAPS-1 and SPO-124ΔC are enzymes consisting of the polypeptide after this portion. .
(3) Mass spectrometry of mature protease PFUS
The mass spectrometry of the purified enzymes, NAPS-1 and SPO-124ΔC, was carried out using an APl300 quadrupole triple mass spectrometer (manufactured by Perkin Elmer Cyex). Since the molecular weight of NAPS-1 is estimated to be 43,744 Da, the mature protease PFUS produced by Bacillus subtilis DB104 / pNAPS1 is the 133th Ala to 552th amino acid sequence of the protease PFUS shown in SEQ ID NO: 15 of the sequence table. It was found to be an enzyme consisting of polypeptides up to Thr. In addition, since the molecular weight of SPO-124ΔC is estimated to be 42,906 Da, the mature protease PFUS produced by Bacillus subtilis DB104 / pSPO124ΔC is the 133th amino acid sequence of the protease PFUS shown in SEQ ID NO: 15 of the sequence table. Was found to be an enzyme consisting of the amino acid sequence shown in SEQ ID NO: 2 of the sequence from Ala to the 544th Ser.

权利要求:
Claims (22)
[1" claim-type="Currently amended] A protease consisting of an amino acid sequence from which one or more amino acid residues are deleted from the C-terminus of the amino acid sequence shown in SEQ ID NO: 4 of the sequence table and having heat resistant protease activity.
[2" claim-type="Currently amended] The protease of claim 1, wherein the protease consists of the amino acid sequence shown as SEQ ID NO: 1 in the Sequence Listing.
[3" claim-type="Currently amended] A protease consisting of an amino acid sequence in which 1 to several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1 of the sequence table and has heat resistant protease activity.
[4" claim-type="Currently amended] A gene encoding a protein having heat resistant protease activity, consisting of an amino acid sequence deleted from at least one amino acid from the C-terminus of the amino acid sequence shown in SEQ ID NO: 4 of the Sequence Listing.
[5" claim-type="Currently amended] The protease gene according to claim 4, which encodes the amino acid sequence shown in SEQ ID NO: 1 in the sequence table.
[6" claim-type="Currently amended] The protease gene according to claim 5, comprising the nucleotide sequence shown in SEQ ID NO: 2 of the sequence table.
[7" claim-type="Currently amended] A protease gene encoding a protein having heat resistant protease activity, consisting of an amino acid sequence in which one to several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing.
[8" claim-type="Currently amended] A protease gene which hybridizes under prolonged conditions with the protease gene according to claim 6 and codes for a protein having heat resistant protease activity.
[9" claim-type="Currently amended] Equation I:
SIG-Ala-Gly-Gly-Asn-PRO [Ⅰ] [Wherein SIG represents the amino acid sequence of the signal peptide derived from subtilisin and PRO represents the amino acid sequence of the protein to be expressed] Gene encoding the amino acid sequence represented by.
[10" claim-type="Currently amended] 10. The gene of claim 9, wherein the SIG is an amino acid sequence as set forth in SEQ ID NO: 3 in the Sequence Listing.
[11" claim-type="Currently amended] 10. The gene of claim 9, wherein PRO is an amino acid sequence of a heat resistant protease derived from Bacillus subtilis.
[12" claim-type="Currently amended] 12. The gene of claim 11, wherein PRO is the amino acid sequence of a protease from Pyrococcus furiosus.
[13" claim-type="Currently amended] 13. The gene according to claim 12, wherein the PRO contains the amino acid sequence of the protease according to claim 1.
[14" claim-type="Currently amended] The gene according to claim 13, which is contained in a plasmid selected from pSPO124 and pSPO124ΔC.
[15" claim-type="Currently amended] 10. The gene of claim 9, wherein the PRO comprises the amino acid sequence of the protease according to claim 2.
[16" claim-type="Currently amended] A method for producing a protein, comprising culturing a bacterium of Bacillus into which the gene according to any one of claims 9 to 15 is introduced, and recovering a desired protein from the culture.
[17" claim-type="Currently amended] The method of claim 16, wherein the genus Bacillus bacteria is Bacillus subtilis.
[18" claim-type="Currently amended] The method according to claim 16, wherein the gene according to any one of claims 9 to 15 is introduced into Bacillus bacteria by a plasmid vector.
[19" claim-type="Currently amended] The method for producing a protein according to claim 18, wherein a plasmid selected from pSPO124 and pSPO124ΔC is introduced into Bacillus bacteria.
[20" claim-type="Currently amended] The method for producing a protein according to claim 19, comprising culturing Bacillus subtilis DB104 / pSPO124ΔC FERM P-16227 and recovering a desired protein from the culture.
[21" claim-type="Currently amended] The plasmid vector into which the gene of any one of Claims 9-15 was inserted.
[22" claim-type="Currently amended] 22. The plasmid vector of claim 21 selected from pSPO124 and pSPO124ΔC.

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Minton1995|Molecular genetics of clostridial neurotoxins

---

US5037760A|1991-08-06|Secretory signal selection vectors for extracellular protein synthesis bacilli

---

EP0493398B1|1999-12-08|Alkaline proteolytic enzyme and method of production

---

FI106315B|2001-01-15|Process for improving the stability of enzymes and stabilized enzymes

---

FI104326B|1999-12-31|Transformed prokaryotic host cells

---

US5891701A|1999-04-06|Nucleic acids encoding a polypeptide having protease activity

---

Silen et al.1989|Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity.

---

JP3210315B2|2001-09-17|Protease deficiency

同族专利:

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公开号 | 公开日

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CN1190494C|2005-02-23|

---

EP0994191A4|2003-04-23|

---

US20050084934A1|2005-04-21|

---

CN1311075C|2007-04-18|

---

EP0994191B1|2006-03-01|

---

WO1998056926A1|1998-12-17|

---

TW530088B|2003-05-01|

---

US7144719B2|2006-12-05|

---

US6783970B2|2004-08-31|

---

EP0994191A1|2000-04-19|

---

CN1260002A|2000-07-12|

---

US6358726B1|2002-03-19|

---

KR100530598B1|2005-11-23|

---

AT318913T|2006-03-15|

---

CN1657614A|2005-08-24|

---

DE69833652T2|2006-09-21|

---

AU7550098A|1998-12-30|

---

JP3601835B2|2004-12-15|

---

DE69833652D1|2006-04-27|

---

US20020132335A1|2002-09-19|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1997-06-10|Priority to JP15196997

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1997-06-10|Priority to JP1997/151969

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1998-06-04|Application filed by 오미야 히사시, 다까라 슈조 가부시키가이샤

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1998-06-04|Priority to PCT/JP1998/002465

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2001-02-26|Publication of KR20010013540A

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2005-11-23|Application granted

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2005-11-23|Publication of KR100530598B1

优先权:

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申请号 | 申请日 | 专利标题

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JP15196997|1997-06-10|

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JP1997/151969|1997-06-10|

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PCT/JP1998/002465|WO1998056926A1|1997-06-10|1998-06-04|System for expressing hyperthermostable protein|