CN113637085A - 融合dna聚合酶突变体及其在等温扩增中的应用 - Google Patents
融合dna聚合酶突变体及其在等温扩增中的应用 Download PDFInfo
- Publication number
- CN113637085A CN113637085A CN202010394538.0A CN202010394538A CN113637085A CN 113637085 A CN113637085 A CN 113637085A CN 202010394538 A CN202010394538 A CN 202010394538A CN 113637085 A CN113637085 A CN 113637085A
- Authority
- CN
- China
- Prior art keywords
- dna polymerase
- amplification
- leu
- mutant
- asp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 title claims abstract description 141
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 title claims abstract description 141
- 230000004927 fusion Effects 0.000 title claims abstract description 59
- 238000011901 isothermal amplification Methods 0.000 title claims abstract description 24
- 230000003321 amplification Effects 0.000 claims abstract description 55
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 55
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 26
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 26
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 25
- 238000006073 displacement reaction Methods 0.000 claims abstract description 22
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 230000035772 mutation Effects 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 14
- 101000844752 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) DNA-binding protein 7d Proteins 0.000 claims description 10
- 239000000523 sample Substances 0.000 claims description 10
- 238000006467 substitution reaction Methods 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 150000001768 cations Chemical class 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 239000012472 biological sample Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 30
- 108090000623 proteins and genes Proteins 0.000 abstract description 24
- 238000001514 detection method Methods 0.000 abstract description 11
- 230000010076 replication Effects 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 51
- 102000004190 Enzymes Human genes 0.000 description 29
- 108090000790 Enzymes Proteins 0.000 description 29
- 229940088598 enzyme Drugs 0.000 description 29
- 239000013615 primer Substances 0.000 description 29
- 238000006243 chemical reaction Methods 0.000 description 27
- 239000012634 fragment Substances 0.000 description 18
- 235000001014 amino acid Nutrition 0.000 description 17
- 108010017826 DNA Polymerase I Proteins 0.000 description 12
- 102000004594 DNA Polymerase I Human genes 0.000 description 12
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 12
- 210000003811 finger Anatomy 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 239000013598 vector Substances 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 239000013078 crystal Substances 0.000 description 7
- 230000006820 DNA synthesis Effects 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- 108010005233 alanylglutamic acid Proteins 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 238000006116 polymerization reaction Methods 0.000 description 5
- 210000003813 thumb Anatomy 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 241001148556 Mycoplasma ovipneumoniae Species 0.000 description 4
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 108010034529 leucyl-lysine Proteins 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 230000004568 DNA-binding Effects 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 241000204031 Mycoplasma Species 0.000 description 3
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 3
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000002457 bidirectional effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229910001437 manganese ion Inorganic materials 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000037048 polymerization activity Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108020001580 protein domains Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- OTCJMMRQBVDQRK-DCAQKATOSA-N Arg-Asp-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O OTCJMMRQBVDQRK-DCAQKATOSA-N 0.000 description 2
- LTZIRYMWOJHRCH-GUDRVLHUSA-N Asn-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N LTZIRYMWOJHRCH-GUDRVLHUSA-N 0.000 description 2
- SPWXXPFDTMYTRI-IUKAMOBKSA-N Asp-Ile-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SPWXXPFDTMYTRI-IUKAMOBKSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108020001019 DNA Primers Proteins 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 241000701832 Enterobacteria phage T3 Species 0.000 description 2
- 101900234631 Escherichia coli DNA polymerase I Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108060002716 Exonuclease Proteins 0.000 description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 2
- NCWOMXABNYEPLY-NRPADANISA-N Glu-Ala-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O NCWOMXABNYEPLY-NRPADANISA-N 0.000 description 2
- GVNNAHIRSDRIII-AJNGGQMLSA-N Ile-Lys-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N GVNNAHIRSDRIII-AJNGGQMLSA-N 0.000 description 2
- KVMULWOHPPMHHE-DCAQKATOSA-N Leu-Glu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KVMULWOHPPMHHE-DCAQKATOSA-N 0.000 description 2
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 2
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 2
- RZHLIPMZXOEJTL-AVGNSLFASA-N Lys-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N RZHLIPMZXOEJTL-AVGNSLFASA-N 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 241000589500 Thermus aquaticus Species 0.000 description 2
- BVOVIGCHYNFJBZ-JXUBOQSCSA-N Thr-Leu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O BVOVIGCHYNFJBZ-JXUBOQSCSA-N 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 108010038633 aspartylglutamate Proteins 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 229910001429 cobalt ion Inorganic materials 0.000 description 2
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- -1 etc.) Substances 0.000 description 2
- 102000013165 exonuclease Human genes 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 2
- 108010017391 lysylvaline Proteins 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000013643 reference control Substances 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 239000012536 storage buffer Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- COEXAQSTZUWMRI-STQMWFEESA-N (2s)-1-[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound C([C@H](N)C(=O)NCC(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 COEXAQSTZUWMRI-STQMWFEESA-N 0.000 description 1
- 102000040352 A family Human genes 0.000 description 1
- 108091072132 A family Proteins 0.000 description 1
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 1
- DKJPOZOEBONHFS-ZLUOBGJFSA-N Ala-Ala-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O DKJPOZOEBONHFS-ZLUOBGJFSA-N 0.000 description 1
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 1
- LGQPPBQRUBVTIF-JBDRJPRFSA-N Ala-Ala-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LGQPPBQRUBVTIF-JBDRJPRFSA-N 0.000 description 1
- ODWSTKXGQGYHSH-FXQIFTODSA-N Ala-Arg-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O ODWSTKXGQGYHSH-FXQIFTODSA-N 0.000 description 1
- IMMKUCQIKKXKNP-DCAQKATOSA-N Ala-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCN=C(N)N IMMKUCQIKKXKNP-DCAQKATOSA-N 0.000 description 1
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 1
- GWFSQQNGMPGBEF-GHCJXIJMSA-N Ala-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N GWFSQQNGMPGBEF-GHCJXIJMSA-N 0.000 description 1
- BUDNAJYVCUHLSV-ZLUOBGJFSA-N Ala-Asp-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O BUDNAJYVCUHLSV-ZLUOBGJFSA-N 0.000 description 1
- NKJBKNVQHBZUIX-ACZMJKKPSA-N Ala-Gln-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKJBKNVQHBZUIX-ACZMJKKPSA-N 0.000 description 1
- FBHOPGDGELNWRH-DRZSPHRISA-N Ala-Glu-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FBHOPGDGELNWRH-DRZSPHRISA-N 0.000 description 1
- VBRDBGCROKWTPV-XHNCKOQMSA-N Ala-Glu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N VBRDBGCROKWTPV-XHNCKOQMSA-N 0.000 description 1
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 1
- WMYJZJRILUVVRG-WDSKDSINSA-N Ala-Gly-Gln Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O WMYJZJRILUVVRG-WDSKDSINSA-N 0.000 description 1
- CKLDHDOIYBVUNP-KBIXCLLPSA-N Ala-Ile-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O CKLDHDOIYBVUNP-KBIXCLLPSA-N 0.000 description 1
- QJABSQFUHKHTNP-SYWGBEHUSA-N Ala-Ile-Trp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QJABSQFUHKHTNP-SYWGBEHUSA-N 0.000 description 1
- CCDFBRZVTDDJNM-GUBZILKMSA-N Ala-Leu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CCDFBRZVTDDJNM-GUBZILKMSA-N 0.000 description 1
- ZKEHTYWGPMMGBC-XUXIUFHCSA-N Ala-Leu-Leu-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O ZKEHTYWGPMMGBC-XUXIUFHCSA-N 0.000 description 1
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 1
- MLNSNVLOEIYJIU-ZUDIRPEPSA-N Ala-Leu-Thr-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MLNSNVLOEIYJIU-ZUDIRPEPSA-N 0.000 description 1
- QUIGLPSHIFPEOV-CIUDSAMLSA-N Ala-Lys-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O QUIGLPSHIFPEOV-CIUDSAMLSA-N 0.000 description 1
- RAAWHFXHAACDFT-FXQIFTODSA-N Ala-Met-Asn Chemical compound CSCC[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CC(N)=O)C(O)=O RAAWHFXHAACDFT-FXQIFTODSA-N 0.000 description 1
- IHRGVZXPTIQNIP-NAKRPEOUSA-N Ala-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C)N IHRGVZXPTIQNIP-NAKRPEOUSA-N 0.000 description 1
- JAQNUEWEJWBVAY-WBAXXEDZSA-N Ala-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 JAQNUEWEJWBVAY-WBAXXEDZSA-N 0.000 description 1
- BTRULDJUUVGRNE-DCAQKATOSA-N Ala-Pro-Lys Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O BTRULDJUUVGRNE-DCAQKATOSA-N 0.000 description 1
- YHBDGLZYNIARKJ-GUBZILKMSA-N Ala-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N YHBDGLZYNIARKJ-GUBZILKMSA-N 0.000 description 1
- VJVQKGYHIZPSNS-FXQIFTODSA-N Ala-Ser-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N VJVQKGYHIZPSNS-FXQIFTODSA-N 0.000 description 1
- DYXOFPBJBAHWFY-JBDRJPRFSA-N Ala-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N DYXOFPBJBAHWFY-JBDRJPRFSA-N 0.000 description 1
- YXXPVUOMPSZURS-ZLIFDBKOSA-N Ala-Trp-Leu Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](C)N)=CNC2=C1 YXXPVUOMPSZURS-ZLIFDBKOSA-N 0.000 description 1
- MTDDMSUUXNQMKK-BPNCWPANSA-N Ala-Tyr-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N MTDDMSUUXNQMKK-BPNCWPANSA-N 0.000 description 1
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 1
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 1
- XPSGESXVBSQZPL-SRVKXCTJSA-N Arg-Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XPSGESXVBSQZPL-SRVKXCTJSA-N 0.000 description 1
- MUXONAMCEUBVGA-DCAQKATOSA-N Arg-Arg-Gln Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(N)=O)C(O)=O MUXONAMCEUBVGA-DCAQKATOSA-N 0.000 description 1
- FEZJJKXNPSEYEV-CIUDSAMLSA-N Arg-Gln-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FEZJJKXNPSEYEV-CIUDSAMLSA-N 0.000 description 1
- KBBKCNHWCDJPGN-GUBZILKMSA-N Arg-Gln-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KBBKCNHWCDJPGN-GUBZILKMSA-N 0.000 description 1
- RYRQZJVFDVWURI-SRVKXCTJSA-N Arg-Gln-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N RYRQZJVFDVWURI-SRVKXCTJSA-N 0.000 description 1
- OGUPCHKBOKJFMA-SRVKXCTJSA-N Arg-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N OGUPCHKBOKJFMA-SRVKXCTJSA-N 0.000 description 1
- YVTHEZNOKSAWRW-DCAQKATOSA-N Arg-Lys-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O YVTHEZNOKSAWRW-DCAQKATOSA-N 0.000 description 1
- BNYNOWJESJJIOI-XUXIUFHCSA-N Arg-Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)N BNYNOWJESJJIOI-XUXIUFHCSA-N 0.000 description 1
- MNBHKGYCLBUIBC-UFYCRDLUSA-N Arg-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CCCNC(N)=N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MNBHKGYCLBUIBC-UFYCRDLUSA-N 0.000 description 1
- OVQJAKFLFTZDNC-GUBZILKMSA-N Arg-Pro-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O OVQJAKFLFTZDNC-GUBZILKMSA-N 0.000 description 1
- ADPACBMPYWJJCE-FXQIFTODSA-N Arg-Ser-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O ADPACBMPYWJJCE-FXQIFTODSA-N 0.000 description 1
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 1
- SUMJNGAMIQSNGX-TUAOUCFPSA-N Arg-Val-Pro Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N1CCC[C@@H]1C(O)=O SUMJNGAMIQSNGX-TUAOUCFPSA-N 0.000 description 1
- QEYJFBMTSMLPKZ-ZKWXMUAHSA-N Asn-Ala-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O QEYJFBMTSMLPKZ-ZKWXMUAHSA-N 0.000 description 1
- QISZHYWZHJRDAO-CIUDSAMLSA-N Asn-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N QISZHYWZHJRDAO-CIUDSAMLSA-N 0.000 description 1
- QYXNFROWLZPWPC-FXQIFTODSA-N Asn-Glu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QYXNFROWLZPWPC-FXQIFTODSA-N 0.000 description 1
- GQRDIVQPSMPQME-ZPFDUUQYSA-N Asn-Ile-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O GQRDIVQPSMPQME-ZPFDUUQYSA-N 0.000 description 1
- ZMUQQMGITUJQTI-CIUDSAMLSA-N Asn-Leu-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ZMUQQMGITUJQTI-CIUDSAMLSA-N 0.000 description 1
- HFPXZWPUVFVNLL-GUBZILKMSA-N Asn-Leu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFPXZWPUVFVNLL-GUBZILKMSA-N 0.000 description 1
- HPNDKUOLNRVRAY-BIIVOSGPSA-N Asn-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N)C(=O)O HPNDKUOLNRVRAY-BIIVOSGPSA-N 0.000 description 1
- JZLFYAAGGYMRIK-BYULHYEWSA-N Asn-Val-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O JZLFYAAGGYMRIK-BYULHYEWSA-N 0.000 description 1
- HPNDBHLITCHRSO-WHFBIAKZSA-N Asp-Ala-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(O)=O HPNDBHLITCHRSO-WHFBIAKZSA-N 0.000 description 1
- VPPXTHJNTYDNFJ-CIUDSAMLSA-N Asp-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N VPPXTHJNTYDNFJ-CIUDSAMLSA-N 0.000 description 1
- TVVYVAUGRHNTGT-UGYAYLCHSA-N Asp-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O TVVYVAUGRHNTGT-UGYAYLCHSA-N 0.000 description 1
- HRGGPWBIMIQANI-GUBZILKMSA-N Asp-Gln-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HRGGPWBIMIQANI-GUBZILKMSA-N 0.000 description 1
- HSWYMWGDMPLTTH-FXQIFTODSA-N Asp-Glu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HSWYMWGDMPLTTH-FXQIFTODSA-N 0.000 description 1
- JNNVNVRBYUJYGS-CIUDSAMLSA-N Asp-Leu-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O JNNVNVRBYUJYGS-CIUDSAMLSA-N 0.000 description 1
- ZQFZEBRNAMXXJV-KKUMJFAQSA-N Asp-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O ZQFZEBRNAMXXJV-KKUMJFAQSA-N 0.000 description 1
- GMXSSZUVDNPRMA-FXQIFTODSA-N Cys-Arg-Asp Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GMXSSZUVDNPRMA-FXQIFTODSA-N 0.000 description 1
- KABHAOSDMIYXTR-GUBZILKMSA-N Cys-Glu-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)N KABHAOSDMIYXTR-GUBZILKMSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 101710096438 DNA-binding protein Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- YNNXQZDEOCYJJL-CIUDSAMLSA-N Gln-Arg-Asp Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)CN=C(N)N YNNXQZDEOCYJJL-CIUDSAMLSA-N 0.000 description 1
- MWLYSLMKFXWZPW-ZPFDUUQYSA-N Gln-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCC(N)=O MWLYSLMKFXWZPW-ZPFDUUQYSA-N 0.000 description 1
- JHPFPROFOAJRFN-IHRRRGAJSA-N Gln-Glu-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O JHPFPROFOAJRFN-IHRRRGAJSA-N 0.000 description 1
- SMLDOQHTOAAFJQ-WDSKDSINSA-N Gln-Gly-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SMLDOQHTOAAFJQ-WDSKDSINSA-N 0.000 description 1
- SXGMGNZEHFORAV-IUCAKERBSA-N Gln-Lys-Gly Chemical compound C(CCN)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N SXGMGNZEHFORAV-IUCAKERBSA-N 0.000 description 1
- XZUUUKNKNWVPHQ-JYJNAYRXSA-N Gln-Phe-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O XZUUUKNKNWVPHQ-JYJNAYRXSA-N 0.000 description 1
- OZEQPCDLCDRCGY-SOUVJXGZSA-N Gln-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCC(=O)N)N)C(=O)O OZEQPCDLCDRCGY-SOUVJXGZSA-N 0.000 description 1
- MQJDLNRXBOELJW-KKUMJFAQSA-N Gln-Pro-Phe Chemical compound N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(O)=O MQJDLNRXBOELJW-KKUMJFAQSA-N 0.000 description 1
- ZGHMRONFHDVXEF-AVGNSLFASA-N Gln-Ser-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ZGHMRONFHDVXEF-AVGNSLFASA-N 0.000 description 1
- JJKKWYQVHRUSDG-GUBZILKMSA-N Glu-Ala-Lys Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O JJKKWYQVHRUSDG-GUBZILKMSA-N 0.000 description 1
- ATRHMOJQJWPVBQ-DRZSPHRISA-N Glu-Ala-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ATRHMOJQJWPVBQ-DRZSPHRISA-N 0.000 description 1
- KKCUFHUTMKQQCF-SRVKXCTJSA-N Glu-Arg-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O KKCUFHUTMKQQCF-SRVKXCTJSA-N 0.000 description 1
- SRZLHYPAOXBBSB-HJGDQZAQSA-N Glu-Arg-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SRZLHYPAOXBBSB-HJGDQZAQSA-N 0.000 description 1
- GCYFUZJHAXJKKE-KKUMJFAQSA-N Glu-Arg-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GCYFUZJHAXJKKE-KKUMJFAQSA-N 0.000 description 1
- ZOXBSICWUDAOHX-GUBZILKMSA-N Glu-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O ZOXBSICWUDAOHX-GUBZILKMSA-N 0.000 description 1
- PCBBLFVHTYNQGG-LAEOZQHASA-N Glu-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N PCBBLFVHTYNQGG-LAEOZQHASA-N 0.000 description 1
- XHUCVVHRLNPZSZ-CIUDSAMLSA-N Glu-Gln-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O XHUCVVHRLNPZSZ-CIUDSAMLSA-N 0.000 description 1
- PVBBEKPHARMPHX-DCAQKATOSA-N Glu-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCC(O)=O PVBBEKPHARMPHX-DCAQKATOSA-N 0.000 description 1
- WLIPTFCZLHCNFD-LPEHRKFASA-N Glu-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)N)C(=O)O WLIPTFCZLHCNFD-LPEHRKFASA-N 0.000 description 1
- HNVFSTLPVJWIDV-CIUDSAMLSA-N Glu-Glu-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HNVFSTLPVJWIDV-CIUDSAMLSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- AUTNXSQEVVHSJK-YVNDNENWSA-N Glu-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O AUTNXSQEVVHSJK-YVNDNENWSA-N 0.000 description 1
- QJCKNLPMTPXXEM-AUTRQRHGSA-N Glu-Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O QJCKNLPMTPXXEM-AUTRQRHGSA-N 0.000 description 1
- OAGVHWYIBZMWLA-YFKPBYRVSA-N Glu-Gly-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)NCC(O)=O OAGVHWYIBZMWLA-YFKPBYRVSA-N 0.000 description 1
- LRPXYSGPOBVBEH-IUCAKERBSA-N Glu-Gly-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O LRPXYSGPOBVBEH-IUCAKERBSA-N 0.000 description 1
- XIKYNVKEUINBGL-IUCAKERBSA-N Glu-His-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(O)=O XIKYNVKEUINBGL-IUCAKERBSA-N 0.000 description 1
- DVLZZEPUNFEUBW-AVGNSLFASA-N Glu-His-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCC(=O)O)N DVLZZEPUNFEUBW-AVGNSLFASA-N 0.000 description 1
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 1
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-N 0.000 description 1
- FBEJIDRSQCGFJI-GUBZILKMSA-N Glu-Leu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FBEJIDRSQCGFJI-GUBZILKMSA-N 0.000 description 1
- AQNYKMCFCCZEEL-JYJNAYRXSA-N Glu-Lys-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AQNYKMCFCCZEEL-JYJNAYRXSA-N 0.000 description 1
- JHSRJMUJOGLIHK-GUBZILKMSA-N Glu-Met-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)O)N JHSRJMUJOGLIHK-GUBZILKMSA-N 0.000 description 1
- UERORLSAFUHDGU-AVGNSLFASA-N Glu-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N UERORLSAFUHDGU-AVGNSLFASA-N 0.000 description 1
- UDEPRBFQTWGLCW-CIUDSAMLSA-N Glu-Pro-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O UDEPRBFQTWGLCW-CIUDSAMLSA-N 0.000 description 1
- HZISRJBYZAODRV-XQXXSGGOSA-N Glu-Thr-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O HZISRJBYZAODRV-XQXXSGGOSA-N 0.000 description 1
- CQGBSALYGOXQPE-HTUGSXCWSA-N Glu-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O CQGBSALYGOXQPE-HTUGSXCWSA-N 0.000 description 1
- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 1
- XCLCVBYNGXEVDU-WHFBIAKZSA-N Gly-Asn-Ser Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O XCLCVBYNGXEVDU-WHFBIAKZSA-N 0.000 description 1
- JMQFHZWESBGPFC-WDSKDSINSA-N Gly-Gln-Asp Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O JMQFHZWESBGPFC-WDSKDSINSA-N 0.000 description 1
- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 description 1
- ZQIMMEYPEXIYBB-IUCAKERBSA-N Gly-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN ZQIMMEYPEXIYBB-IUCAKERBSA-N 0.000 description 1
- DENRBIYENOKSEX-PEXQALLHSA-N Gly-Ile-His Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 DENRBIYENOKSEX-PEXQALLHSA-N 0.000 description 1
- COVXELOAORHTND-LSJOCFKGSA-N Gly-Ile-Val Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O COVXELOAORHTND-LSJOCFKGSA-N 0.000 description 1
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 1
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 1
- WZSHYFGOLPXPLL-RYUDHWBXSA-N Gly-Phe-Glu Chemical compound NCC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(O)=O)C(O)=O WZSHYFGOLPXPLL-RYUDHWBXSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- ZVXMEWXHFBYJPI-LSJOCFKGSA-N Gly-Val-Ile Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZVXMEWXHFBYJPI-LSJOCFKGSA-N 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- CTGZVVQVIBSOBB-AVGNSLFASA-N His-His-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O CTGZVVQVIBSOBB-AVGNSLFASA-N 0.000 description 1
- JUCZDDVZBMPKRT-IXOXFDKPSA-N His-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N)O JUCZDDVZBMPKRT-IXOXFDKPSA-N 0.000 description 1
- NKVZTQVGUNLLQW-JBDRJPRFSA-N Ile-Ala-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)O)N NKVZTQVGUNLLQW-JBDRJPRFSA-N 0.000 description 1
- WECYRWOMWSCWNX-XUXIUFHCSA-N Ile-Arg-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(O)=O WECYRWOMWSCWNX-XUXIUFHCSA-N 0.000 description 1
- IDAHFEPYTJJZFD-PEFMBERDSA-N Ile-Asp-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N IDAHFEPYTJJZFD-PEFMBERDSA-N 0.000 description 1
- XLCZWMJPVGRWHJ-KQXIARHKSA-N Ile-Glu-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N XLCZWMJPVGRWHJ-KQXIARHKSA-N 0.000 description 1
- LPFBXFILACZHIB-LAEOZQHASA-N Ile-Gly-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)O)N LPFBXFILACZHIB-LAEOZQHASA-N 0.000 description 1
- YNMQUIVKEFRCPH-QSFUFRPTSA-N Ile-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)O)N YNMQUIVKEFRCPH-QSFUFRPTSA-N 0.000 description 1
- KLBVGHCGHUNHEA-BJDJZHNGSA-N Ile-Leu-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)O)N KLBVGHCGHUNHEA-BJDJZHNGSA-N 0.000 description 1
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 1
- USXAYNCLFSUSBA-MGHWNKPDSA-N Ile-Phe-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N USXAYNCLFSUSBA-MGHWNKPDSA-N 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- PZWBBXHHUSIGKH-OSUNSFLBSA-N Ile-Thr-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PZWBBXHHUSIGKH-OSUNSFLBSA-N 0.000 description 1
- YCKPUHHMCFSUMD-IUKAMOBKSA-N Ile-Thr-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCKPUHHMCFSUMD-IUKAMOBKSA-N 0.000 description 1
- AUIYHFRUOOKTGX-UKJIMTQDSA-N Ile-Val-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N AUIYHFRUOOKTGX-UKJIMTQDSA-N 0.000 description 1
- YWCJXQKATPNPOE-UKJIMTQDSA-N Ile-Val-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YWCJXQKATPNPOE-UKJIMTQDSA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 1
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 1
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 description 1
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 1
- HBJZFCIVFIBNSV-DCAQKATOSA-N Leu-Arg-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(N)=O)C(O)=O HBJZFCIVFIBNSV-DCAQKATOSA-N 0.000 description 1
- POJPZSMTTMLSTG-SRVKXCTJSA-N Leu-Asn-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N POJPZSMTTMLSTG-SRVKXCTJSA-N 0.000 description 1
- WGNOPSQMIQERPK-GARJFASQSA-N Leu-Asn-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N WGNOPSQMIQERPK-GARJFASQSA-N 0.000 description 1
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 1
- KTFHTMHHKXUYPW-ZPFDUUQYSA-N Leu-Asp-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KTFHTMHHKXUYPW-ZPFDUUQYSA-N 0.000 description 1
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-N 0.000 description 1
- GPICTNQYKHHHTH-GUBZILKMSA-N Leu-Gln-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GPICTNQYKHHHTH-GUBZILKMSA-N 0.000 description 1
- APFJUBGRZGMQFF-QWRGUYRKSA-N Leu-Gly-Lys Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN APFJUBGRZGMQFF-QWRGUYRKSA-N 0.000 description 1
- KEVYYIMVELOXCT-KBPBESRZSA-N Leu-Gly-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KEVYYIMVELOXCT-KBPBESRZSA-N 0.000 description 1
- KOSWSHVQIVTVQF-ZPFDUUQYSA-N Leu-Ile-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O KOSWSHVQIVTVQF-ZPFDUUQYSA-N 0.000 description 1
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 1
- KYIIALJHAOIAHF-KKUMJFAQSA-N Leu-Leu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 KYIIALJHAOIAHF-KKUMJFAQSA-N 0.000 description 1
- ZGUMORRUBUCXEH-AVGNSLFASA-N Leu-Lys-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZGUMORRUBUCXEH-AVGNSLFASA-N 0.000 description 1
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 1
- AIRUUHAOKGVJAD-JYJNAYRXSA-N Leu-Phe-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIRUUHAOKGVJAD-JYJNAYRXSA-N 0.000 description 1
- JIHDFWWRYHSAQB-GUBZILKMSA-N Leu-Ser-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JIHDFWWRYHSAQB-GUBZILKMSA-N 0.000 description 1
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 1
- DAYQSYGBCUKVKT-VOAKCMCISA-N Leu-Thr-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O DAYQSYGBCUKVKT-VOAKCMCISA-N 0.000 description 1
- XZNJZXJZBMBGGS-NHCYSSNCSA-N Leu-Val-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XZNJZXJZBMBGGS-NHCYSSNCSA-N 0.000 description 1
- HKCCVDWHHTVVPN-CIUDSAMLSA-N Lys-Asp-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O HKCCVDWHHTVVPN-CIUDSAMLSA-N 0.000 description 1
- QQUJSUFWEDZQQY-AVGNSLFASA-N Lys-Gln-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCCN QQUJSUFWEDZQQY-AVGNSLFASA-N 0.000 description 1
- IRRZDAIFYHNIIN-JYJNAYRXSA-N Lys-Gln-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IRRZDAIFYHNIIN-JYJNAYRXSA-N 0.000 description 1
- GJJQCBVRWDGLMQ-GUBZILKMSA-N Lys-Glu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O GJJQCBVRWDGLMQ-GUBZILKMSA-N 0.000 description 1
- ULUQBUKAPDUKOC-GVXVVHGQSA-N Lys-Glu-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ULUQBUKAPDUKOC-GVXVVHGQSA-N 0.000 description 1
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 1
- GQFDWEDHOQRNLC-QWRGUYRKSA-N Lys-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN GQFDWEDHOQRNLC-QWRGUYRKSA-N 0.000 description 1
- JYXBNQOKPRQNQS-YTFOTSKYSA-N Lys-Ile-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JYXBNQOKPRQNQS-YTFOTSKYSA-N 0.000 description 1
- ONPDTSFZAIWMDI-AVGNSLFASA-N Lys-Leu-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ONPDTSFZAIWMDI-AVGNSLFASA-N 0.000 description 1
- RBEATVHTWHTHTJ-KKUMJFAQSA-N Lys-Leu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O RBEATVHTWHTHTJ-KKUMJFAQSA-N 0.000 description 1
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 1
- YUAXTFMFMOIMAM-QWRGUYRKSA-N Lys-Lys-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O YUAXTFMFMOIMAM-QWRGUYRKSA-N 0.000 description 1
- PLDJDCJLRCYPJB-VOAKCMCISA-N Lys-Lys-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PLDJDCJLRCYPJB-VOAKCMCISA-N 0.000 description 1
- MGKFCQFVPKOWOL-CIUDSAMLSA-N Lys-Ser-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)O)N MGKFCQFVPKOWOL-CIUDSAMLSA-N 0.000 description 1
- PLOUVAYOMTYJRG-JXUBOQSCSA-N Lys-Thr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PLOUVAYOMTYJRG-JXUBOQSCSA-N 0.000 description 1
- JHNOXVASMSXSNB-WEDXCCLWSA-N Lys-Thr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JHNOXVASMSXSNB-WEDXCCLWSA-N 0.000 description 1
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 1
- WINFHLHJTRGLCV-BZSNNMDCSA-N Lys-Tyr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=C(O)C=C1 WINFHLHJTRGLCV-BZSNNMDCSA-N 0.000 description 1
- IKXQOBUBZSOWDY-AVGNSLFASA-N Lys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CCCCN)N IKXQOBUBZSOWDY-AVGNSLFASA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 1
- HUKLXYYPZWPXCC-KZVJFYERSA-N Met-Ala-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HUKLXYYPZWPXCC-KZVJFYERSA-N 0.000 description 1
- ZAJNRWKGHWGPDQ-SDDRHHMPSA-N Met-Arg-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N ZAJNRWKGHWGPDQ-SDDRHHMPSA-N 0.000 description 1
- TUSOIZOVPJCMFC-FXQIFTODSA-N Met-Asp-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O TUSOIZOVPJCMFC-FXQIFTODSA-N 0.000 description 1
- DJDFBVNNDAUPRW-GUBZILKMSA-N Met-Glu-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O DJDFBVNNDAUPRW-GUBZILKMSA-N 0.000 description 1
- RNAGAJXCSPDPRK-KKUMJFAQSA-N Met-Glu-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 RNAGAJXCSPDPRK-KKUMJFAQSA-N 0.000 description 1
- DGNZGCQSVGGYJS-BQBZGAKWSA-N Met-Gly-Asp Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O DGNZGCQSVGGYJS-BQBZGAKWSA-N 0.000 description 1
- LRALLISKBZNSKN-BQBZGAKWSA-N Met-Gly-Ser Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LRALLISKBZNSKN-BQBZGAKWSA-N 0.000 description 1
- FWAHLGXNBLWIKB-NAKRPEOUSA-N Met-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCSC FWAHLGXNBLWIKB-NAKRPEOUSA-N 0.000 description 1
- HGAJNEWOUHDUMZ-SRVKXCTJSA-N Met-Leu-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O HGAJNEWOUHDUMZ-SRVKXCTJSA-N 0.000 description 1
- HAQLBBVZAGMESV-IHRRRGAJSA-N Met-Lys-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O HAQLBBVZAGMESV-IHRRRGAJSA-N 0.000 description 1
- CNAGWYQWQDMUGC-IHRRRGAJSA-N Met-Phe-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CNAGWYQWQDMUGC-IHRRRGAJSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010017301 Mycoplasma pneumoniae CARDS toxin Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- DFEVBOYEUQJGER-JURCDPSOSA-N Phe-Ala-Ile Chemical compound N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O DFEVBOYEUQJGER-JURCDPSOSA-N 0.000 description 1
- UHRNIXJAGGLKHP-DLOVCJGASA-N Phe-Ala-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O UHRNIXJAGGLKHP-DLOVCJGASA-N 0.000 description 1
- MQWISMJKHOUEMW-ULQDDVLXSA-N Phe-Arg-His Chemical compound C([C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=CC=C1 MQWISMJKHOUEMW-ULQDDVLXSA-N 0.000 description 1
- JIYJYFIXQTYDNF-YDHLFZDLSA-N Phe-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N JIYJYFIXQTYDNF-YDHLFZDLSA-N 0.000 description 1
- WLYPRKLMRIYGPP-JYJNAYRXSA-N Phe-Lys-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 WLYPRKLMRIYGPP-JYJNAYRXSA-N 0.000 description 1
- MMJJFXWMCMJMQA-STQMWFEESA-N Phe-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CC=CC=C1 MMJJFXWMCMJMQA-STQMWFEESA-N 0.000 description 1
- VDGTVWFMRXVQCT-GUBZILKMSA-N Pro-Glu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 VDGTVWFMRXVQCT-GUBZILKMSA-N 0.000 description 1
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 1
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 1
- XQPHBAKJJJZOBX-SRVKXCTJSA-N Pro-Lys-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O XQPHBAKJJJZOBX-SRVKXCTJSA-N 0.000 description 1
- RFWXYTJSVDUBBZ-DCAQKATOSA-N Pro-Pro-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RFWXYTJSVDUBBZ-DCAQKATOSA-N 0.000 description 1
- KHRLUIPIMIQFGT-AVGNSLFASA-N Pro-Val-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHRLUIPIMIQFGT-AVGNSLFASA-N 0.000 description 1
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- LVVBAKCGXXUHFO-ZLUOBGJFSA-N Ser-Ala-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O LVVBAKCGXXUHFO-ZLUOBGJFSA-N 0.000 description 1
- FCRMLGJMPXCAHD-FXQIFTODSA-N Ser-Arg-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O FCRMLGJMPXCAHD-FXQIFTODSA-N 0.000 description 1
- HEQPKICPPDOSIN-SRVKXCTJSA-N Ser-Asp-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HEQPKICPPDOSIN-SRVKXCTJSA-N 0.000 description 1
- MUARUIBTKQJKFY-WHFBIAKZSA-N Ser-Gly-Asp Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MUARUIBTKQJKFY-WHFBIAKZSA-N 0.000 description 1
- KCGIREHVWRXNDH-GARJFASQSA-N Ser-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N KCGIREHVWRXNDH-GARJFASQSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- 240000002033 Tacca leontopetaloides Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- DFTCYYILCSQGIZ-GCJQMDKQSA-N Thr-Ala-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O DFTCYYILCSQGIZ-GCJQMDKQSA-N 0.000 description 1
- GFDUZZACIWNMPE-KZVJFYERSA-N Thr-Ala-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O GFDUZZACIWNMPE-KZVJFYERSA-N 0.000 description 1
- RKDFEMGVMMYYNG-WDCWCFNPSA-N Thr-Gln-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O RKDFEMGVMMYYNG-WDCWCFNPSA-N 0.000 description 1
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 1
- MPUMPERGHHJGRP-WEDXCCLWSA-N Thr-Gly-Lys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O MPUMPERGHHJGRP-WEDXCCLWSA-N 0.000 description 1
- JKGGPMOUIAAJAA-YEPSODPASA-N Thr-Gly-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O JKGGPMOUIAAJAA-YEPSODPASA-N 0.000 description 1
- AYCQVUUPIJHJTA-IXOXFDKPSA-N Thr-His-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O AYCQVUUPIJHJTA-IXOXFDKPSA-N 0.000 description 1
- MEJHFIOYJHTWMK-VOAKCMCISA-N Thr-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)O MEJHFIOYJHTWMK-VOAKCMCISA-N 0.000 description 1
- TZJSEJOXAIWOST-RHYQMDGZSA-N Thr-Lys-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N TZJSEJOXAIWOST-RHYQMDGZSA-N 0.000 description 1
- MGJLBZFUXUGMML-VOAKCMCISA-N Thr-Lys-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MGJLBZFUXUGMML-VOAKCMCISA-N 0.000 description 1
- UXUAZXWKIGPUCH-RCWTZXSCSA-N Thr-Met-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCSC)C(O)=O UXUAZXWKIGPUCH-RCWTZXSCSA-N 0.000 description 1
- XKWABWFMQXMUMT-HJGDQZAQSA-N Thr-Pro-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XKWABWFMQXMUMT-HJGDQZAQSA-N 0.000 description 1
- VTMGKRABARCZAX-OSUNSFLBSA-N Thr-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O VTMGKRABARCZAX-OSUNSFLBSA-N 0.000 description 1
- NDLHSJWPCXKOGG-VLCNGCBASA-N Thr-Trp-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N)O NDLHSJWPCXKOGG-VLCNGCBASA-N 0.000 description 1
- BZTSQFWJNJYZSX-JRQIVUDYSA-N Thr-Tyr-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O BZTSQFWJNJYZSX-JRQIVUDYSA-N 0.000 description 1
- RPECVQBNONKZAT-WZLNRYEVSA-N Thr-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H]([C@@H](C)O)N RPECVQBNONKZAT-WZLNRYEVSA-N 0.000 description 1
- XGFYGMKZKFRGAI-RCWTZXSCSA-N Thr-Val-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XGFYGMKZKFRGAI-RCWTZXSCSA-N 0.000 description 1
- WKCFCVBOFKEVKY-HSCHXYMDSA-N Trp-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N WKCFCVBOFKEVKY-HSCHXYMDSA-N 0.000 description 1
- HKIUVWMZYFBIHG-KKUMJFAQSA-N Tyr-Arg-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O HKIUVWMZYFBIHG-KKUMJFAQSA-N 0.000 description 1
- NZFCWALTLNFHHC-JYJNAYRXSA-N Tyr-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NZFCWALTLNFHHC-JYJNAYRXSA-N 0.000 description 1
- FNWGDMZVYBVAGJ-XEGUGMAKSA-N Tyr-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC1=CC=C(C=C1)O)N FNWGDMZVYBVAGJ-XEGUGMAKSA-N 0.000 description 1
- JKUZFODWJGEQAP-KBPBESRZSA-N Tyr-Gly-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O JKUZFODWJGEQAP-KBPBESRZSA-N 0.000 description 1
- NOOMDULIORCDNF-IRXDYDNUSA-N Tyr-Gly-Phe Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NOOMDULIORCDNF-IRXDYDNUSA-N 0.000 description 1
- ARJASMXQBRNAGI-YESZJQIVSA-N Tyr-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N ARJASMXQBRNAGI-YESZJQIVSA-N 0.000 description 1
- XDGPTBVOSHKDFT-KKUMJFAQSA-N Tyr-Met-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O XDGPTBVOSHKDFT-KKUMJFAQSA-N 0.000 description 1
- IEWKKXZRJLTIOV-AVGNSLFASA-N Tyr-Ser-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O IEWKKXZRJLTIOV-AVGNSLFASA-N 0.000 description 1
- LUMQYLVYUIRHHU-YJRXYDGGSA-N Tyr-Ser-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LUMQYLVYUIRHHU-YJRXYDGGSA-N 0.000 description 1
- ABSXSJZNRAQDDI-KJEVXHAQSA-N Tyr-Val-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ABSXSJZNRAQDDI-KJEVXHAQSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 1
- JFAWZADYPRMRCO-UBHSHLNASA-N Val-Ala-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JFAWZADYPRMRCO-UBHSHLNASA-N 0.000 description 1
- CVUDMNSZAIZFAE-UHFFFAOYSA-N Val-Arg-Pro Natural products NC(N)=NCCCC(NC(=O)C(N)C(C)C)C(=O)N1CCCC1C(O)=O CVUDMNSZAIZFAE-UHFFFAOYSA-N 0.000 description 1
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 1
- TZVUSFMQWPWHON-NHCYSSNCSA-N Val-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N TZVUSFMQWPWHON-NHCYSSNCSA-N 0.000 description 1
- SYOMXKPPFZRELL-ONGXEEELSA-N Val-Gly-Lys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N SYOMXKPPFZRELL-ONGXEEELSA-N 0.000 description 1
- WJVLTYSHNXRCLT-NHCYSSNCSA-N Val-His-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N WJVLTYSHNXRCLT-NHCYSSNCSA-N 0.000 description 1
- ZIGZPYJXIWLQFC-QTKMDUPCSA-N Val-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N)O ZIGZPYJXIWLQFC-QTKMDUPCSA-N 0.000 description 1
- CPGJELLYDQEDRK-NAKRPEOUSA-N Val-Ile-Ala Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](C)C(O)=O CPGJELLYDQEDRK-NAKRPEOUSA-N 0.000 description 1
- GVJUTBOZZBTBIG-AVGNSLFASA-N Val-Lys-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N GVJUTBOZZBTBIG-AVGNSLFASA-N 0.000 description 1
- WBAJDGWKRIHOAC-GVXVVHGQSA-N Val-Lys-Gln Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O WBAJDGWKRIHOAC-GVXVVHGQSA-N 0.000 description 1
- DIOSYUIWOQCXNR-ONGXEEELSA-N Val-Lys-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O DIOSYUIWOQCXNR-ONGXEEELSA-N 0.000 description 1
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-N 0.000 description 1
- HPANGHISDXDUQY-ULQDDVLXSA-N Val-Lys-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HPANGHISDXDUQY-ULQDDVLXSA-N 0.000 description 1
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 1
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 1
- VIKZGAUAKQZDOF-NRPADANISA-N Val-Ser-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O VIKZGAUAKQZDOF-NRPADANISA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- HVRRJRMULCPNRO-BZSNNMDCSA-N Val-Trp-Arg Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 HVRRJRMULCPNRO-BZSNNMDCSA-N 0.000 description 1
- PFMSJVIPEZMKSC-DZKIICNBSA-N Val-Tyr-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PFMSJVIPEZMKSC-DZKIICNBSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010038850 arginyl-isoleucyl-tyrosine Proteins 0.000 description 1
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical group 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 108010054812 diprotin A Proteins 0.000 description 1
- 108010054813 diprotin B Proteins 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 1
- 108010062266 glycyl-glycyl-argininal Proteins 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 108010041601 histidyl-aspartyl-glutamyl-leucine Proteins 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 108010027338 isoleucylcysteine Proteins 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 108010045397 lysyl-tyrosyl-lysine Proteins 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 108010024607 phenylalanylalanine Proteins 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 101150055096 polA gene Proteins 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 108010079317 prolyl-tyrosine Proteins 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 108010005652 splenotritin Proteins 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
Abstract
本发明公开了一种融合DNA聚合酶突变体及其在等温扩增中的应用，为来自基于Bacillus stearothermophilus的DNA聚合酶改造的突变体或衍生物，野生型DNA聚合酶中，定点氨基酸突变的位点和氨基酸为Arg596、Asp598、Gly600、Ile716、Asp718、Try719中的两种或多种，称之为新型融合DNA聚合酶突变体，所述新型融合DNA聚合酶突变体具有高链置换活性的优点，在恒温条件下即可加速解开DNA双螺旋结构，解链效率更高，用于恒温扩增时其实现目标基因的高效快速复制，用于核酸扩增检测速度更快，效率更高。
Description
技术领域
本发明属于DNA聚合酶突变及其扩增技术，具体涉及一种新型融合高解链扩增性能的DNA聚合酶突变体或衍生物，及其在链置换等温扩增中的应用。
背景技术
以聚合酶链反应（PCR）扩增为基础的分子诊断在特异性、灵敏度和检测时间方面可以满足必要的诊断要求；但其涉及多个操作步骤，需要具有较强操作技能的专业技术人员来进行样品制备，DNA扩增和检测。另外，PCR需要精确的热循环仪来进行PCR反应。而等温核酸扩增技术与传统的聚合酶链反应相比不需要热循环，更适合在特定的恒定温度下进行即时检测，等温核酸扩增代表了一种极具潜力的技术，该技术可以针对传染病提供快速，灵敏和特定的分子诊断 (Caliendo AM, 2013)。在绝大多数基于恒温扩增方法中，有效的靶标扩增取决于扩增步骤中使用的DNA聚合酶的固有链置换活性 (Craw P, 2012) (Gill P,2008)。链置换反应是指一种DNA聚合酶在延伸新链的过程中如果遇到下游DNA链，可以继续延伸反应并同时将下游双链剥离而产生游离的单链，这种链置换能力替代了传统上通过物理高温实现的双链解离。等温扩增以其独特的自身优势开辟了广阔的市场。DNA聚合酶基于其氨基酸序列和结构同源性已被分为七个家族（A，B，C，D，X，Y，RT） (Burgers PM, 2001)。这些不同的家族具有执行其在核酸代谢中不同生物学作用所需的独特结构和功能特性；其中A家族DNA聚合酶主要存在于原核生物中，来自原核生物的DNA聚合酶I就属于该家族。DNA聚合酶I高解析度的晶体结构已经被确定包括Taq聚合酶I (Kim, 1995) (Li, 1998)，大肠杆菌聚合酶I (Beese, 1993) (Ollis, 1985)，Bst聚合酶 (Kiefer, 1998)和噬菌体T7DNA聚合酶 (Doublie, 1998)，它的结构可以用人的右手来描述，具有三个子域，分别称为拇指（thumb），手指（finger）和手掌（palm）。手指（finger）亚域主要功能是结合刚进入的核苷酸和与单链模板相互作用，手掌（palm）亚域催化氨基酸残基和结合刚进入的dNTP，拇指（thumb）亚域则结合双链DNA。
一种具有高聚合活性和高链置换活性的DNA聚合酶对于等温核酸扩增是非常关键的，然而目前只能选择那些具有固有链置换活性的DNA聚合酶，这些酶能够在特定温度下进行DNA合成，但是对于其扩增聚合能力和DNA链置换能力方面仍有待改善。
发明内容
野生型来自Bacillus stearothermophilus的Bst DNA聚合酶I因其最适温度在60℃左右和固有的链置换活性而被广泛用于等温扩增，并且在临床快速检测病毒感染中起着关键作用。与其他DNA聚合酶I类似，Bst DNA聚合酶I有三个独立的子域，分别执行生物学功能：（I）5'-3'核酸外切酶活性（位于N端），（II）5'-3'DNA聚合酶活性（位于C端）和（III）3'-5'核酸外切酶活性。结构域II和III位于Bst DNA聚合酶的C末端，称为大片段（LargeFragment, LF）。由于Bst DNA聚合酶满足高灵敏度，聚合活性和热稳定性的所有要求，因此已被广泛应用于快速检测的等温扩增过程中，但是效率以及链置换能力还需要进一步提升。
本发明采用如下技术方案：
一种融合DNA聚合酶突变体，所述融合DNA聚合酶突变体包括融合Sso7d结构域与基于野生型DNA聚合酶基础上改造的突变体或衍生物；所述野生型DNA聚合酶为来自Bacillus stearothermophilus的DNA聚合酶；所述野生型DNA聚合酶经过定点氨基酸突变得到所述基于野生型DNA聚合酶基础上改造的突变体或衍生物；所述野生型DNA聚合酶中，定点氨基酸突变的位点和氨基酸为Arg596、Asp598、Gly600、Ile716、Asp718、Try719中的两种或多种。
本发明中，所述融合结构域为融合Sso7d结构域；所述野生型DNA聚合酶为来自Bacillus stearothermophilus（Bst）的DNA聚合酶；基于野生型DNA聚合酶的突变体为在野生型DNA聚合酶的氨基酸序列上进行至少两个氨基酸位点取代；氨基酸位点为Arg596、Asp598、Gly600、Ile716、Asp718、Try719，其中字母代表通用氨基酸，数字代表位于野生型DNA聚合酶的序列位置。
本发明中，所述定点氨基酸突变为Arg596、Asp598、Gly600、Ile716、Asp718、Try719中的至少两种分别进行定点氨基酸突变，优选的，所述定点氨基酸突变采用以下（1）～（6）种取代方式中的至少两种进行取代：
（1）596位R（Arg）被取代为K（Lys）、G（Gly）或者E（Glu）；
（2）598位D（Asp）被取代为V（Val）或者E（Glu）；
（3）600位G（Gly）被取代为K（Lys）、R（Arg）或者E（Glu）；
（4）716位I（Ile）被取代为V（Val）、F（Phe）或者L（Leu）；
（5）718位D（Asp）被取代为K（Lys）、A（Ala）、T（Thr）或者E（Glu）；
（6）719位Y（Try）被取代为F（Phe）。
上述新型融合DNA聚合酶突变体在等温扩增中的应用。
本发明公开了一种核酸等温扩增体系，包括上述新型融合DNA聚合酶突变体、扩增模板与常规扩增试剂。常规扩增试剂比如引物对、探针、二价阳离子（镁离子、锰离子、钴离子等）、Tris缓冲液等，都为本领域常规试剂。
本发明公开了一种核酸等温扩增用试剂盒，包括上述新型融合DNA聚合酶突变体与常规组分。常规组分比如引物对、探针、二价阳离子（镁离子、锰离子、钴离子等）、Tris缓冲液等，都为本领域常规试剂。
一种核酸等温扩增的方法，以核酸为模板、上述新型融合DNA聚合酶突变体为链置换扩增所必须的酶，在常规扩增试剂中，等温扩增，完成核酸等温扩增。常规扩增试剂比如引物对、探针、二价阳离子（镁离子、锰离子、钴离子等）、Tris缓冲液等，都为本领域常规试剂。具体扩增程序以及其他组分与现有Bst DNA聚合酶一致。
本发明的创造性在于公开了新型融合DNA聚合酶突变体，替换现有DNA聚合酶，改善了扩增聚合能力和DNA链置换能力。
本发明公开的新型融合DNA聚合酶突变体具有氨基酸序列，可以通过常规原核或真核生物表达纯化制备而得；因此本发明要求保护上述新型融合DNA聚合酶突变体的氨基酸序列。
野生型DNA聚合酶在续进性和抑制剂耐受性方面的改善可以显著提高酶在各种实践中的应用。续进性是酶催化反应而不与DNA模板解离的能力，这是DNA长时间合成的关键特征，它决定了DNA产物的数量和质量。本发明公开的新型融合DNA聚合酶突变体，具有至少2处定点氨基酸突变，与未突变野生型DNA聚合酶（来自Bacillus stearothermophilus（Bst）的DNA聚合酶）相比，具有高链置换活性的优点，在恒温条件下即可加速解开DNA双螺旋结构，解链效率更高，用于恒温扩增时其实现目标基因的高效快速复制，用于核酸扩增检测速度更快，效率更高。
本发明DNA聚合酶改造工程中一个有效的策略是构建融合DNA结构域蛋白，它来自天然蛋白并具有所需特征的蛋白结构域，与其他DNA结合结构域/完整蛋白的结合大大提高了酶的续进性，改造后的聚合酶能够比野生DNA聚合酶合成更长的DNA片段，本发明利用融合设计的策略将来自嗜热古细菌Sulfolobus solfataricus的Sso7d蛋白与Bst DNA聚合酶连接，获得的融合DNA聚合酶突变体续进性大大提高，除此之外，DNA结合蛋白的连接还增强了融合DNA聚合酶突变体在等温扩增中对几种常见DNA污染物的抑制剂耐受性，例如：尿素，全血，肝素，EDTA，NaCl和乙醇等。
Bst DNA聚合酶活性位点中有四个保守氨基酸残基，分别是Gly600，Arg702，Lys706和Asp830，对DNA引物末端和dNTP结合催化聚合酶反应具有极其重要的作用，对其进行多种定点氨基酸突变，从而影响聚合活性和解链活性。DNA聚合反应最重要的特征之一就是保真度。该酶的X射线晶体结构揭示了其与模板，引物和刚进入dNTP 的相互作用可能会影响保真度。DNA聚合酶的三个结构域共同形成一个与模板引物结合的U型裂缝，裂口的底部包含许多聚合酶中高度保守且对催化重要的羧酸残基，由“手指”和“拇指”子域形成的裂口壁还包含许多高度保守的残基，据推测具有重要功能。通过对Bst聚合酶的结构和生化研究表明，聚合酶结构域中的氨基酸可能通过错误的插入引发起始错误。这些残基是手指子域螺旋O中的残基，该残基面对裂口并被认为与刚进入dNTP（Arg702，Lys706和Phe710）或单链模板（Try714）接触。如图1所示。在这些位置上对氨基酸进行突变后聚合酶特性发生了实质上的改变，包括改变对正确/错误核苷酸的辨别力。Y714F突变酶具有与野生型相似的掺入特性，而Y714S聚合酶比野生型能够更有效的插入错误的核苷酸，因此核苷酸错误插入和链滑动机制对于聚合酶非保真性受具有相似亚域结构的DNA聚合酶独特结构原件变化的不同影响。
DNA聚合酶I的手指亚结构域中存在的Ser769，Phe771和Arg841共同参与促进了链置换DNA合成，其中Ser769和Phe771一起通过形成瓣状结构参与链分离，Arg841通过与模板链相互作用来实现最佳的链分离和DNA合成。Ser769和Phe771氨基酸残基组成的O1-螺旋，与O和O2螺旋一起形成3-螺旋束结构，在DNA和RNA聚合酶中都采用了该3-螺旋束基序来实现链分离功能。Phe771相当于Bacillus stearothermophilus Bst的催化活性DNA结合二元复合物中的Tyr719，手指亚结构域的O和O1-螺旋中的残基对于聚合酶功能具有重要影响，O1-螺旋中特别存在的残基对于链置换合成至关重要。
通过对Bst DNA聚合酶的三维结构研究表明，与718位相对应的氨基酸残基位于短的α-螺旋上，即O1-螺旋。该α-螺旋由44个氨基酸残基组成，是指向拇指亚结构的DNA聚合酶I大片段的手指亚结构域的一部分。根据右手规则，可以将其与通过食指和中指尖端的线进行比较。O1-螺旋的三维空间中相邻的二级结构元素是O和O2螺旋，它们一起形成3-螺旋束。序列分析发现其具有高度序列同一性，718位天冬氨酸具有保守性。通过定点突变将天冬氨酸残基更改为丙氨酸残基，与野生型相比，D718A变体链置换活性增加了2.5倍。
DNA聚合酶的三维结构表明，D718侧链与位于O2螺旋上的R729侧链之间的相互作用是在被覆盖的氨基酸序列内唯一直接相互作用的3-螺旋束。R729的NH1与D718的OD1之间存在盐桥，除此之外，3-螺旋束中的相互作用是基于骨架原子之间的氢键或疏水相互作用，用非阴离子侧链取代Asp侧链会破坏与3-螺旋束基序中Arg侧链形成的盐桥，从而提供一个更灵活的区域。聚合酶Arg729处于保守状态，因此观察到的酶链置换活性增加可能是由于柔韧性的局部增加所致。
本发明公开的新型融合DNA聚合酶突变体置换DNA合成效率可达到相比较野生型其链置换活性明显增加，大大提升了扩增效率，在基于链置换反应的核酸扩增技术中大大提高了链置换扩增活性，增加了检测的灵敏度和扩增速度。
附图说明
图1为Bst DNA聚合酶活性中心图；
图2为pETsso-BstPol表达载体示意图；
图3为新型融合DNA聚合酶与NEB Bst DNA聚合酶3.0、野生型DNA聚合酶延伸对比度图；
图4为新型融合DNA聚合酶与对照酶针对绵羊肺炎支原体基因的LAMP扩增结果；
图5为新型融合DNA聚合酶与对照酶针对肺炎支原体毒素基因M129的LAMP扩增结果；
图6为新型融合DNA聚合酶与NEB Bst DNA 聚合酶3.0不同用量条件下的切口延伸扩增技术检测低拷贝数模板对比结果；
图7为新型融合DNA聚合酶与对照酶的切口延伸扩增技术检测低拷贝数模板结果。
具体实施方式
本发明公开了一种新型融合DNA聚合酶突变体，具有至少2处定点氨基酸突变，具有高链置换活性的优点，具体的，对Bacillus stearothermophilus DNA聚合酶（野生型BstDNA聚合酶）进行了人为改造，将DNA结合结构域Sso7d蛋白与Bst DNA聚合酶融合，并对保守位点Arg596，Asp598，Gly600，Ile716，Asp718，Try719中的至少两种分别进行定向取代；在恒温条件下即可加速解开DNA双螺旋结构，解链效率更高，用于恒温扩增时其实现目标基因的高效快速复制，用于核酸扩增检测速度更快，效率更高。
在野生型Bst DNA聚合酶氨基酸序列的基础上，本发明采用以下（1）～（6）种取代方式中的至少两种进行取代（数字位置为野生型DNA聚合酶全长位置）：
（1）596位R被取代为K、G或者E；
（2）598位D被取代为V或者E；
（3）600位G被取代为K、R或者E；
（4）716位I被取代为V、F或者L；
（5）718位D被取代为K、A、T或者E；
（6）719位Y被取代为F。
本发明涉及的具体方法，比如克隆与PCR扩增实验均为常见分子生物常规实验，所采用的实验条件和方法在非特别说明情况下，均参考《分子克隆实验指南（第四版）》 进行（科学出版社，作者J.萨姆布鲁克、M.R.格林，译者为贺福初）。
下面结合实施例与附图进一步说明，涉及的原料、具体方法都为本领域常规技术；本发明新型融合DNA聚合酶突变体可以通过化学合成法制备双链DNA基因，并克隆至表达载体，转化大肠杆菌，诱导表达和纯化制备相应的融合DNA聚合酶突变体，具体在本发明给出序列的基础上，为常规技术；也可以通过野生型Bst DNA聚合酶大片段定向突变、测序得到，具体在本发明给出序列的基础上，为常规技术。在本发明部分对比实验中，融合DNA聚合酶突变体（或对照的野生型DNA聚合酶，NEB DNA聚合酶 3.0等）的加入量一样，为平行实验。
实施例一
以野生型DNA聚合酶（由GenBank: U33536.1核酸对应编码氨基酸序列纯化而得，称为野生型Bst DNA聚合酶）为基础，经过基因工程定向突变取代，得到本发明新型融合DNA聚合酶突变体。
其中野生型Bst DNA聚合酶的氨基酸序列为：
MKKKLVLIDGNSVAYRAFFALPLLHNDKGIHTNAVYGFTMMLNKILAEEQPTHLLVAFDAGKTTFRHETFQEYKGGRQQTPPELSEQFPLLRELLKAYRIPAYELDHYEADDIIGTLAARAEQEGFEVKIISGDRDLTQLASRHVTVDITKKGITDIEPYTPETVREKYGLTPEQIVDLKGLMGDKSDNIPGVPGIGEKTAVKLLKQFGTVENVLASIDEVKGEKLKENLRQHRDLALLSKQLASICRDAPVELSLDDIVYEGQDREKVIALFKELGFQSFLEKMAAPAAEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK
新型融合DNA聚合酶突变体具体取代为：
Sso7d蛋白结构域的蛋白氨基酸序列为:
matvkfkykgeekevdiskikkvwrvgkmisftydegggktgrgavsekdapkellqmlekqkk
对应碱基序列为：
CCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCGCAACAGTAAAGTTCAAGTACAAGGGAGAAGAGAAGGAAGTAGATATAAGTAAGATAAAGAAGGTATGGAGAGTAGGCAAAATGATAAGTTTCACCTATGATGAGGGTGGAGGAAAGACTGGTAGAGGAGCTGTAAGCGAGAAAGACGCTCCAAAAGAACTACTACAAATGTTAGAAAAGCAAAAGAAACATATG
Sso7d蛋白结构域的C端与基于野生型DNA聚合酶基础上改造的突变体或衍生物N端融合，得到本发明新型融合DNA聚合酶突变体。
将上述Sso7d蛋白结构域的DNA双链片段分别通过克隆位点Nco I和Nde I克隆至pET28a载体上（所采用的限制性内切酶均采用New England Biolabs产品，下同）。经测序验证克隆正确后，作为pETsso 载体，用于下游克隆。
设计如下引物：
分别利用双向引物Bst-fv1-F/Bst-fv1-R和双向引物Bst-fv3-F/Bst-fv3-R针对野生型Bacillus stearothermophilus基因组进行扩增。PCR扩增体系：模板DNA 1 μL，F-引物2μL（母液浓度10pmol/uL），R-引物2 μL（母液浓度10pmol/uL），dNTP (25 mM each) 0.6 μL，10×pfu buffer 5 μL，pfu DNA polymerase 0.5 μL，DMSO 2.5 μL，用ddH2O补充至50 μL，混匀。
PCR扩增反应参数：94 ℃预变性5 min；94 ℃变性30 s，52 ℃退火30s，72 ℃延伸1 min，30个循环；72 ℃终延伸10 min。
扩增后，通过1.5％琼脂糖凝胶电泳，使用DNA凝胶提取试剂盒（Sangon，Shanghai）纯化。根据Sangon DNA凝胶回收试剂盒说明书对PCR扩增产物进行纯化。扩增后分别回收900bp和444bp的PCR产物，将回收后产物作为模板，并将片段分别利用Nde I/EcoRI双酶切和EcoR I/Xho I双酶切进行片段消化，并将pETsso 载体进行Nde I/Xho I双酶切。分别回收两个酶切片段和酶切载体，并用T4 DNA连接酶将双片段连接，具体连接方式为常规技术，比如：
用酶切后质粒和外源PCR产物片段，按如下所述设立连接反应体系：将0.1 μg载体DNA转移到无菌的EP管中，加3 倍摩尔的外源DNA。加水补至8.0 μL，于45 ℃加温5 分钟以便重新退火的粘端解链，将混合物冷却至0 ℃。加入：10×T4 DNA连接酶缓冲液 1 μL，T4 DNA连接酶1 μL，于22 ℃温育2 小时。
将连接产物转化E.coli TOP10感受态细胞：取50 μL冰浴上融化的感受态细胞，加入连接产物，轻轻混匀，在冰浴上放置30分钟。42℃水浴热激45秒，然后快速将管转移到冰浴中静置2分钟。向每个离心管中加入500 μL无菌的LB培养基，混匀至于37℃，200rpm培养一个小时，使细菌复苏。吸取100 μL菌液转移到LB平板固体培养基（含30 μg/ml Kan）表面，用涂布棒涂布均匀，于37 ℃恒温箱倒置静置过夜培养。
挑取5个克隆进行测序验证所获得目标片段均与预期片段大小一致后，标记质粒载体为pETsso-Bstf13。该载体进一步采用Apa I和EcoR I双酶切后，载体片段回收，用于下一步PCR扩增的克隆载体。
利用双向引物Bst-fv2-F/Bst-fv2-R针对模板进行扩增（反应条件与Bst-fv1-F/Bst-fv1-R相同）。扩增后回收410bp条带，用EcoR I单酶切回收后并克隆至上述同样双酶切线性化的pETsso-Bstf13载体上，所得到混合连接产物编号命名为pETsso-BstPol，其示意图见图2。将混合连接产物进行转化，所采用的感受态细胞为One Shot® BL21 Star™(DE3) E. coli（Thermo Fisher，目录号：C6010-03），操作步骤与方法严格参考使用说明书。
将转化菌落进行铺平板（15cm x 15cm），共计100只平板。37°C培养过夜后，挑取单菌落50个96孔的深孔培养板上进行培养，每个孔0.8ml培养基，培养4个小时后将培养板取出，分别对应孔位保种，每个50 ul菌液；剩余培养基加入终浓度为1 mmol/L IPTG诱导后继续培养3小时；保种菌液另行培养后，抽提质粒并进行一代sanger测序，确定对应的DNA聚合酶突变序列。
将96孔深孔板取出后进行整版离心，5000 rpm 离心5 min，移液器吸出上清后，PBS缓冲液洗涤并通过反复冻融裂解菌体，每一个孔中加入500 ul裂解液（20 mM TrispH7.5，250 mM NaCl，10 mM咪唑，0.2 mg/ml溶菌酶，1mg/mlDOC）溶解目标蛋白。高速离心后将上清液转移至新的96孔深孔板，每个孔中加入Ni-NTA孵育1 h，重组蛋白充分结合至Ni介质上后，分别用含咪唑的洗脱缓冲液（20 mM Tris pH7.5，250 mM NaCl，150 mM咪唑）进行洗脱，并加入50%甘油保存，所获得构建表达纯化的突变型酶液称之为新型融合DNA聚合酶突变体，并与野生型DNA聚合酶（由GenBank: U33536.1核酸对应编码氨基酸序列纯化而得）和商品化NEB Bst DNA聚合酶 3.0或（和）野生型DNA聚合酶进行对比。
实施例二
本发明在野生型DNA聚合酶的基础上，融合Sso7d蛋白以及定点突变，得到新型融合DNA聚合酶突变体，比如：
596位R被取代为K；598位D被取代为E；600位G被取代为K；716位I被取代为V；718位D被取代为E；719位Y被取代为F。
以上六个位置上，包含任意两个或两个以上的突变点组合，并融合了Sso7d蛋白结构域，除此6个位置氨基酸外，未标明的其他位置，均与野生型氨基酸一致，即为本发明所述新型融合DNA聚合酶突变体。
根据实施例一获得的DNA测序结果，选取对应其突变氨基酸如下所示位置，进行活性测试：
采用单链聚合法进行DNA聚合酶活性的测试。利用单链引物合成延伸，并通过SybrGreen I进行监测双链DNA合成的效率，来检测DNA聚合酶的聚合效率。配置聚合酶反应缓冲液，2X 缓冲液成分为，40 mM Tris-HCl、20 mM (NH4)2SO4、300 mM KCl、4 mM MgSO4、0.2%Tween20（pH 8.8）混合液，将100×SYBR Green I浓缩液加入到扩增延伸反应体系，使终浓度为1×。延伸引物M13AP终浓度为0.2umol/L，单链M13mp18 DNA模板（New EnglandBiolabs目录号：N4040S）添加0.25ug/反应，加入dNTPs，最终浓度为300µM。将新型融合DNA聚合酶突变体通过通用酶存储缓冲液（100 mM KCl、10 mM Tris-HCl、0.1 mM EDTA、1 mMDTT、0.1% Triton® X-100、50% Glycerol，pH 7.4）稀释100倍后，取1ul加入反应体系中。酶的活性对照品采用 0.01活性单位Bst 3.0 DNA聚合酶（New England Biolabs目录号：M0374L）作为参考对比。所有聚合酶的加入蛋白浓度一样，为平行实验。
反应通过96孔板进行，并将酶加至反应冠盖，加样完成盖上盖子后，将整版倒置混合，并瞬时离心15秒，之后上机反应，延伸反应均在65°C下进行。通过荧光定量PCR仪器（苏州雅瑞科技有限公司，型号：MA-6000，温度设置65℃恒温,30秒扫描一次，共20分钟）读取FAM/Sybr Green I荧光，荧光值均为线性延伸，得到线性斜率。根据扩增结果，以参考对照品bst 3.0 DNA聚合酶和野生型DNA聚合酶作为参考，20分钟反应终止时，本发明酶都高于对照参考品酶活线性斜率，斜率越高，代表信号增益越强，DNA合成的效率越高，酶活越高；重复上述检测，均与上次结果一致，参见图3，为本发明新型DNA聚合酶突变体和对照酶扩增延伸线图，测试三次。
引物M13AP的序列为：CATCCAATAAATCATACA。
实施例三
以实施例一制备的新型融合DNA聚合酶突变体与Bst 3.0 DNA聚合酶（New EnglandBiolabs目录号：M0374L）对比，进行LAMP等温扩增实验，以确定酶的解链与扩增能力。
LAMP引物与扩增条件参考文献进行(张双翔等. 中国兽医学报. 2013(33). 362-366)。针对绵羊肺炎支原体（Mo）16S rRNA基因，引物设计所选区域的外引物F3/B3与内引物FIP/BIP，引物序列见表1，Flc(Blc)与F2(B2)之间以“TTTT”连接。引物由上海捷瑞生物工程有限公司合成。
表1 绵羊肺炎支原体（Mo）16S rRNA基因LAMP引物序列
LAMP等温扩增实验流程：
1.采用2X 缓冲液成分为，40 mM Tris-HCl、20 mM (NH4)2SO4、300 mM KCl、4 mMMgSO4、0.2% Tween® 20（pH 8.8）混合液。
2.下列组分按顺序配置反应混合液（按2.5ml体系配制）：
3.用移液器吹打混匀，并短暂离心后，均匀分装至96孔PCR板中。
4.将以下试剂加入到冠盖上，瞬时离心将酶混入PCR板后，混匀并离心，酶缓冲液的加入量为1μl，酶终浓度320 U/ml。
5.63℃恒温孵育1小时。
6.通过荧光定量PCR仪器（苏州雅瑞科技有限公司，型号：MA-6000，温度设置65℃恒温,60秒扫描一次，共45分钟）读取FAM/Sybr Green I荧光，根据扩增曲线判断酶的功能。曲线采用相对相对荧光值显示方式。
图4为本发明新型融合DNA聚合酶与对照酶针对绵羊肺炎支原体基因的LAMP扩增结果；结果显示，本发明酶突变体扩增效率均优于NEB Bst DNA聚合酶 3.0，扩增速度明显快于NEB Bst DNA聚合酶 3.0。
实施例四
以实施例一的新型融合DNA聚合酶突变体与野生型 DNA聚合酶对比，采用超微量分光光度计（宝予德 BIO-DL，Micro Drop 2000）蛋白定量后，通过通用酶存储缓冲液（100 mMKCl、10 mM Tris-HCl、0.1 mM EDTA、1 mM DTT、0.1% Triton® X-100、50% Glycerol，pH7.4）稀释至相同蛋白浓度（0.1mg/ml）后，进一步测试LAMP扩增实验，以验证其扩增效率。
选用能够引起社区获得性呼吸窘迫综合征(CARDS)的肺炎支原体毒素基因M129(GenBank ID：DQ447750.1) 及其对应的LAMP引物（表2）（参考文献：Brianna，L.Journal ofClinical Microbiology.2015. DOI: 10.1128/jcm.01431-15）。LAMP反应以25μL的体积进行，反应条件与加样参考实施例三进行。恒温反应时间和为65℃恒温45分钟，每60秒荧光扫描一次。
表2 用于肺炎支原体CARDS毒素基因的LAMP的引物
反应结果如下：本发明的新型融合DNA聚合酶突变体效果（参考因素：扩增时间和荧光高度）均优于野生型DNA聚合酶；图5为本发明新型融合DNA聚合酶与对照酶针对肺炎支原体毒素基因M129的LAMP扩增结果。
实施例五
采用链切口延伸扩增是一种基于DNA聚合酶的等温扩增方法，对比实施例一的新型融合DNA聚合酶突变体与NEB Bst DNA 聚合酶 3.0的低拷贝数DNA检测差异。
参考专利申请202010160768.0，其中使用的引物、探针为经过2’-O-methyl-修饰，且探针序列5端加Fam、3端加BHQ1。
上游引物序列mMPF：5‘-TAATACTAATGAGTCGAGGA mC mU mU mA TT mG G mA mAmG-3’；
下游引物序列mMPR：5‘-CAGCGACAGAGTCACCAAACAA mA mAA mC mG A mC mA-3’；
探针序列mMPBP：5'- Fam-CTGCGTAT mU mU C mC TACCA mA mA mG mG mC TACGCAG -BHQ1- 3' ；
其中，反应管中包含上游引物终反应浓度为0.5uM，下游0.2uM，并向制备好的反应管中分别加入肺炎支原体10拷贝，每个反应中添加1ul。反应的缓冲液终浓度如下表所示：
另添加dNTPs 终浓度0.4mM；锰离子终浓度，2mM；并将本发明酶或商品化NEB Bst DNA聚合酶加至管盖。盖好盖子后颠倒摇动混匀后放入恒温震荡仪，温度设置为55℃，2000RPM/分钟震荡30s；之后转移至恒温荧光扩增仪上，在55℃条件下继续反应15min，每30s采集一次荧光信号值。本发明新型融合DNA聚合酶突变体的用量为3ug/反应。设置参考对照为NEBBst DNA聚合酶 3.0 并采用不同用量梯度（分别为4，10，20，25，40，60个活性单位）进行对比。
检测结果见图6，本发明新型融合DNA聚合酶突变体均可检测出10拷贝的阳性支原体DNA，而采用NEB Bst DNA 聚合酶3.0 则无法检测到阳性。
实施例六
采用本发明其他三个新型融合DNA聚合酶突变体进行测试，以NEB Bst DNA聚合酶3.0的25活性单位/20活性单位做对比，其它条件均与实施例五一致，测试结果均显示，3ug/反应、5ug/反应下，本发明新型融合DNA聚合酶突变体均可以检测30拷贝的阳性支原体DNA。
检测结果见图7，本发明新型融合DNA聚合酶与对照酶的切口延伸扩增技术检测低拷贝数模板结果。
参考文献：
AstatkeGrindley, N. D. F., and Joyce, C. M.M.,. (1995). J. Biol. Chem,270，1945-1954.
BeeseL.S.,Derbyshire,V.&Steitz,T.A. (1993). Structure of DNA polymerase IKlenow fragment bound to duplex DNA.Science. 260,352-355.
Burgers PMEV, Bruford E, Blanco L, Burtis KC, Christman MF, Copeland WC,Friedberg EC, Hanaoka F, Hinkle DC, Lawrence CW, Nakanishi M, Ohmori H,Prakash L, Prakash S, Reynaud CA, Sugino A, Todo T, Wang Z, Weill JC,Woodgate R.Koonin. (2001). Eukaryotic DNA polymerases: proposal for a revisednomenclature. J Biol Chem. 276(47);43487-90.
Caliendo AMDN, Ginocchio CC, Hanson KE, May L, Quinn TC, Tenover FC,Alland D, Blaschke AJ, Bonomo RA, Carroll KC, Ferraro MJ, Hirschhorn LR,Joseph WP, Karchmer T, MacIntyre AT, Reller LB, Jackson AF.Gilbert. (2013).Infectious diseases Society of a. better tests, better care: improveddiagnostics for infectious diseases. Clin Infect Dis. . 57(Suppl 3):S139–70.
张双翔等. 中国兽医学报. 2013(33). 362-366.
Craw PW.Balachandran. (2012). Isothermal nucleic acid amplificationtechnologies for point-of-care diagnostics: a critical review. Lab Chip. 12(14):2469–86.
de VegaL´azaro,J.M., Menc´ıa,M., Blanco,L. and Salas,MM.,. (2010). 107,16506–16511.
DoublieS.,Tabor,S.,Long,A.M.,Richardson,C.C.&Ellenberger,T. (1998).Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Aresolution(see comments).Nature,. 391,251-258.
Gill PA.Ghaemi. (2008). Nucleic acid isothermal amplificationtechnologies: a review. Nucleosides Nucleotides Nucleic Acids. . 27(3):224–43.
JoyceC.M.&Grindley,N.D. (1984). Method for determining whether a gene ofEscherichia coli is essential: application to the polA gene.J.Bacteriol. 158,636-643.
JoyceM., and Steitz, T. A.C. (1995). J. Bacteriol, 177, 6321–6329 .
Juliette B. BellA. Eckert, Catherine M. Joycei, and Thomas A.KunkelKristin. (1997). Base Miscoding and Strand Misalignment Errors byMutator Klenow Polymerases with Amino Acid Substitutions at Tyrosine 766 inthe O Helix of the Fingers Subdomain. 7345-7351.
Kamalendra SinghAashishSrivastava,SmitaS.Patel,andMukundJ.Modak. (2007).Participation of the Fingers Subdomain of Escherichia coli DNAPolymerase I inthe Strand Displacement Synthesisof DNA. 10594-10604.
Kaushik NVN, Modak MJ.Pandey. (1996). Significance of the O-helixresidues of Escherichia coli DNA polymerase I in DNA synthesis: dynamics ofthe dNTP binding pocket. 35(22):7256-66.
Kiefer JRC, Hansen CJ, Basehore SL, Hogrefe HH, Braman JC, Beese LS.Mao.(1997). Crystal structure of a thermostable Bacillus DNA polymerase I largefragment at 2.1 a resolution. Structure. . 5(1):95–108. .
KieferJ.R.,Mao,C.,Braman,J.C.&Beese,L.S. (1998). Visualizing DNAreplication in a catalytically active Bacillus DNA polymerase crystal(seecomments).Nature. 391,304-307.
KimW.&Steitz.,T.A.Y.,Eom,S.H.,Wang,J.,Lee,D.-S.,Suh,S. (1995). Crystalstructure of Thermus aquaticus DNA polymerase.Nature,. 376,612-616.
LiKorolev, S. & Waksman, G.Y.,. (1998). Crystal structures of open andclosed formas of binary and ternary compleses of the large fragment ofThermus aquaticus DNA polymerase I: structural basis for nucletideincorporation. EMBO J. 17,7514-7525.
M. G. RiggsTudor, M. Sivaram, and S. H. McDonoughS. (1996). “Constructionof single amino acid substitution mutants of cloned Bacillusstearothermophilus DNA polymerase I which lack 5' →3'exonuclease activity,”Biochimica et Biophysica Acta,vol.1307,no.2,pp. 178–186.
OllisL., Brick, P., Hamlin, R., Xuong, N. G. & Steitz, T. A.D. (1985).Structure of large fragment of Escherichia coli DNA polymerase I complexedwith dTMP. Nature. 313,762-766.
S.-M. PhangTeo, E. Lo, and V. W. Thi WongC.-Y. (1995). “Cloning andcomplete sequence of the DNA polymerase-encoding gene (BstpolI) andcharacterisation of the Klenow-like fragment fromBacillusstearothermophilus,”Gene,vol.163,no.1,pp. 65– 68.
Singh KA, Patel SS, Modak MJ.Srivastava. (2007). Participation of thefingers subdomain of Escherichia coli DNA polymerase I in the stranddisplacement synthesis of DNA. 282(14):10594-604.
WangProsen,D.E., Mei,L., Sullivan,J.C., Finney,M. and Vander Horn,P.B.Y.,. (2004). A novel strategy to engineer DNA polymerases for enhancedprocessivity and improved performance in vitro. Nucleic Acids Res. 1197-1207.
Y.XuJoyceO.Potapova,A.E.Leschziner,N.D.F.Grindley,andC.M. (2001).“Contacts between the 5'nuclease of DNA PolymeraseI and Its DNA substrate,”Journal of Biological Chemistry,vol.276, no.32,pp. 30167–30177.
Yi MaZhang, Meng Wang, Yanghui Ou, Jufang Wang, andShan LiBeilei. (2016).Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase Ifrom Geobacillus stearothermophilus by Site-Directed Mutagenesis at theActive Site.
Yvonne PiotrowskiKumari Gurung and Atle Noralf LarsenMan. (2019).Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacementactivity of A-family prokaryotic DNA polymerases . 20(1):31.
参考专利：
CN 101180390A
CN 102257136 A
US 8,828,700 B2
US 9,157,073 B1
US 9,890,336 B2
US 2016/0145588 A1
CN 1123328A
CN202010160768.0。
序列表
<110> 苏州先达基因科技有限公司
<120> 融合DNA聚合酶突变体及其在等温扩增中的应用
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 876
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Lys Lys Lys Leu Val Leu Ile Asp Gly Asn Ser Val Ala Tyr Arg
1 5 10 15
Ala Phe Phe Ala Leu Pro Leu Leu His Asn Asp Lys Gly Ile His Thr
20 25 30
Asn Ala Val Tyr Gly Phe Thr Met Met Leu Asn Lys Ile Leu Ala Glu
35 40 45
Glu Gln Pro Thr His Leu Leu Val Ala Phe Asp Ala Gly Lys Thr Thr
50 55 60
Phe Arg His Glu Thr Phe Gln Glu Tyr Lys Gly Gly Arg Gln Gln Thr
65 70 75 80
Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu Leu Arg Glu Leu Leu Lys
85 90 95
Ala Tyr Arg Ile Pro Ala Tyr Glu Leu Asp His Tyr Glu Ala Asp Asp
100 105 110
Ile Ile Gly Thr Leu Ala Ala Arg Ala Glu Gln Glu Gly Phe Glu Val
115 120 125
Lys Ile Ile Ser Gly Asp Arg Asp Leu Thr Gln Leu Ala Ser Arg His
130 135 140
Val Thr Val Asp Ile Thr Lys Lys Gly Ile Thr Asp Ile Glu Pro Tyr
145 150 155 160
Thr Pro Glu Thr Val Arg Glu Lys Tyr Gly Leu Thr Pro Glu Gln Ile
165 170 175
Val Asp Leu Lys Gly Leu Met Gly Asp Lys Ser Asp Asn Ile Pro Gly
180 185 190
Val Pro Gly Ile Gly Glu Lys Thr Ala Val Lys Leu Leu Lys Gln Phe
195 200 205
Gly Thr Val Glu Asn Val Leu Ala Ser Ile Asp Glu Val Lys Gly Glu
210 215 220
Lys Leu Lys Glu Asn Leu Arg Gln His Arg Asp Leu Ala Leu Leu Ser
225 230 235 240
Lys Gln Leu Ala Ser Ile Cys Arg Asp Ala Pro Val Glu Leu Ser Leu
245 250 255
Asp Asp Ile Val Tyr Glu Gly Gln Asp Arg Glu Lys Val Ile Ala Leu
260 265 270
Phe Lys Glu Leu Gly Phe Gln Ser Phe Leu Glu Lys Met Ala Ala Pro
275 280 285
Ala Ala Glu Gly Glu Lys Pro Leu Glu Glu Met Glu Phe Ala Ile Val
290 295 300
Asp Val Ile Thr Glu Glu Met Leu Ala Asp Lys Ala Ala Leu Val Val
305 310 315 320
Glu Val Met Glu Glu Asn Tyr His Asp Ala Pro Ile Val Gly Ile Ala
325 330 335
Leu Val Asn Glu His Gly Arg Phe Phe Met Arg Pro Glu Thr Ala Leu
340 345 350
Ala Asp Ser Gln Phe Leu Ala Trp Leu Ala Asp Glu Thr Lys Lys Lys
355 360 365
Ser Met Phe Asp Ala Lys Arg Ala Val Val Ala Leu Lys Trp Lys Gly
370 375 380
Ile Glu Leu Arg Gly Val Ala Phe Asp Leu Leu Leu Ala Ala Tyr Leu
385 390 395 400
Leu Asn Pro Ala Gln Asp Ala Gly Asp Ile Ala Ala Val Ala Lys Met
405 410 415
Lys Gln Tyr Glu Ala Val Arg Ser Asp Glu Ala Val Tyr Gly Lys Gly
420 425 430
Val Lys Arg Ser Leu Pro Asp Glu Gln Thr Leu Ala Glu His Leu Val
435 440 445
Arg Lys Ala Ala Ala Ile Trp Ala Leu Glu Gln Pro Phe Met Asp Asp
450 455 460
Leu Arg Asn Asn Glu Gln Asp Gln Leu Leu Thr Lys Leu Glu Gln Pro
465 470 475 480
Leu Ala Ala Ile Leu Ala Glu Met Glu Phe Thr Gly Val Asn Val Asp
485 490 495
Thr Lys Arg Leu Glu Gln Met Gly Ser Glu Leu Ala Glu Gln Leu Arg
500 505 510
Ala Ile Glu Gln Arg Ile Tyr Glu Leu Ala Gly Gln Glu Phe Asn Ile
515 520 525
Asn Ser Pro Lys Gln Leu Gly Val Ile Leu Phe Glu Lys Leu Gln Leu
530 535 540
Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Ser Thr Ser Ala Asp Val
545 550 555 560
Leu Glu Lys Leu Ala Pro His His Glu Ile Val Glu Asn Ile Leu His
565 570 575
Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Leu Leu
580 585 590
Lys Val Val Arg Pro Asp Thr Gly Lys Val His Thr Met Phe Asn Gln
595 600 605
Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Ala Glu Pro Asn Leu Gln
610 615 620
Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe
625 630 635 640
Val Pro Ser Glu Pro Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln
645 650 655
Ile Glu Leu Arg Val Leu Ala His Ile Ala Asp Asp Asp Asn Leu Ile
660 665 670
Glu Ala Phe Gln Arg Asp Leu Asp Ile His Thr Lys Thr Ala Met Asp
675 680 685
Ile Phe His Val Ser Glu Glu Glu Val Thr Ala Asn Met Arg Arg Gln
690 695 700
Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly
705 710 715 720
Leu Ala Gln Asn Leu Asn Ile Thr Arg Lys Glu Ala Ala Glu Phe Ile
725 730 735
Glu Arg Tyr Phe Ala Ser Phe Pro Gly Val Lys Gln Tyr Met Glu Asn
740 745 750
Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Leu His
755 760 765
Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg Asn Phe Asn Val Arg
770 775 780
Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Ser Ala
785 790 795 800
Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Leu Ala Ala Arg Leu Lys
805 810 815
Glu Glu Gln Leu Gln Ala Arg Leu Leu Leu Gln Val His Asp Glu Leu
820 825 830
Ile Leu Glu Ala Pro Lys Glu Glu Ile Glu Arg Leu Cys Glu Leu Val
835 840 845
Pro Glu Val Met Glu Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val
850 855 860
Asp Tyr His Tyr Gly Pro Thr Trp Tyr Asp Ala Lys
865 870 875
<210> 2
<211> 64
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp
1 5 10 15
Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe
20 25 30
Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu
35 40 45
Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys
50 55 60
<210> 3
<211> 236
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccatgggcag cagccatcat catcatcatc acagcagcgg cgcaacagta aagttcaagt 60
acaagggaga agagaaggaa gtagatataa gtaagataaa gaaggtatgg agagtaggca 120
aaatgataag tttcacctat gatgagggtg gaggaaagac tggtagagga gctgtaagcg 180
agaaagacgc tccaaaagaa ctactacaaa tgttagaaaa gcaaaagaaa catatg 236
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aatcatatgg attttgcgat cgcc 24
<210> 5
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
attgaattcg ggcccttcaa tatacgttga ctg 33
<210> 6
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctgctgaaag tggtgrracc cgwwacgrrg aaagtgcaca cgatgttc 48
<210> 7
<211> 81
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aatgaattca gccgcttctt tgcgcgtaat gttcaagttt tgcgccagac cawathyact 60
aangccgtac acgatgccaa a 81
<210> 8
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gctgaattca ttgagcgata ttttgc 26
<210> 9
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aatctcgagt tatttggcgt cgtaccac 28
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
catccaataa atcataca 18
<210> 11
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
attgagatac ggcccaga 18
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gccgtcactt tctaataagg t 21
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cagcagtaag gaatattcca ca 22
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acttcatcct gcactctgtg tc 22
<210> 15
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ccgtcaagac taaatcattt cc 22
<210> 16
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttagggatgt aaactgctgt tg 22
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ccacctagtg atttggaaga 20
<210> 18
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ggacaaagaa gattttcgaa gtt 23
<210> 19
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gctgaacatc aacaaagaag gtgcattgtt gatgaatgta ctaccca 47
<210> 20
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ataccccaca attaagtggt tgattcatag aatatctgtc catctgg 47
<210> 21
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
taatactaat gagtcgagga mcmmmattmg gmamamg 37
<210> 22
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
cagcgacaga gtcaccaaac aamamaamcm gamcma 36
<210> 23
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
amctgcgtat mmcmctacca mamamgmgmc tacgcagbh 39
Claims (10)
1.一种融合DNA聚合酶突变体，其特征在于，所述融合DNA聚合酶突变体包括融合Sso7d结构域与基于野生型DNA聚合酶基础上改造的突变体或衍生物；
所述野生型DNA聚合酶为来自Bacillus stearothermophilus的DNA聚合酶；
所述野生型DNA聚合酶经过定点氨基酸突变得到所述基于野生型DNA聚合酶基础上改造的突变体或衍生物；
所述野生型DNA聚合酶中，定点氨基酸突变的位点和氨基酸为Arg596、Asp598、Gly600、Ile716、Asp718、Try719中的两种或多种。
2.根据权利要求1所述融合DNA聚合酶突变体，其特征在于，所述定点氨基酸突变采用以下（1）～（6）种取代方式中的至少两个氨基酸位点进行取代：
（1）596位R被取代为K、G或者E；
（2）598位D被取代为V或者E；
（3）600位G被取代为K、R或者E；
（4）716位I被取代为V、F或者L；
（5）718位D被取代为K、A、T或者E；
（6）719位Y被取代为F。
3.权利要求1所述融合DNA聚合酶突变体在等温扩增中的应用。
4.根据权利要求3所述的应用，其特征在于，所述等温扩增为基于链置换反应的核酸等温扩增。
5.一种核酸等温扩增体系，其特征在于，包括权利要求1所述融合DNA聚合酶突变体与常规扩增试剂。
6.根据权利要求5所述核酸等温扩增体系，其特征在于，常规扩增试剂包括引物组、探针、二价阳离子、Tris缓冲液。
7.一种核酸等温扩增用试剂盒，其特征在于，包括权利要求1所述融合DNA聚合酶突变体与常规组分。
8.根据权利要求7所述核酸等温扩增用试剂盒，用于样本中是否包括特定生物样本，如DNA或RNA等。
9.编码权利要求1所述融合DNA聚合酶突变体的氨基酸序列。
10.一种核酸等温扩增的方法，其特征在于，以核酸为模板，在权利要求1所述融合DNA聚合酶突变体的存在下，在常规扩增试剂中，完成核酸等温扩增。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010394538.0A CN113637085B (zh) | 2020-05-11 | 2020-05-11 | 融合dna聚合酶突变体及其在等温扩增中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010394538.0A CN113637085B (zh) | 2020-05-11 | 2020-05-11 | 融合dna聚合酶突变体及其在等温扩增中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113637085A true CN113637085A (zh) | 2021-11-12 |
| CN113637085B CN113637085B (zh) | 2024-01-30 |
Family
ID=78415558
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010394538.0A Active CN113637085B (zh) | 2020-05-11 | 2020-05-11 | 融合dna聚合酶突变体及其在等温扩增中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113637085B (zh) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060275792A1 (en) * | 2004-11-15 | 2006-12-07 | Lee Jun E | Enhancement of nucleic acid amplification using double-stranded DNA binding proteins |
| GB0803628D0 (en) * | 2008-02-28 | 2008-04-02 | Genesys Ltd | Enzyme |
| WO2013033528A1 (en) * | 2011-09-01 | 2013-03-07 | Jennifer Ong | Compositions and methods relating to variant dna polymerases and synthetic dna polymerases |
| US20130323793A1 (en) * | 2012-06-04 | 2013-12-05 | New England Biolabs, Inc. | Compositions and Methods for Reducing Background DNA Amplification |
| WO2014210416A1 (en) * | 2013-06-27 | 2014-12-31 | New England Biolabs, Inc. | Helicase suppression of non-template amplification |
| US8993298B1 (en) * | 2012-08-31 | 2015-03-31 | New England Biolabs, Inc. | DNA polymerases |
| US20160160193A1 (en) * | 2014-08-27 | 2016-06-09 | New England Biolabs, Inc. | Fusion Polymerase and Method for Using the Same |
-
2020
- 2020-05-11 CN CN202010394538.0A patent/CN113637085B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060275792A1 (en) * | 2004-11-15 | 2006-12-07 | Lee Jun E | Enhancement of nucleic acid amplification using double-stranded DNA binding proteins |
| GB0803628D0 (en) * | 2008-02-28 | 2008-04-02 | Genesys Ltd | Enzyme |
| WO2013033528A1 (en) * | 2011-09-01 | 2013-03-07 | Jennifer Ong | Compositions and methods relating to variant dna polymerases and synthetic dna polymerases |
| US20130323793A1 (en) * | 2012-06-04 | 2013-12-05 | New England Biolabs, Inc. | Compositions and Methods for Reducing Background DNA Amplification |
| US8993298B1 (en) * | 2012-08-31 | 2015-03-31 | New England Biolabs, Inc. | DNA polymerases |
| WO2014210416A1 (en) * | 2013-06-27 | 2014-12-31 | New England Biolabs, Inc. | Helicase suppression of non-template amplification |
| US20160160193A1 (en) * | 2014-08-27 | 2016-06-09 | New England Biolabs, Inc. | Fusion Polymerase and Method for Using the Same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113637085B (zh) | 2024-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11603547B2 (en) | Modified type A DNA polymerases | |
| JP5241493B2 (ja) | Ｄｎａ結合タンパク質−ポリメラーゼのキメラ | |
| JP6150847B2 (ja) | キメラｄｎａポリメラーゼ | |
| US10035993B2 (en) | DNA polymerases and related methods | |
| CA2888148C (en) | Dna polymerases with improved activity | |
| CA2858264C (en) | Dna polymerases with improved activity | |
| JP2006513726A (ja) | Ｄｎａポリメラーゼブレンドおよびその使用 | |
| EP2164955B1 (en) | Mutant dna polymerases and related methods | |
| US20130149748A1 (en) | Dna polymerases with improved activity | |
| EP2788479A1 (en) | Dna polymerases with improved activity | |
| CA2839964A1 (en) | Dna polymerases with improved activity | |
| Oscorbin et al. | Bst polymerase—a humble relative of Taq polymerase | |
| CN113637085B (zh) | 融合dna聚合酶突变体及其在等温扩增中的应用 | |
| WO2023082266A1 (zh) | 嵌合dna聚合酶及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |