JPWO2020113036A5 - - Google Patents
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- JPWO2020113036A5 JPWO2020113036A5 JP2021529774A JP2021529774A JPWO2020113036A5 JP WO2020113036 A5 JPWO2020113036 A5 JP WO2020113036A5 JP 2021529774 A JP2021529774 A JP 2021529774A JP 2021529774 A JP2021529774 A JP 2021529774A JP WO2020113036 A5 JPWO2020113036 A5 JP WO2020113036A5
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- 239000012491 analyte Substances 0.000 claims description 52
- 102000004965 antibodies Human genes 0.000 claims description 33
- 108090001123 antibodies Proteins 0.000 claims description 33
- 239000000758 substrate Substances 0.000 claims description 18
- 235000001014 amino acid Nutrition 0.000 claims description 15
- 150000001413 amino acids Chemical group 0.000 claims description 14
- 229920001184 polypeptide Polymers 0.000 claims description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 9
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 9
- 230000000295 complement Effects 0.000 claims description 8
- 239000004475 Arginine Substances 0.000 claims description 6
- 150000001412 amines Chemical class 0.000 claims description 6
- 238000004020 luminiscence type Methods 0.000 claims description 6
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 6
- 102000011931 Nucleoproteins Human genes 0.000 claims description 5
- 108010061100 Nucleoproteins Proteins 0.000 claims description 5
- 108010090804 Streptavidin Proteins 0.000 claims description 5
- 231100000765 Toxin Toxicity 0.000 claims description 5
- 239000000427 antigen Substances 0.000 claims description 5
- 108091007172 antigens Proteins 0.000 claims description 5
- 102000038129 antigens Human genes 0.000 claims description 5
- 230000027455 binding Effects 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 229940079593 drugs Drugs 0.000 claims description 5
- 239000003446 ligand Substances 0.000 claims description 5
- 125000005647 linker group Chemical group 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- 235000000346 sugar Nutrition 0.000 claims description 5
- 239000003053 toxin Substances 0.000 claims description 5
- 108020003112 toxins Proteins 0.000 claims description 5
- 102000004127 Cytokines Human genes 0.000 claims description 4
- 108090000695 Cytokines Proteins 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 4
- 125000003282 alkyl amino group Chemical group 0.000 claims description 4
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 4
- 150000002148 esters Chemical class 0.000 claims description 4
- 150000008163 sugars Chemical class 0.000 claims description 4
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 3
- 229960005261 Aspartic Acid Drugs 0.000 claims description 2
- MTCFGRXMJLQNBG-UWTATZPHSA-N D-serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 claims description 2
- 229960002989 Glutamic Acid Drugs 0.000 claims description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 2
- 239000004473 Threonine Substances 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims description 2
- 235000013922 glutamic acid Nutrition 0.000 claims description 2
- 239000004220 glutamic acid Substances 0.000 claims description 2
- 150000003141 primary amines Chemical class 0.000 claims description 2
- 238000000225 bioluminescence resonance energy transfer Methods 0.000 claims 3
- 238000000034 method Methods 0.000 claims 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims 1
- 238000006467 substitution reaction Methods 0.000 claims 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N Rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 4
- 239000002831 pharmacologic agent Substances 0.000 description 4
- -1 sulfo-NHS compound Chemical class 0.000 description 3
- 241001438449 Silo Species 0.000 description 2
- 241000711975 Vesicular stomatitis virus Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000000269 nucleophilic Effects 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 241000932822 Tinaroo virus Species 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 125000000511 arginine group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000004404 heteroalkyl group Chemical group 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
Description
配列
WT OgLuc(配列番号1)
MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVTINGVTGWRLCENILA
WT OgLuc Lg(配列番号2)
MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPD
WT OgLuc β9(配列番号3)
GSLLFRVTIN
WT OgLuc β10(配列番号4)
GVTGWRLCENILA
NanoLuc(配列番号5)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA
NanoLuc Lg(配列番号6)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPD
NanoLuc β9(配列番号7)
GSLLFRVTINV
NanoLuc β10(配列番号8)
GVTGWRLCERILA
LgBiT(配列番号9)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN
SmBiT(配列番号10)
VTGYRLFEEIL
HiBiT(pep86)(配列番号11)
VSGWRLFKKIS
LgTrip(3546)(配列番号12)
MKHHHHHHVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD
SmTrip9(配列番号13)
GSMLFRVTINS
β9/β10ジペプチド(配列番号14)
GSMLFRVTINSVSGWRLFKKIS
Pep521(配列番号15)
GKMLFRVTINSWK
Pep693(配列番号16)
GRMLFRVTINSWR
Pep840(配列番号17)
GKLLFVVVIEKYK
Pep895(配列番号18)
GRLLFVVVIERYR
Pep760(配列番号19)
KKMLFRVTIQKWK
Pep929(配列番号20)
RRMLFRVTIQRWR
VS-HiBiT(Pep289)(配列番号21)
VSVSGWRLFKKIS
Pep692(配列番号22)
VSVSGWRLFRRIS
Pep691(配列番号23)
VSGWRLFRRIS
Pep759(配列番号24)
DKLLFTVTIEKYK
Pep824(配列番号25)
DRLLFTVTIERYR
Pep521-C(配列番号26)
GKMLFRVTINSWKC
Pep693-C(配列番号27)
GRMLFRVTINSWRC
Pep840-C(配列番号28)
GKLLFTVTIEKYKC
Pep895-C(配列番号29)
GRLLFTVTIERYRC
Pep760-C(配列番号30)
KKMLFRVTIQKWKC
Pep929-C(配列番号31)
RRMLFRVTIQRWRC
VS-HiBiT-C(Pep289)(配列番号32)
VSVSGWRLFKKISC
Pep692-C(配列番号33)
VSVSGWRLFRRISC
Pep691-C(配列番号34)
VSGWRLFRRISC
Pep759-C(配列番号35)
DKLLFTVTIEKYKC
Pep824-C(配列番号36)
DRLLFTVTIERYRC
Pep937(配列番号37)
VSGWRLFRRISC
Pep938(配列番号38)
GRMLFRVTINSWRC
Pep939(配列番号39)
GRLLFTVTIERYRC
本発明のまた別の態様は、以下のとおりであってもよい。
〔1〕スルホn-ヒドロキシスクシンイミジルエステル(スルホ-SE)基に連結されたペプチドを含む組成物であって、前記ペプチドがシステイン残基もリジン残基も含まない、前記組成物。
〔2〕前記スルホ-SEが前記ペプチドのN末端に連結されている、前記〔1〕に記載の組成物。
〔3〕前記スルホ-SEが前記ペプチドのC末端に連結されている、前記〔1〕に記載の組成物。
〔4〕前記スルホ-SEが前記ペプチドのアミノ酸側鎖に連結されている、前記〔1〕に記載の組成物。
〔5〕前記ペプチドが、セリン、スレオニン、チロシン、グルタミン酸、アルギニン、ヒスチジン、トリプトファン及びアスパラギン酸から選択される少なくとも1つの非アルキルアミノ酸を含む、前記〔1〕に記載の組成物。
〔6〕前記少なくとも1つの反応性非アルキルアミノ酸がaまたはチロシンである、前記〔5〕に記載の組成物。
〔7〕前記少なくとも1つの反応性求核性アミノ酸がアルギニンである、前記〔5〕に記載の組成物。
〔8〕前記スルホ-SE基が非ペプチドリンカー基によって前記ペプチドに連結されている、前記〔1〕に記載の組成物。
〔9〕前記リンカー基がアルキルまたはヘテロアルキル鎖を含む、前記〔8〕に記載の組成物。
〔10〕前記リンカーが1つ以上の側鎖置換基を含む、前記〔8〕に記載の組成物。
〔11〕前記ペプチドの長さが4~50アミノ酸である、前記〔1〕に記載の組成物。
〔12〕前記ペプチドの長さが8~20アミノ酸である、前記〔11〕に記載の組成物。
〔13〕前記スルホ-SEが前記ペプチドのN末端に結合している、前記〔1〕に記載の組成物。
〔14〕前記スルホ-SEがリンカー基を介して前記ペプチドのN末端に結合している、前記〔13〕に記載の組成物。
〔15〕前記ペプチドが蛍光団結合体または発色団結合体を含む、前記〔1〕に記載の組成物。
〔16〕前記ペプチドが生体分子複合体の構成要素である、前記〔1〕に記載の組成物。
〔17〕前記ペプチドが生体分子複合体の構成要素である、前記〔14〕に記載の組成物。
〔18〕配列番号10(SmBiT)と比較して前記ペプチドが5個以下の置換基を含む、前記〔17〕に記載の組成物。
〔19〕配列番号1の1つ以上のリジンがアルギニンに置き換わっている、前記〔17〕に記載の組成物。
〔20〕前記ペプチドがPep691(配列番号23)を含む、前記〔19〕に記載の組成物。
〔21〕前記ペプチドがSmBiT(配列番号10)を含む、前記〔19〕に記載の組成物。
〔22〕前記ペプチドが蛍光団と結合体化されている、前記〔18〕に記載の組成物。
〔23〕前記ペプチドが、アルギニンに結合体化された蛍光団を含む、前記〔22〕に記載の組成物。
〔24〕前記ペプチドが、配列番号23に結合体化された蛍光団を含む、前記〔23〕に記載の組成物。
〔25〕前記ペプチドが、配列番号10に結合体化された蛍光団を含む、前記〔23〕に記載の組成物。
〔26〕生体分子にペプチドで標識付けする方法であって、
前記スルホ-SE基と前記生体分子上のアミンとが反応するような条件の下で前記生体分子を前記〔1〕~〔25〕のうちの1項に記載の組成物に接触させること
を含む、前記方法。
〔27〕前記スルホ-SE基と前記生体分子上のアミンとが反応するような条件の下で前記〔14〕に記載のペプチド組成物が前記生体分子に接触する、前記〔24〕に記載の方法。
〔28〕前記アミンが第一級アミンである、前記〔26〕または前記〔27〕に記載の方法。
〔29〕前記生体分子が、抗原、抗体、抗体断片、ナノボディ、darpin、非抗体タンパク質、受容体、リガンド、毒素、サイトカイン、核酸、核タンパク質複合体、ペプチド、アミノ酸、糖、薬物及びストレプトアビジンからなる群から選択される、前記〔14〕に記載の方法。
〔30〕ペプチドにスルホ-SE部分で標識付けする方法であって、
スルホ-NHS化合物のヒドロキシと前記ペプチドの末端アミンとが反応するような条件の下で前記ペプチドを前記スルホ-NHS化合物に接触させること
を含み、前記ペプチドがシステイン残基もリジン残基も含まない、前記方法。
〔31〕前記ペプチドが少なくとも1つの反応性求核性アミノ酸を含む、前記〔20〕に記載の方法。
〔32〕前記〔1〕~〔25〕のうちの1項に記載のペプチドで標識付けされた生体分子を含む、組成物。
〔33〕前記〔32〕に記載の組成物を被分析物に接触させることを含む方法。
〔34〕前記被分析物が、抗原、抗体、抗体断片、ナノボディ、darpin、非抗体タンパク質、受容体、リガンド、毒素、サイトカイン、核酸、核タンパク質複合体、ペプチド、アミノ酸、糖、薬物及びストレプトアビジンからなる群から選択される、前記〔33〕に記載の方法。
〔35〕前記被分析物が、前記生体分子上の前記ペプチドとの生物発光複合体を形成できる相補性ポリペプチドに連結されている、前記〔35〕に記載の方法。
〔36〕前記生物発光複合体を前記生物発光複合体の基質に接触させること、及び発光を検出することをさらに含む、前記〔35〕に記載の方法。
〔37〕前記〔1〕~〔25〕のうちの1項に記載のペプチドで標識付けされた被分析物を含む組成物。
〔38〕前記〔37〕に記載の組成物を生体分子に接触させることを含む方法。
〔39〕前記生体分子が、前記被分析物上の前記ペプチドとの生物発光複合体を形成できる相補性ポリペプチドに連結されている、前記〔38〕に記載の方法。
〔40〕前記生物発光複合体を前記生物発光複合体の基質に接触させること、ならびに発光、蛍光及び/またはBRETを検出することをさらに含む、前記〔39〕に記載の方法。
〔41〕前記〔1〕~〔25〕のうちの1項に記載の第1のペプチドで標識付けされた被分析物と、前記〔1〕~〔25〕のうちの1項に記載の第2のペプチドで標識付けされた生体分子とを含む組成物であって、前記第1及び第2のペプチドが、相補性ポリペプチドの存在下で生物発光複合体を形成できるものである、前記組成物。
〔42〕前記〔41〕に記載の前記被分析物及び前記生体分子を前記相補性ポリペプチドに接触させること、ならびに前記生物発光複合体を形成することを含む、方法。
〔43〕前記生物発光複合体を前記生物発光複合体の基質に接触させること、及び発光を検出することをさらに含む、前記〔42〕に記載の方法。
〔44〕前記ペプチドの1つ以上が蛍光団結合体化ペプチドまたは発色団結合体化ペプチドである、前記〔26〕~〔31〕、前記〔33〕~〔36〕、前記〔38〕~〔40〕及び前記〔42〕~〔43〕のうちの1項に記載の方法。
〔45〕前記生物発光複合体から前記蛍光団または発色団への蛍光/光及び/またはBRETを検出することをさらに含む、前記〔44〕に記載の方法。
〔46〕生体分子1つあたりの標識付け数が生体分子1つあたりの蛍光団分子または発色団分子の数によって算出される、前記〔45〕に記載の方法。
〔47〕前記蛍光団分子が、FAM、TAMRA、ROX、siloローダミン、BODIPY、TOM、Dyomics染料または炭素ローダミンであるが、それらの蛍光団に限定されない、前記〔44〕に記載の方法。
〔48〕(a)配列番号1(SmBiT)標識被分析物生体分子とLgBiT標識被分析物生体分子特異抗体との生物発光複合体を形成すること、
(b)前記生物発光複合体を前記被分析物に接触させること、
(c)前記生物発光複合体を前記生物発光複合体の基質に接触させること、及び
(d)前記生物発光複合体から発せられた光を検出すること
を含む方法。
〔49〕(a)被分析物を、配列番号1(SmBiT)標識被分析物特異抗体、及びLgBiT標識被分析物特異抗体に接触させ、生物発光複合体を形成すること、
(b)前記生物発光複合体を前記生物発光複合体の基質に接触させること、ならびに
(c)前記生物発光複合体から発せられた光を検出すること
を含む方法。
〔50〕(a)被分析物を、
配列番号1(SmBiT)標識被分析物特異抗体、
配列番号11(HiBiT)標識被分析物特異抗体、ならびに
HiBiT及びSmBiTとの生物発光複合体を形成できるポリペプチド
に接触させること、
(b)前記生物発光複合体を前記生物発光複合体の基質に接触させること、ならびに
(c)前記生物発光複合体から発せられた光を検出すること
を含む方法。
〔50〕(a)被分析物を、
配列番号1(SmBiT)標識被分析物特異抗体、
配列番号11(HiBiT)標識被分析物特異抗体、ならびに
HiBiT及びSmBiTとの生物発光複合体を形成できるポリペプチド
に接触させること、
(b)前記生物発光複合体を前記生物発光複合体の基質に接触させること、ならびに
(c)前記生物発光複合体から発せられた光を検出すること
を含む方法。
〔51〕前記〔1〕~〔25〕のうちの1項に記載の組成物で標識付けされた生体分子を被分析物に接触させることを含む方法。
〔52〕前記被分析物が、抗原、抗体、非抗体タンパク質、受容体、リガンド、毒素、サイトカイン、核酸、ペプチド、アミノ酸、糖、薬物、核タンパク質複合体、ビオチン及びストレプトアビジンからなる群から選択される、前記〔51〕に記載の方法。
〔53〕前記被分析物生体分子が配列番号1(SmBiT)で標識付けされている、前記〔51〕に記載の方法。
〔54〕(a)生物発光複合体を、配列番号1(SmBiT)標識被分析物生体分子、及びLgBiT標識被分析物生体分子特異抗体から形成する;
(b)前記生物発光複合体を前記被分析物に接触させる;
(c)前記生物発光複合体を前記生物発光複合体の基質に接触させる;ならびに
(d)前記生物発光複合体から発せられた光を検出する、
前記〔51〕に記載の方法。
〔55〕(a)配列番号1(SmBiT)標識被分析物特異抗体とLgBiT標識被分析物特異抗体とから生物発光複合体を形成する;
(b)前記生物発光複合体を前記被分析物に接触させる;
(c)前記生物発光複合体を前記生物発光複合体の基質に接触させる;ならびに
(d)前記生物発光複合体から発せられた光を検出する、
前記〔51〕に記載の方法。
〔56〕(a)配列番号10(SmBiT)標識抗体または受容体、及び配列番号11(HiBiT)標識抗体または受容体に被分析物を接触させる;
(b)前記被分析物をLgBiTに接触させて生物発光複合体を形成する;
(c)前記生物発光複合体を前記生物発光複合体の基質に接触させる;ならびに
(d)前記生物発光複合体から発せられた光を検出する、
前記〔51〕に記載の方法。
〔57〕a)SmBiTまたはHiBiT標識被分析物生体分子をHiBiTまたはSmBiT標識被分析物生体分子特異抗体に接触させる;
(b)LgBiTに接触させて生物発光複合体を形成する;
(c)被分析物に接触させる、
(d)前記生物発光複合体を前記生物発光複合体の基質に接触させる;ならびに
(e)前記生物発光複合体から発せられた光を検出する、
前記〔51〕に記載の方法。
〔58〕前記ペプチドが蛍光団結合体化ペプチドまたは発色団結合体化ペプチドである、前記〔57〕に記載の方法。
〔59〕生体分子1つあたりの標識付け数が生体分子1つあたりの蛍光団分子または発色団分子の数によって算出される、前記〔58〕に記載の方法。
〔60〕前記蛍光団分子が、FAM、TAMRA、ROX、siloローダミン、BODIPY、TOM、Dyomics染料または炭素ローダミンであるが、それらの蛍光団に限定されない、前記〔58〕に記載の方法。
〔61〕(a)蛍光団結合体化配列番号1(SmBiT)標識被分析物生体分子とLgBiT標識被分析物生体分子特異抗体とから生物発光複合体を形成する;
(b)前記生物発光複合体を前記被分析物に接触させる;
(c)前記生物発光複合体を前記生物発光複合体の基質に接触させる;ならびに
(d)前記生物発光複合体から発せられた光を検出する、
前記〔51〕に記載の方法。
〔62〕(a)蛍光団結合体化配列番号1(SmBiT)標識被分析物特異抗体とLgBiT標識被分析物特異抗体との両方に被分析物を接触させて生物発光複合体を形成する、
(b)前記生物発光複合体を前記生物発光複合体の基質に接触させる;及び
(c)前記生物発光複合体から発せられた光を検出する、
前記〔51〕に記載の方法。
〔63〕(a)配列番号10(SmBiT)ペプチド及び配列番号11(HiBiT)ペプチドのうちの1つが蛍光団結合体化ペプチドであり;
(b)配列番号10(SmBiT)及び配列番号11(HiBiT)で標識付けされた抗体もしくは受容体または組合せに前記被分析物が接触し、前記ペプチドの1つが蛍光団結合体化ペプチドであり;
(b)LgBiTに接触させて生物発光複合体を形成し;
(c)前記生物発光複合体を前記生物発光複合体の基質に接触させ;
(d)前記生物発光複合体から発せられた光を検出する、
前記〔51〕に記載の方法。
〔64〕蛍光団と結合体化された配列番号10(SmBiT)または配列番号11(HiBiT)で被分析物生体分子または被分析物特異抗体が標識付けされており、
(a)配列番号10(SmBiT)または配列番号11(HiBiT)で標識付けされた被分析物生体分子が、配列番号11(HiBiT)または配列番号10(SmBiT)で標識付けされた被分析物生体分子特異抗体に接触し、前記ペプチドの1つが、前記蛍光団と結合体化されたペプチドであり、
(b)LgBiTに接触させて生物発光複合体を形成し、
(c)前記被分析物に接触させ、
(d)前記生物発光複合体を前記生物発光複合体の基質に接触させ、
(e)前記生物発光複合体から発せられた光を検出する、
方法。
Sequence WT OgLuc (SEQ ID NO: 1)
MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVTINGVTGWRLCENILA
WT OgLuc Lg (SEQ ID NO: 2)
MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPINPGIAVFDGKQITTVTGTLWNGPDLINKIYDER
WT OgLuc β9 (SEQ ID NO:3)
GSLL FRVTIN
WT OgLuc β10 (SEQ ID NO:4)
GVTGWRLCENILA
NanoLuc (SEQ ID NO: 5)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPINYEGIAVFDGKKKITVTGTLWNGNPDVLGTILGLTLANGLIDERLL
NanoLuc Lg (SEQ ID NO: 6)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPINYEGIAVFDGKKITTVTGTLWNGPDKIIDERLL
NanoLuc β9 (SEQ ID NO:7)
GSLL FRV TINV
NanoLuc β10 (SEQ ID NO: 8)
GVTGWRL CERIL A
LgBiT (SEQ ID NO: 9)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPINYEGIAVFDGKKITTVTGTLWVTFRNGSLIIDERLL
SmBiT (SEQ ID NO: 10)
VTG YRL FEEIL
HiBiT (pep86) (SEQ ID NO: 11)
VSGW RLFKKIS
LgTrip (3546) (SEQ ID NO: 12)
MKHHHHHHVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKITTTGPDTLWNGNKI
SmTrip9 (SEQ ID NO: 13)
GSML FRV TINS
β9/β10 dipeptide (SEQ ID NO: 14)
GSML FRV TINS VSGWRLFKKIS
Pep521 (SEQ ID NO: 15)
GKML FRV T IN SWK
Pep693 (SEQ ID NO: 16)
GRML FRV TINSWR
Pep840 (SEQ ID NO: 17)
GKLLFVVIEKYK
Pep895 (SEQ ID NO: 18)
GRLLFVVVIERYR
Pep760 (SEQ ID NO: 19)
KKMLFRVTIQKWK
Pep929 (SEQ ID NO: 20)
RRMLFRVTIQRWR
VS-HiBiT (Pep289) (SEQ ID NO:21)
VSVSGWRLFKKIS
Pep692 (SEQ ID NO:22)
VSVSGWRLFRRIS
Pep691 (SEQ ID NO:23)
VSGWRLFRRIS
Pep759 (SEQ ID NO:24)
DKLL FTV TIEKYK
Pep824 (SEQ ID NO:25)
DRLL FTV TIERY R
Pep521-C (SEQ ID NO:26)
GKML FRVTIN SWKC
Pep693-C (SEQ ID NO:27)
GRML FRV T IN SW RC
Pep840-C (SEQ ID NO:28)
GKLLFTVTIEKYKC
Pep895-C (SEQ ID NO:29)
GRLL FTV TIERYRC
Pep760-C (SEQ ID NO:30)
KKMLFRVTIQKWKC
Pep929-C (SEQ ID NO:31)
RRML FRVTIQRWRC
VS-HiBiT-C (Pep289) (SEQ ID NO:32)
VSV SGW RLF KKISC
Pep692-C (SEQ ID NO:33)
VSV SGWRLFRRISC
Pep691-C (SEQ ID NO:34)
VSGWRLFRRISC
Pep759-C (SEQ ID NO:35)
DKLLFTVTIEKYKC
Pep824-C (SEQ ID NO:36)
DRLL FTV TIERYRC
Pep937 (SEQ ID NO:37)
VSGWRLFRRISC
Pep938 (SEQ ID NO:38)
GRML FRV T IN SW RC
Pep939 (SEQ ID NO:39)
GRLL FTV TIERYRC
Another aspect of the present invention may be as follows.
[1] A composition comprising a peptide linked to a sulfo n-hydroxysuccinimidyl ester (sulfo-SE) group, wherein said peptide contains neither cysteine nor lysine residues.
[2] The composition according to [1] above, wherein the sulfo-SE is linked to the N-terminus of the peptide.
[3] The composition according to [1] above, wherein the sulfo-SE is linked to the C-terminus of the peptide.
[4] The composition according to [1] above, wherein the sulfo-SE is linked to an amino acid side chain of the peptide.
[5] The composition according to [1] above, wherein the peptide contains at least one non-alkyl amino acid selected from serine, threonine, tyrosine, glutamic acid, arginine, histidine, tryptophan and aspartic acid.
[6] The composition according to [5] above, wherein the at least one reactive non-alkyl amino acid is a or tyrosine.
[7] The composition according to [5] above, wherein the at least one reactive nucleophilic amino acid is arginine.
[8] The composition of [1] above, wherein the sulfo-SE group is linked to the peptide by a non-peptide linker group.
[9] The composition of [8] above, wherein the linker group comprises an alkyl or heteroalkyl chain.
[10] The composition of [8] above, wherein the linker comprises one or more side chain substituents.
[11] The composition of [1] above, wherein the peptide has a length of 4 to 50 amino acids.
[12] The composition of [11] above, wherein the peptide has a length of 8 to 20 amino acids.
[13] The composition of [1] above, wherein the sulfo-SE is bound to the N-terminus of the peptide.
[14] The composition of [13] above, wherein the sulfo-SE is bound to the N-terminus of the peptide via a linker group.
[15] The composition of [1] above, wherein the peptide comprises a fluorophore conjugate or a chromophore conjugate.
[16] The composition of [1] above, wherein the peptide is a component of a biomolecular complex.
[17] The composition of [14] above, wherein the peptide is a component of a biomolecular complex.
[18] The composition of [17] above, wherein the peptide contains 5 or less substituents compared to SEQ ID NO: 10 (SmBiT).
[19] The composition of [17] above, wherein one or more lysines in SEQ ID NO: 1 are replaced with arginine.
[20] The composition of [19] above, wherein the peptide comprises Pep691 (SEQ ID NO: 23).
[21] The composition of [19] above, wherein the peptide comprises SmBiT (SEQ ID NO: 10).
[22] The composition of [18] above, wherein the peptide is conjugated with a fluorophore.
[23] The composition of [22] above, wherein the peptide comprises a fluorophore conjugated to arginine.
[24] The composition of [23] above, wherein the peptide comprises a fluorophore conjugated to SEQ ID NO:23.
[25] The composition of [23] above, wherein the peptide comprises a fluorophore conjugated to SEQ ID NO:10.
[26] A method for labeling a biomolecule with a peptide, comprising:
Contacting the biomolecule with the composition according to any one of [1] to [25] under conditions such that the sulfo-SE group reacts with the amine on the biomolecule.
The above method, comprising
[27] The above-described [24], wherein the peptide composition of [14] is brought into contact with the biomolecule under conditions such that the sulfo-SE group reacts with the amine on the biomolecule. Method.
[28] The method according to [26] or [27], wherein the amine is a primary amine.
[29] the biomolecules are antigens, antibodies, antibody fragments, nanobodies, darpins, non-antibody proteins, receptors, ligands, toxins, cytokines, nucleic acids, nucleoprotein complexes, peptides, amino acids, sugars, drugs and streptavidin The method according to [14] above, which is selected from the group consisting of
[30] A method for labeling a peptide with a sulfo-SE moiety, comprising:
contacting said peptide with said sulfo-NHS compound under conditions such that the hydroxy of said sulfo-NHS compound reacts with the terminal amine of said peptide;
and wherein said peptide contains no cysteine or lysine residues.
[31] The method of [20] above, wherein the peptide contains at least one reactive nucleophilic amino acid.
[32] A composition comprising a biomolecule labeled with the peptide of any one of [1] to [25] above.
[33] A method comprising contacting the composition of [32] above with an analyte.
[34] the analyte is an antigen, antibody, antibody fragment, nanobody, darpin, non-antibody protein, receptor, ligand, toxin, cytokine, nucleic acid, nucleoprotein complex, peptide, amino acid, sugar, drug and streptavidin The method according to [33] above, which is selected from the group consisting of
[35] The method of [35] above, wherein the analyte is linked to a complementary polypeptide capable of forming a bioluminescent complex with the peptide on the biomolecule.
[36] The method of [35] above, further comprising contacting the bioluminescent complex with a substrate for the bioluminescent complex and detecting luminescence.
[37] A composition comprising an analyte labeled with the peptide of any one of [1] to [25] above.
[38] A method comprising contacting the composition of [37] above with a biomolecule.
[39] The method of [38] above, wherein the biomolecule is linked to a complementary polypeptide capable of forming a bioluminescent complex with the peptide on the analyte.
[40] The method of [39] above, further comprising contacting the bioluminescent complex with a substrate for the bioluminescent complex and detecting luminescence, fluorescence and/or BRET.
[41] an analyte labeled with the first peptide according to any one of the above [1] to [25], and the first peptide according to one of the above [1] to [25]; and two peptide-labeled biomolecules, wherein said first and second peptides are capable of forming a bioluminescent complex in the presence of a complementary polypeptide. thing.
[42] A method comprising contacting the analyte and the biomolecule of [41] with the complementary polypeptide, and forming the bioluminescent complex.
[43] The method of [42] above, further comprising contacting the bioluminescent complex with a substrate for the bioluminescent complex and detecting luminescence.
[44] the above [26] to [31], the above [33] to [36], the above [38] to [40], wherein at least one of the peptides is a fluorophore-conjugated peptide or a chromophore-conjugated peptide; and the method according to one of the above [42] to [43].
[45] The method of [44] above, further comprising detecting fluorescence/light and/or BRET from the bioluminescent complex to the fluorophore or chromophore.
[46] The method of [45] above, wherein the number of labels per biomolecule is calculated by the number of fluorophore molecules or chromophore molecules per biomolecule.
[47] The method of [44] above, wherein the fluorophore molecule is FAM, TAMRA, ROX, silo rhodamine, BODIPY, TOM, Dyomics dyes or carbon rhodamine, but is not limited to these fluorophores.
[48] (a) forming a bioluminescent complex between a SEQ ID NO: 1 (SmBiT)-labeled analyte biomolecule and an LgBiT-labeled analyte biomolecule-specific antibody;
(b) contacting the bioluminescent complex with the analyte;
(c) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(d) detecting light emitted from said bioluminescent complex;
method including.
[49] (a) contacting an analyte with a SEQ ID NO: 1 (SmBiT)-labeled analyte-specific antibody and an LgBiT-labeled analyte-specific antibody to form a bioluminescent complex;
(b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(c) detecting light emitted from said bioluminescent complex;
method including.
[50] (a) the analyte,
SEQ ID NO: 1 (SmBiT)-labeled analyte-specific antibody,
SEQ ID NO: 11 (HiBiT)-labeled analyte-specific antibody, and
Polypeptides capable of forming bioluminescent complexes with HiBiT and SmBiT
to come into contact with
(b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(c) detecting light emitted from said bioluminescent complex;
method including.
[50] (a) the analyte,
SEQ ID NO: 1 (SmBiT)-labeled analyte-specific antibody,
SEQ ID NO: 11 (HiBiT)-labeled analyte-specific antibody, and
Polypeptides capable of forming bioluminescent complexes with HiBiT and SmBiT
to come into contact with
(b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(c) detecting light emitted from said bioluminescent complex;
method including.
[51] A method comprising contacting an analyte with a biomolecule labeled with the composition according to any one of [1] to [25] above.
[52] the analyte is selected from the group consisting of antigens, antibodies, non-antibody proteins, receptors, ligands, toxins, cytokines, nucleic acids, peptides, amino acids, sugars, drugs, nucleoprotein complexes, biotin and streptavidin The method according to [51] above.
[53] The method of [51] above, wherein the analyte biomolecule is labeled with SEQ ID NO: 1 (SmBiT).
[54] (a) forming a bioluminescent complex from a SEQ ID NO: 1 (SmBiT)-labeled analyte biomolecule and an LgBiT-labeled analyte biomolecule-specific antibody;
(b) contacting the bioluminescent complex with the analyte;
(c) contacting said bioluminescent complex with a substrate for said bioluminescent complex; and
(d) detecting light emitted from the bioluminescent complex;
The method according to [51] above.
[55] (a) forming a bioluminescent complex from SEQ ID NO: 1 (SmBiT)-labeled analyte-specific antibody and LgBiT-labeled analyte-specific antibody;
(b) contacting the bioluminescent complex with the analyte;
(c) contacting said bioluminescent complex with a substrate for said bioluminescent complex; and
(d) detecting light emitted from the bioluminescent complex;
The method according to [51] above.
[56] (a) contacting an analyte with SEQ ID NO: 10 (SmBiT)-labeled antibody or receptor and SEQ ID NO: 11 (HiBiT)-labeled antibody or receptor;
(b) contacting the analyte with LgBiT to form a bioluminescent complex;
(c) contacting said bioluminescent complex with a substrate for said bioluminescent complex; and
(d) detecting light emitted from the bioluminescent complex;
The method according to [51] above.
[57] a) contacting a SmBiT- or HiBiT-labeled analyte biomolecule with a HiBiT- or SmBiT-labeled analyte biomolecule-specific antibody;
(b) contacting with LgBiT to form a bioluminescent complex;
(c) contacting the analyte;
(d) contacting said bioluminescent complex with a substrate for said bioluminescent complex; and
(e) detecting light emitted from said bioluminescent complex;
The method according to [51] above.
[58] The method of [57] above, wherein the peptide is a fluorophore-conjugated peptide or a chromophore-conjugated peptide.
[59] The method of [58] above, wherein the number of labels per biomolecule is calculated by the number of fluorophore molecules or chromophore molecules per biomolecule.
[60] The method of [58] above, wherein the fluorophore molecule is FAM, TAMRA, ROX, silo rhodamine, BODIPY, TOM, Dyomics dyes or carbon rhodamine, but is not limited to these fluorophores.
[61] (a) forming a bioluminescent complex from a fluorophore-conjugated SEQ ID NO: 1 (SmBiT)-labeled analyte biomolecule and an LgBiT-labeled analyte biomolecule-specific antibody;
(b) contacting the bioluminescent complex with the analyte;
(c) contacting said bioluminescent complex with a substrate for said bioluminescent complex; and
(d) detecting light emitted from the bioluminescent complex;
The method according to [51] above.
[62] (a) contacting the analyte with both the fluorophore-conjugated SEQ ID NO: 1 (SmBiT)-labeled analyte-specific antibody and the LgBiT-labeled analyte-specific antibody to form a bioluminescent complex;
(b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(c) detecting light emitted from said bioluminescent complex;
The method according to [51] above.
[63] (a) one of SEQ ID NO: 10 (SmBiT) peptide and SEQ ID NO: 11 (HiBiT) peptide is a fluorophore-conjugated peptide;
(b) contacting said analyte with an antibody or receptor or combination labeled with SEQ ID NO: 10 (SmBiT) and SEQ ID NO: 11 (HiBiT), one of said peptides being a fluorophore-conjugated peptide;
(b) contacting with LgBiT to form a bioluminescent complex;
(c) contacting the bioluminescent complex with a substrate for the bioluminescent complex;
(d) detecting light emitted from the bioluminescent complex;
The method according to [51] above.
[64] the analyte biomolecule or analyte-specific antibody is labeled with SEQ ID NO: 10 (SmBiT) or SEQ ID NO: 11 (HiBiT) conjugated with a fluorophore;
(a) Analyte biomolecules labeled with SEQ ID NO: 10 (SmBiT) or SEQ ID NO: 11 (HiBiT) are analyte biomolecules labeled with SEQ ID NO: 11 (HiBiT) or SEQ ID NO: 10 (SmBiT) contacted with a molecule-specific antibody, one of said peptides being a peptide conjugated with said fluorophore;
(b) contacting with LgBiT to form a bioluminescent complex;
(c) contacting the analyte;
(d) contacting the bioluminescent complex with a substrate for the bioluminescent complex;
(e) detecting light emitted from said bioluminescent complex;
Method.
Claims (21)
前記スルホ-SE基と前記生体分子上のアミンとが反応するような条件の下で前記生体分子を請求項1に記載の組成物に接触させること
を含む、前記方法。 A method of labeling a biomolecule with a peptide, comprising:
3. The method comprising contacting the biomolecule with the composition of claim 1 under conditions such that the sulfo-SE groups and amines on the biomolecule react.
(b)前記生物発光複合体を前記生物発光複合体の基質に接触させる工程、及び
(c)発光を検出する工程
を含む、方法。 (a) contacting the composition of claim 14 with an analyte, wherein said analyte is a complementary polypeptide capable of forming a bioluminescent complex with said peptide on said biomolecule; linked process,
(b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(c) detecting luminescence
A method, including
(b)前記生物発光複合体を前記生物発光複合体の基質に接触させる工程、及び
(c)発光、蛍光及び/又は生物発光共鳴エネルギー移動(BRET)を検出する工程
を含む、方法。 (a) contacting the composition of claim 17 with a biomolecule, wherein said biomolecule is linked to a complementary polypeptide capable of forming a bioluminescent complex with said peptide on said analyte; being done, process,
(b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(c) detecting luminescence, fluorescence and/or bioluminescence resonance energy transfer (BRET);
A method, including
第2のスルホn-ヒドロキシスクシンイミジルエステル(スルホ-SE)基に連結された第2のペプチドで標識付けされた生体分子とを含む組成物であって、
前記第1のペプチドがシステイン残基もリジン残基も含まず、
前記第2のペプチドがシステイン残基もリジン残基も含まず、
前記第1及び第2のペプチドが、相補性ポリペプチドの存在下で生物発光複合体を形成できるものである、前記組成物。 an analyte labeled with a first peptide linked to a first sulfo n-hydroxysuccinimidyl ester (sulfo-SE) group ;
a biomolecule labeled with a second peptide linked to a second sulfo n-hydroxysuccinimidyl ester (sulfo-SE) group , wherein
wherein the first peptide contains no cysteine or lysine residues;
wherein the second peptide does not contain cysteine or lysine residues;
Said composition, wherein said first and second peptides are capable of forming a bioluminescent complex in the presence of complementary polypeptides.
(b)前記生物発光複合体を前記生物発光複合体の基質に接触させる工程、及び
(c)発光を検出する工程
を含む、方法。 (a) contacting the analyte and the biomolecule of claim 19 with the complementary polypeptide to form the bioluminescent complex ;
(b) contacting the bioluminescent complex with a substrate for the bioluminescent complex; and
(c) detecting luminescence
A method, including
前記生物発光複合体から前記蛍光団又は発色団への蛍光/光及び/又はBRETを検出する工程を更に含む、
請求項20に記載の方法。 wherein the first peptide or the second peptide is a fluorophore-conjugated peptide or a chromophore-conjugated peptide;
detecting fluorescence/light and/or BRET from said bioluminescent complex to said fluorophore or chromophore;
21. The method of claim 20 .
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US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
WO1988001649A1 (en) | 1986-09-02 | 1988-03-10 | Genex Corporation | Single polypeptide chain binding molecules |
US5260203A (en) | 1986-09-02 | 1993-11-09 | Enzon, Inc. | Single polypeptide chain binding molecules |
FR2918664B1 (en) * | 2007-07-11 | 2009-10-02 | Commissariat Energie Atomique | TRIFUNCTIONAL PEPTIDE PSEUDO REAGENT, USES THEREOF AND APPLICATIONS. |
AU2010242771B2 (en) | 2009-05-01 | 2015-01-22 | Promega Corporation | Synthetic Oplophorus luciferases with enhanced light output |
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US9056885B2 (en) | 2011-11-21 | 2015-06-16 | Promega Corporation | Carboxy X rhodamine analogs |
US10067149B2 (en) | 2012-12-12 | 2018-09-04 | Promega Corporation | Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer |
CA3174484A1 (en) | 2012-12-12 | 2014-06-19 | Promega Corporation | Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer |
PT2970412T (en) * | 2013-03-15 | 2022-09-13 | Promega Corp | Activation of bioluminescence by structural complementation |
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