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    • 61. 发明公开
    • 3가 금속 매개 균질 발광 근접 분석
    • 多金属介质均质光致亲和性测定
    • KR1020070099519A
    • 2007-10-09
    • KR1020077007650
    • 2005-09-19
    • 노바티스 백신즈 앤드 다이아그노스틱스 인코포레이티드
    • 쿠오리차드씨.더즈티모티디.마카쥬스키티모티디.
    • C12Q1/48C12Q1/00G01N33/53
    • G01N33/542C12Q1/42C12Q1/44C12Q1/485
    • An in vitro protein kinase assay technology that (1) exhibits a high assay signal to background ratio (S/B) and range (S-B); (2) is homogenous; (3) is non-radioactive; and (4) does not require a phospho-specific antibody involves complexing a trivalent metal ion (e.g. Ga3+, Fe3+, Al3+, In3+, Ru3+, Sc3+, Y3+) to the surface of amplified luminescent proximity assay acceptor or donor beads, e.g., via a suitable linker such as nitrilotriacetic acid (NTA; also referred to as carboxymethyl-lysine), iminodiacetic acid (IDA), or an appropriately substituted N-containing heterocycle, for example a triazoheterocycle, for example a triazocyclononaneononane, such as 1-propylamino-4-acetato-1,4,7-triazacyclononane. A protein (or constituent part) or other kinase substrate is bound to the surface of the other of an amplified luminescent proximity assay acceptor or donor bead and, if phosphorylated, brought into proximity with the trivalent metal ion-complexed acceptor bead to generate a luminescent signal. Presence of a kinase inhibitor inhibits phosphorylation and therefore signal generation and, in this way, is detectable. As the invention described herein recognizes the presence or absence of phosphate groups on a protein, (or constituent part), or other biological macromolecule (e.g., mono, di, or trinucleotides, cyclic nucleotides or phosphate substituted inositols), it is broadly applicable to any phosphorlylation or dephosphorylation reaction enzymes and provides a highly robust and flexible assay format for protein kinases and other enzyme classes, including lipid kinases, phosphatases, phosphodiesterases and others.
    • (1)表现出高测定信号与背景比(S / B)和范围(S-B)的体外蛋白激酶测定技术; (2)是均匀的; (3)是非放射性的; 和(4)不需要磷酸特异性抗体涉及将三价金属离子(例如,Ga 3+,Fe 3+,Al 3+,In 3+,Ru 3+,Sc 3+,Y 3+)与扩增的发光亲和力测定受体或供体珠的表面络合, 适当的接头如次氮基三乙酸(NTA;也称羧甲基赖氨酸),亚氨基二乙酸(IDA)或适当取代的含N的杂环,例如三氮杂环壬烷,例如三氮杂环壬烷,例如1-丙基氨基 - 4-乙酸基-1,4,7-三氮杂环。 蛋白质(或组成部分)或其他激酶底物与扩增的发光亲和测定受体或供体小珠的另一个的表面结合,并且如果磷酸化,则与三价金属离子络合的受体珠接近以产生发光 信号。 激酶抑制剂的存在抑制磷酸化并因此产生信号,并且以这种方式可检测。 如本文所述的发明认识到蛋白质(或组成部分)或其他生物大分子(例如单,二或三核苷酸,环状核苷酸或磷酸酯取代的肌醇)上磷酸基团的存在或不存在,广泛适用于 任何磷酸化或去磷酸化反应酶,并为蛋白激酶和其他酶类提供了强大而灵活的测定形式,包括脂质激酶,磷酸酶,磷酸二酯酶等。
    • 63. 发明公开
    • 세포내 맥아당의 정량적 측정을 위한 맥아당 바이오센서
    • MALTOSE生物传感器用于在细胞中定量测定麦芽糖
    • KR1020070013024A
    • 2007-01-30
    • KR1020050067353
    • 2005-07-25
    • 한국생명공학연구원
    • 이승구송재준하재석이영미
    • C12Q1/00C12Q1/68C12N15/09
    • C07K14/195C07K2319/60G01N33/533G01N33/542G01N33/582G01N33/536
    • Maltose biosensors for quantitative measurement of maltose in a cell are provided to improve accuracy of maltose measurement by increasing FRET(fluorescence resonance energy transfer) sensing signal through mutation of genes associated with connection between a maltose-binding protein(MBP) and an energy donor protein, so that the biosensor is applicable to other biosensors for measuring periplasmic binding protein family. The maltose biosensor for quantitative measurement of maltose in the cell, CMYB-LH, prepared by connecting CFP(cyan fluorescence protein) and MBP through Leu-His linkage has the amino acid sequence of SEQ ID NO:11. The maltose biosensor for quantitative measurement of maltose in the cell, CMYB-QI, prepared by connecting CFP and MBP through Gln-Ile linkage has the amino acid sequence of SEQ ID NO:12. The maltose biosensor for quantitative measurement of maltose in the cell, CMYBLH/W62L, prepared by connecting CFP and MBP having a W62L mutation through Leu-His linkage has the amino acid sequence of SEQ ID NO:13.
    • 提供了用于定量测量细胞中麦芽糖的麦芽糖生物传感器,以通过与麦芽糖结合蛋白(MBP)和能量供体蛋白之间的连接相关基因的突变增加FRET(荧光共振能量转移)感测信号来提高麦芽糖测量的准确度 ,使得生物传​​感器适用于其他用于测量周质结合蛋白家族的生物传感器。 通过连接CFP(青色荧光蛋白)和MBP通过Leu-His键制备的用于定量测量细胞中的麦芽糖的麦芽糖生物传感器CMYB-LH具有SEQ ID NO:11的氨基酸序列。 通过连接CFP和MBP通过Gln-Ile连接制备的用于定量测量细胞中麦芽糖的麦芽糖生物传感器CMYB-QI具有SEQ ID NO:12的氨基酸序列。 通过连接具有W62L突变的CFP和MBP通过Leu-His键制备的用于定量测量细胞中的麦芽糖的麦芽糖生物传感器CMYBLH / W62L具有SEQ ID NO:13的氨基酸序列。