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1. 发明授权

US5804418A Methods for preparing nucleotide integrases 失效
标题翻译：核苷酸整合酶的制备方法
公开(公告)号：US5804418A
公开(公告)日：1998-09-08
申请号：US752238
申请日：1996-11-19
申请人： Alan Marc Lambowitz , Georg Mohr , Roland Saldanha , Manabu Matsuura
发明人： Alan Marc Lambowitz , Georg Mohr , Roland Saldanha , Manabu Matsuura
IPC分类号： C12N9/22 , C12P21/02 , C12N9/12 , C12N15/54 , C12P19/34
CPC分类号： C12N9/22
摘要： The present invention provides new, improved, and easily manipulable methods for making nucleotide integrases. In one embodiment, the nucleotide integrase is prepared by introducing a DNA molecule which comprises a group II intron DNA sequence into a host cell. The group II intron DNA sequence is then expressed in the host cell such that RNP particles having nucleotide integrase activity are formed in the cell. Such RNP particles comprise an exiced group II intron RNA encoded by the introduced DNA molecule and a group II intron-encoded protein encoded by the introduced DNA molecule. Thereafter, the nucleotide integrase is isolated from the cell. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as "exogenous RNA", with a group II intron-encoded protein. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as "exogenous RNA", with an RNA-protein complex which comprises a group II intron-encoded protein. Preferably, the exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises the group II intron sequence. Preferably, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule which comprises the open reading frame sequence of a group II intron, and then expressing the the open reading sequence in the host cell such that the group II intron-encoded protein encoded by the open reading frame sequence is formed in the cell. Preferably, the RNA-protein complex is made by introducing into a host cell a DNA molecule comprising a group II intron DNA sequence which encodes a splicing-defective group II intron RNA. The present invention also relates to a nucleotide integrase and an improved method for making RNA-protein complexes for use in preparing nucleotide integrases in vitro.
摘要翻译：本发明提供了用于制备核苷酸整合酶的新的，改进的和易于操作的方法。在一个实施方案中，通过将包含II组内含子DNA序列的DNA分子引入宿主细胞来制备核苷酸整合酶。然后在宿主细胞中表达第II组内含子DNA序列，使得在细胞中形成具有核苷酸整合酶活性的RNP颗粒。这样的RNP颗粒包含由导入的DNA分子编码的经过II组的内含子核糖核酸和由引入的DNA分子编码的II组内含子编码的蛋白质。此后，从细胞中分离出核苷酸整合酶。在另一个实施方案中，核苷酸整合酶通过将体外切割的II组内含子RNA（以下称为“外源RNA”）与II组内含子编码的蛋白质组合来制备。在另一个实施方案中，核苷酸整合酶通过将体外包含第II组内含子编码蛋白质的RNA-蛋白质复合物的切割的II组内含子RNA（以下称为“外源RNA”）组合而制备。优选地，外源RNA通过体外转录包含II组内含子序列的DNA分子来制备。优选地，II组内含子编码的蛋白质通过将包含第II组内含子的开放阅读框序列的DNA分子引入宿主细胞，然后在宿主细胞中表达开放阅读序列使得组II 由开放阅读框序列编码的内含子编码的蛋白质在细胞中形成。优选地，通过将包含编码剪接缺陷型II组内含子RNA的II组内含子DNA序列的DNA分子导入宿主细胞来制备RNA-蛋白质复合物。本发明还涉及核苷酸整合酶和用于制备用于在体外制备核苷酸整合酶的RNA-蛋白复合物的改进方法。

2. 发明授权

US6001608A Methods of making an RNP particle having nucleotide integrase activity 失效
标题翻译：制备具有核苷酸整合酶活性的RNP颗粒的方法
公开(公告)号：US6001608A
公开(公告)日：1999-12-14
申请号：US85603
申请日：1998-05-27
申请人： Alan M. Lambowitz , Georg Mohr , Roland Saldanha , Manabu Matsuura , Clifford James Beall , Jiam Yang , Steven Zimmerly , Huatao Guo
发明人： Alan M. Lambowitz , Georg Mohr , Roland Saldanha , Manabu Matsuura , Clifford James Beall , Jiam Yang , Steven Zimmerly , Huatao Guo
IPC分类号： C12N9/22 , C12P19/34 , C07H21/04 , C12N9/12 , C12P21/06
CPC分类号： C12N9/22
摘要： Methods for preparing nucleotide integrases are provided. The nucleotide integrases are prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as "exogenous RNA", with a group II intron-encoded protein. The exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises a group II intron sequence. In one embodiment, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule that comprises at least the open reading frame sequence of a group II intron and then expressing the open reading frame sequence in the host cell. The DNA molecule may comprise the open reading frame sequence operably linked to a promoter, preferably an inducible promoter. Thereafter, the cell is fractionated and the protein is recovered and combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity. In another embodiment, the DNA molecule comprise a group II intron sequence that encodes both a group II intron RNA as well as a group II intron encoded protein. The DNA molecule is then expressed in the host cell to provide RNP particles that comprise the group II intron-encoded protein bound to the group II intron RNA. Thereafter, the RNP particles comprising the group II intron-encoded protein and the group II intron RNA are isolated from the cell and treated with a nuclease to remove the RNA and to provide the group II-intron encoded protein. The group II intron-encoded protein is then combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity.
摘要翻译：提供了制备核苷酸整合酶的方法。核苷酸整合是通过将体外切割的II组内含子RNA（以下称为“外源RNA”）与II组内含子编码的蛋白质组合来制备的。通过体外转录包含II组内含子序列的DNA分子制备外源RNA。在一个实施方案中，II组内含子编码的蛋白质是通过向宿主细胞中引入至少包含第II组内含子的开放阅读框序列，然后在宿主细胞中表达开放阅读框序列的DNA分子来制备的。 DNA分子可以包含可操作地连接到启动子，优选诱导型启动子的开放阅读框序列。此后，分离细胞并回收蛋白质并将其与外源RNA组合以提供具有核苷酸整合酶活性的RNP颗粒。在另一个实施方案中，DNA分子包含编码II组内含子RNA以及II组内含子编码蛋白质的II组内含子序列。然后将DNA分子在宿主细胞中表达以提供包含与II组内含子RNA结合的II组内含子编码的蛋白质的RNP颗粒。此后，从细胞中分离包含II组内含子编码的蛋白质和II组内含子RNA的RNP颗粒，并用核酸酶处理以除去RNA并提供II组内含子编码的蛋白质。然后将II组内含子编码的蛋白质在体外与外源RNA组合以提供具有核苷酸整合酶活性的RNP颗粒。

3. 发明授权

US10113156B2 Stabilized reverse transcriptase fusion proteins 有权
公开(公告)号：US10113156B2
公开(公告)日：2018-10-30
申请号：US13254223
申请日：2010-03-04
申请人： Alan M. Lambowitz , Sabine Mohr , Georg Mohr , Eman Ghanem
发明人： Alan M. Lambowitz , Sabine Mohr , Georg Mohr , Eman Ghanem
IPC分类号： C12N9/12 , C12P19/34
摘要： Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

4. 发明申请

US20120009630A1 STABILIZED REVERSE TRANSCRIPTASE FUSION PROTEINS 审中-公开
标题翻译：稳定的逆转录酶融合蛋白
公开(公告)号：US20120009630A1
公开(公告)日：2012-01-12
申请号：US13254223
申请日：2010-03-04
申请人： Alan M. Lambowitz , Sabine Mohr , Georg Mohr , Eman Ghanem
发明人： Alan M. Lambowitz , Sabine Mohr , Georg Mohr , Eman Ghanem
IPC分类号： C12P19/34 , C12N15/63 , C12N9/12
CPC分类号： C12N9/1276 , C07K2319/00 , C07K2319/24 , C12P19/34 , C12Y207/07049
摘要： Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.
摘要翻译：描述了稳定的逆转录酶融合蛋白，包括与稳定蛋白连接的热稳定逆转录酶。将稳定剂蛋白质连接到热稳定逆转录酶稳定融合蛋白并可以帮助其纯化，提供增加的溶解度，允许更长的储存，或允许融合蛋白在更严格的条件下使用，例如较高的温度。稳定的逆转录酶融合蛋白还可以包括稳定蛋白和热稳定逆转录酶之间的接头。稳定的逆转录酶融合蛋白适用于核酸扩增方法，如逆转录聚合酶链反应和涉及cDNA合成的其他应用。

5. 发明授权

US06306596B1 Methods for cleaving single-stranded and double-stranded DNA substrates with nucleotide integrase 失效
公开(公告)号：US06306596B1
公开(公告)日：2001-10-23
申请号：US09257770
申请日：1999-02-25
申请人： Allen M. Lambowitz , Steven Zimmerly , Huatao Guo , Georg Mohr , Clifford James Beall
发明人： Allen M. Lambowitz , Steven Zimmerly , Huatao Guo , Georg Mohr , Clifford James Beall
IPC分类号： C12Q168
CPC分类号： C12N15/10 , C12Q1/68 , C12Q1/6813 , C12Q1/6823 , C12Q2563/119 , C12Q2525/185 , C12Q2521/313
摘要： Methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double-stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate are provided. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing an isolated nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on the top strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate to permit the nucleotide integrase to cleave the top strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that interacts with a first sequence element and a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences for hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.

6. 发明授权

US6027895A Methods for cleaving DNA with nucleotide integrases 失效
公开(公告)号：US6027895A
公开(公告)日：2000-02-22
申请号：US31897
申请日：1998-02-27
申请人： Allen M. Lambowitz , Steven Zimmerly , Huatao Gau , Georg Mohr , Clifford James Beall
发明人： Allen M. Lambowitz , Steven Zimmerly , Huatao Gau , Georg Mohr , Clifford James Beall
IPC分类号： A01N43/04 , C12P19/34 , C12Q1/68
CPC分类号： A01N43/04 , C12P19/34 , C12Q1/68
摘要： The present invention provides new methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double- stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on the one strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate under conditions that permit the nucleotide integrase to cleave the one strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that is capable of binding to a first sequence element and to a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences that are capable of hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate for a time and at a temperature sufficient to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.

7. 发明授权

US4838873A Apparatus for introducing liquids and/or elongated elements into a body cavity 失效
标题翻译：用于将液体和/或细长元件引入体腔的装置
公开(公告)号：US4838873A
公开(公告)日：1989-06-13
申请号：US198591
申请日：1988-05-23
申请人： Jurgen Landskron , Jorn Zahn , Harald Heckmann , Georg Mohr
发明人： Jurgen Landskron , Jorn Zahn , Harald Heckmann , Georg Mohr
IPC分类号： A61M25/00
CPC分类号： A61M25/0014 , Y10S604/905
摘要： The apparatus for medical purposes comprises a sleeve-type catheter hub (10) having a cylindrical longitudinal channel (11) and a flexible catheter capillary (24) which, through an orifice portion (11a) of the longitudinal channel (11) extends into an expanded intermediate section (11c) of the longitudinal channel (11). The catheter capillary (24) includes a thicker head portion (23) tightened in the expanded intermediate section (11c) of the longitudinal channel (11) by an annular shoulder (22) of a radial annular collar (20) which is formed by ultrasonic welding into the wall of the longitudinal channel (11). To obtain a stepless passage of the cylindrical channel (11), an adapter (18) with a conical passage (19) is joined by press fit to the annular collar (20). The catheter capillary (24) is firmly anchored. In addition, the conical passage (19) serves as an insertion aid for a probe or the like.
摘要翻译：用于医疗目的的装置包括具有圆柱形纵向通道（11）和柔性导管毛细管（24）的套筒型导管套管（10），该导管毛细管通过纵向通道（11）的孔口部分（11a）延伸到扩展的纵向通道（11）的中间部分（11c）。导管毛细管（24）包括通过径向环形套环（20）的环形肩部（22）紧固在纵向通道（11）的膨胀的中间部分（11c）中的较厚的头部部分（23），所述环形肩部（22）由超声波形成焊接到纵向通道（11）的壁中。为了获得圆柱形通道（11）的无级通道，具有锥形通道（19）的适配器（18）通过压配合连接到环形套环（20）。导管毛细管（24）牢固地锚定。此外，锥形通道（19）用作探针等的插入辅助件。

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