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    Methods of Protein Destabilization and Uses Thereof
    1.
    发明申请
    Methods of Protein Destabilization and Uses Thereof 审中-公开
    蛋白质不稳定的方法及其用途

    公开(公告)号：US20110191873A1

    公开(公告)日：2011-08-04

    申请号：US12938179

    申请日：2010-11-02

    申请人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    发明人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    IPC分类号： A01K67/00 ,  C12N5/10 ,  C07H21/04 ,  C12N1/00 ,  C07K19/00 ,  A01H5/00 ,  C12Q1/02 ,  C12Q1/37 ,  C12Q1/42 ,  C12Q1/48 ,  C12Q1/66 ,  C12N5/07 ,  C12N5/071 ,  C12N5/04

    CPC分类号： C12Q1/485

    摘要： This invention is directed towards methods of destabilizing proteins in living cells, and their use for the development of novel assays. In one embodiment, the invention comprises the use of non-cleavable multimerized ubiquitin fusion proteins to destabilize a target protein, such as a reporter moiety. In one aspect of this method the constructs also comprises a linker that operatively couples the reporter moiety to the multimerized ubiquitin fusion protein. In this embodiment, enzymatic modification of the linker results in a modulation of the coupling of the reporter protein to the multimerized ubiquitin domains resulting in a change in the stability of the reporter moiety. The level of the reporter moiety in the cell can then be used as a measure of the enzymatic activity in the cell. In another embodiment the invention provides for a generalized way of coordinately regulating the cellular concentration of a plurality of target proteins. In one aspect of this method, the target proteins are operatively coupled to a ubiquitin fusion protein via a linker containing a protease cleavage site. Cleavage of the linker by a protease results in uncoupling of the target protein from the multimerized ubiquitin construct, and results in an increase in the stability and concentration of the target protein.

    摘要翻译： 本发明涉及使活细胞中的蛋白质失稳的方法及其用于开发新的测定方法。 在一个实施方案中，本发明包括使用不可切割的多聚化泛素融合蛋白来使目标蛋白如报道分子部分不稳定。 在该方法的一个方面，构建体还包含使报道部分与多聚化泛素融合蛋白可操作地连接的连接体。 在该实施方案中，接头的酶修饰导致报道蛋白与多聚泛素结构域的偶联的调节，导致报道部分稳定性的变化。 然后可以将细胞中的报告物部分的水平用作细胞中酶活性的量度。 在另一个实施方案中，本发明提供了协调调节多种靶蛋白的细胞浓度的一般化方式。 在该方法的一个方面，靶蛋白通过含有蛋白酶切割位点的接头与泛素融合蛋白可操作地偶联。 通过蛋白酶切割接头导致靶蛋白与多聚化泛素构建体解偶联，并导致靶蛋白的稳定性和浓度增加。

    Methods of protein destabilization and uses thereof
    2.
    发明授权
    Methods of protein destabilization and uses thereof 有权
    蛋白质不稳定的方法及其用途

    公开(公告)号：US07824850B2

    公开(公告)日：2010-11-02

    申请号：US11821562

    申请日：2007-06-22

    申请人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    发明人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    IPC分类号： C12Q1/00 ,  C07K14/00

    CPC分类号： C12Q1/485

    摘要： This invention is directed towards methods of destabilizing proteins in living cells, and their use for the development of novel assays. In one embodiment, the invention comprises the use of non-cleavable multimerized ubiquitin fusion proteins to destabilize a target protein, such as a reporter moiety. In one aspect of this method the constructs also comprises a linker that operatively couples the reporter moiety to the multimerized ubiquitin fusion protein. In this embodiment, enzymatic modification of the linker results in a modulation of the coupling of the reporter protein to the multimerized ubiquitin domains resulting in a change in the stability of the reporter moiety. The level of the reporter moiety in the cell can then be used as a measure of the enzymatic activity in the cell. In another embodiment the invention provides for a generalized way of coordinately regulating the cellular concentration of a plurality of target proteins. In one aspect of this method, the target proteins are operatively coupled to a ubiquitin fusion protein via a linker containing a protease cleavage site. Cleavage of the linker by a protease results in uncoupling of the target protein from the multimerized ubiquitin construct, and results in an increase in the stability and concentration of the target protein.

    摘要翻译： 本发明涉及使活细胞中的蛋白质失稳的方法及其用于开发新的测定方法。 在一个实施方案中，本发明包括使用不可切割的多聚化泛素融合蛋白来使目标蛋白如报道分子部分不稳定。 在该方法的一个方面，构建体还包含使报道部分与多聚化泛素融合蛋白可操作地连接的连接体。 在该实施方案中，接头的酶修饰导致报道蛋白与多聚泛素结构域的偶联的调节，导致报道部分稳定性的变化。 然后可以将细胞中的报告物部分的水平用作细胞中酶活性的量度。 在另一个实施方案中，本发明提供了协调调节多种靶蛋白的细胞浓度的一般化方式。 在该方法的一个方面，靶蛋白通过含有蛋白酶切割位点的接头与泛素融合蛋白可操作地偶联。 通过蛋白酶切割接头导致靶蛋白与多聚化泛素构建体解偶联，并导致靶蛋白的稳定性和浓度增加。

    Methods of protein destabilization and uses thereof
    3.
    发明申请
    Methods of protein destabilization and uses thereof 有权
    蛋白质不稳定的方法及其用途

    公开(公告)号：US20080227129A1

    公开(公告)日：2008-09-18

    申请号：US11821562

    申请日：2007-06-22

    申请人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    发明人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    IPC分类号： C12Q1/66 ,  C12Q1/02 ,  C12Q1/37 ,  C12Q1/48 ,  C12N1/16 ,  C07K14/00 ,  C12N5/06 ,  C12N5/00 ,  C12Q1/42 ,  C12Q1/34

    CPC分类号： C12Q1/485

    摘要： This invention is directed towards methods of destabilizing proteins in living cells, and their use for the development of novel assays. In one embodiment, the invention comprises the use of non-cleavable multimerized ubiquitin fusion proteins to destabilize a target protein, such as a reporter moiety. In one aspect of this method the constructs also comprises a linker that operatively couples the reporter moiety to the multimerized ubiquitin fusion protein. In this embodiment, enzymatic modification of the linker results in a modulation of the coupling of the reporter protein to the multimerized ubiquitin domains resulting in a change in the stability of the reporter moiety. The level of the reporter moiety in the cell can then be used as a measure of the enzymatic activity in the cell. In another embodiment the invention provides for a generalized way of coordinately regulating the cellular concentration of a plurality of target proteins. In one aspect of this method, the target proteins are operatively coupled to a ubiquitin fusion protein via a linker containing a protease cleavage site. Cleavage of the linker by a protease results in uncoupling of the target protein from the multimerized ubiquitin construct, and results in an increase in the stability and concentration of the target protein.

    摘要翻译： 本发明涉及使活细胞中的蛋白质失稳的方法及其用于开发新的测定方法。 在一个实施方案中，本发明包括使用不可切割的多聚化泛素融合蛋白来使目标蛋白如报道分子部分不稳定。 在该方法的一个方面，构建体还包含使报道部分与多聚化泛素融合蛋白可操作地连接的接头。 在该实施方案中，接头的酶修饰导致报道蛋白与多聚泛素结构域的偶联的调节，导致报道部分稳定性的变化。 然后可以将细胞中的报告物部分的水平用作细胞中酶活性的量度。 在另一个实施方案中，本发明提供了协调调节多种靶蛋白的细胞浓度的一般化方式。 在该方法的一个方面，靶蛋白通过含有蛋白酶切割位点的接头与泛素融合蛋白可操作地偶联。 通过蛋白酶切割接头导致靶蛋白与多聚化泛素构建体解偶联，并导致靶蛋白的稳定性和浓度增加。

    Methods of protein destabilization and uses thereof
    4.
    发明授权
    Methods of protein destabilization and uses thereof 有权
    蛋白质不稳定的方法及其用途

    公开(公告)号：US07262005B1

    公开(公告)日：2007-08-28

    申请号：US09498098

    申请日：2000-02-04

    申请人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    发明人： Jeffrey Stack ,  Michael Whitney ,  Andrew B. Cubitt ,  Brian Pollok

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/485

    摘要： This invention is directed towards methods of destabilizing proteins in living cells, and their use for the development of novel assays. In one embodiment, the invention comprises the use of non-cleavable multimerized ubiquitin fusion proteins to destabilize a target protein, such as a reporter moiety. In one aspect of this method the constructs also comprises a linker that operatively couples the reporter moiety to the multimerized ubiquitin fusion protein. In this embodiment, enzymatic modification of the linker results in a modulation of the coupling of the reporter protein to the multimerized ubiquitin domains resulting in a change in the stability of the reporter moiety. The level of the reporter moiety in the cell can then be used as a measure of the enzymatic activity in the cell. In another embodiment the invention provides for a generalized way of coordinately regulating the cellular concentration of a plurality of target proteins. In one aspect of this method, the target proteins are operatively coupled to a ubiquitin fusion protein via a linker containing a protease cleavage site. Cleavage of the linker by a protease results in uncoupling of the target protein from the multimerized ubiquitin construct, and results in an increase in the stability and concentration of the target protein.

    摘要翻译： 本发明涉及使活细胞中的蛋白质失稳的方法及其用于开发新的测定方法。 在一个实施方案中，本发明包括使用不可切割的多聚化泛素融合蛋白来使目标蛋白如报道分子部分不稳定。 在该方法的一个方面，构建体还包含使报道部分与多聚化泛素融合蛋白可操作地连接的接头。 在该实施方案中，接头的酶修饰导致报道蛋白与多聚泛素结构域的偶联的调节，导致报道部分稳定性的变化。 然后可以将细胞中的报告物部分的水平用作细胞中酶活性的量度。 在另一个实施方案中，本发明提供了协调调节多种靶蛋白的细胞浓度的一般化方式。 在该方法的一个方面，靶蛋白通过含有蛋白酶切割位点的接头与泛素融合蛋白可操作地偶联。 通过蛋白酶切割接头导致靶蛋白与多聚化泛素构建体解偶联，并导致靶蛋白的稳定性和浓度增加。

    Somatic hypermutation systems
    6.
    发明申请
    Somatic hypermutation systems 审中-公开
    体细胞超突变体系

    公开(公告)号：US20090075378A1

    公开(公告)日：2009-03-19

    申请号：US12070905

    申请日：2008-02-20

    申请人： Robert A. Horlick ,  Andrew B. Cubitt ,  Peter M. Bowers

    发明人： Robert A. Horlick ,  Andrew B. Cubitt ,  Peter M. Bowers

    IPC分类号： C12N5/06 ,  C12N15/12 ,  C12N15/52 ,  C12N5/08 ,  C12N5/00 ,  C12N1/19 ,  C12N15/85 ,  C12N15/81

    CPC分类号： C07K16/461 ,  C07K16/00 ,  C07K16/22 ,  C07K16/40 ,  C07K2317/21 ,  C07K2317/565 ,  C07K2317/92 ,  C40B50/06

    摘要： The present application relates to somatic hypermutation (SHM) systems and synthetic genes. Synthetic genes can be designed using computer-based approaches to increase or decrease susceptibility of a polynucleotide to somatic hypermutation. Genes of interest are inserted into the vectors and subjected to activation-induced cytidine deaminase to induce somatic hypermutation. Proteins or portions thereof encoded by the modified genes can be introduced into a SHM system for somatic hypermutation and proteins or portions thereof exhibiting a desired phenotype or function can be isolated for in vitro or in vivo diagnostic or therapeutic uses.

    摘要翻译： 本申请涉及体细胞超突变（SHM）系统和合成基因。 可以使用基于计算机的方法设计合成基因以增加或降低多核苷酸对体细胞超突变的易感性。 将感兴趣的基因插入载体中并进行活化诱导的胞苷脱氨酶诱导体细胞超突变。 由修饰的基因编码的蛋白质或其部分可以被引入用于体细胞超突变的SHM系统中，并且可以分离出表现出所需表型或功能的蛋白质或其部分用于体外或体内诊断或治疗用途。