公开(公告)号：US08067155B2

公开(公告)日：2011-11-29

申请号：US12536667

申请日：2009-08-06

申请人： Wei Feng ,  William Raab ,  Philip Achacoso ,  Thomas Wehrman ,  Keith R. Olson

发明人： Wei Feng ,  William Raab ,  Philip Achacoso ,  Thomas Wehrman ,  Keith R. Olson

IPC分类号： C12Q1/00 ,  G01N33/53

CPC分类号： G01N33/566 ,  C12Q1/34 ,  C12Q1/485

摘要： Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of β-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of β-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a β-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

摘要翻译： 提供了激活后检测受体酪氨酸激酶（“RTK”）磷酸化的方法。 该方法使用包含两个融合产物的细胞：（1）融合到β-半乳糖苷酶的小片段的RTK和（2）与β-半乳糖苷酶的大片段融合的磷酸酪氨酸结合肽，其中2个片段与 形成活性酶，以及任选的胞质RTK磷酸化激酶的构建体，当RTK不是自磷酸化时。 为了检测磷酸化，将一个半乳糖苷酶底物加入到细胞中，由此产物形成指示磷酸化的发生。

公开(公告)号：US20120040372A1

公开(公告)日：2012-02-16

申请号：US13280919

申请日：2011-10-25

申请人： Wei Feng ,  William Raab ,  Philip Achacoso ,  Thomas Wehrman ,  Keith R. Olson

发明人： Wei Feng ,  William Raab ,  Philip Achacoso ,  Thomas Wehrman ,  Keith R. Olson

IPC分类号： G01N33/573

CPC分类号： G01N33/566 ,  C12Q1/34 ,  C12Q1/485

摘要： Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation and the modulation of activation by a candidate compound are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of β-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of β-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a β-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

摘要翻译： 提供了激活后受体酪氨酸激酶（“RTK”）磷酸化和候选化合物激活调节的方法。 该方法使用包含两个融合产物的细胞：（1）融合到β-半乳糖苷酶的小片段的RTK和（2）与β-半乳糖苷酶的大片段融合的磷酸酪氨酸结合肽，其中2个片段与 形成活性酶，以及任选的胞质RTK磷酸化激酶的构建体，当RTK不是自磷酸化时。 为了检测磷酸化，将一个半乳糖苷酶底物加入到细胞中，由此产物形成指示磷酸化的发生。

公开(公告)号：US20100041052A1

公开(公告)日：2010-02-18

申请号：US12536667

申请日：2009-08-06

申请人： Wei Feng ,  William Raab ,  Philip Achacoso ,  Thomas Wehrman ,  Keith R. Olson

发明人： Wei Feng ,  William Raab ,  Philip Achacoso ,  Thomas Wehrman ,  Keith R. Olson

IPC分类号： C12Q1/68 ,  G01N33/573

CPC分类号： G01N33/566 ,  C12Q1/34 ,  C12Q1/485

摘要： Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of β-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of β-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a β-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

摘要翻译： 提供了激活后检测受体酪氨酸激酶（“RTK”）磷酸化的方法。 该方法使用包含两个融合产物的细胞：（1）融合到β-半乳糖苷酶的小片段的RTK和（2）与β-半乳糖苷酶的大片段融合的磷酸酪氨酸结合肽，其中2个片段与 形成活性酶，以及任选的胞质RTK磷酸化激酶的构建体，当RTK不是自磷酸化时。 为了检测磷酸化，将一个半乳糖苷酶底物加入到细胞中，由此产物形成指示磷酸化的发生。

公开(公告)号：US08569057B2

公开(公告)日：2013-10-29

申请号：US13531230

申请日：2012-06-22

申请人： Thomas S. Wehrman ,  Daniel Bassoni ,  William Raab

发明人： Thomas S. Wehrman ,  Daniel Bassoni ,  William Raab

IPC分类号： C12N5/02

CPC分类号： C12Q1/34 ,  C07K2319/61 ,  G01N33/5076

摘要： Methods and materials are disclosed for use in an enzyme fragment complementation assay using complementary fragments of β-galactosidase to study the trafficking of proteins in a cell. Compounds that bind to a target peptide have been found to affect protein folding and therefore trafficking. β-Galactosidase fragments, an enzyme donor (ED) and an enzyme acceptor (EA), are fused to a target peptide and to an intracellular compartment protein, wherein the compartment is involved in intracellular trafficking. Contacting the cell with a compound that binds to the target peptide results in enhanced movement of the protein through the cellular trafficking pathway comprised of the endoplasmic reticulum, Golgi apparatus, the plasma membrane, endosomes, etc. Using this approach, compounds that bind to a target peptide and alter its ability to traffic through the normal cellular pathway can be readily detected.

摘要翻译： 公开了用于酶片段互补测定的方法和材料，其使用β-半乳糖苷酶的互补片段来研究细胞中蛋白质的运输。 已经发现结合目标肽的化合物影响蛋白质折叠并因此影响贩运。 β-半乳糖苷酶片段，酶供体（ED）和酶受体（EA），与目标肽和细胞内区室蛋白融合，其中所述隔室参与细胞内运输。 将细胞与结合靶肽的化合物接触导致蛋白质通过由内质网，高尔基体，质膜，内体等构成的细胞运送途径的增强的运动。使用这种方法， 可以容易地检测目标肽并改变其通过正常细胞通路的能力。

公开(公告)号：US20120329075A1

公开(公告)日：2012-12-27

申请号：US13531230

申请日：2012-06-22

申请人： Thomas S. Wehrman ,  Daniel Bassoni ,  William Raab

发明人： Thomas S. Wehrman ,  Daniel Bassoni ,  William Raab

IPC分类号： G01N33/573

CPC分类号： C12Q1/34 ,  C07K2319/61 ,  G01N33/5076

摘要： Methods and materials are disclosed for use in an enzyme fragment complementation assay using complementary fragments of β-galactosidase to study the trafficking of proteins in a cell. Compounds that bind to a target peptide have been found to affect protein folding and therefore trafficking. β-Galactosidase fragments, an enzyme donor (ED) and an enzyme acceptor (EA), are fused to a target peptide and to an intracellular compartment protein, wherein the compartment is involved in intracellular trafficking. Contacting the cell with a compound that binds to the target peptide results in enhanced movement of the protein through the cellular trafficking pathway comprised of the endoplasmic reticulum, Golgi apparatus, the plasma membrane, endosomes, etc. Using this approach, compounds that bind to a target peptide and alter its ability to traffic through the normal cellular pathway can be readily detected.

摘要翻译： 公开了用于酶片段互补测定的方法和材料，其使用β-半乳糖苷酶的互补片段来研究细胞中蛋白质的运输。 已经发现结合目标肽的化合物影响蛋白质折叠并因此影响贩运。 将半乳糖苷酶片段，酶供体（ED）和酶受体（EA）融合到靶肽和细胞内区室蛋白，其中所述隔室参与细胞内运输。 将细胞与结合目标肽的化合物接触导致蛋白质通过由内质网，高尔基体，质膜，内体等组成的细胞运送途径的增强的运动。使用这种方法， 可以容易地检测目标肽并改变其通过正常细胞通路的能力。

公开(公告)号：US08211655B2

公开(公告)日：2012-07-03

申请号：US12704399

申请日：2010-02-11

申请人： Thomas S. Wehrman ,  William Raab ,  Chin Yee Loh

发明人： Thomas S. Wehrman ,  William Raab ,  Chin Yee Loh

IPC分类号： G01N33/567 ,  C07K14/705 ,  C07K19/00 ,  C12N15/62

CPC分类号： G01N33/566 ,  G01N2333/726

摘要： A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of β-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of β-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the β-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.

摘要翻译： 使用表达至少辅助蛋白质的结合结构域和半乳糖苷酶的片段的融合蛋白的遗传构建体的细胞来测定受体的配体活化的方法，内含子相关蛋白的融合蛋白和互补片段的互补片段 半乳糖苷酶和野生型受体。 受体的特征在于通过激动剂激活后与辅助蛋白结合结构域结合，然后与内体相关蛋白结合的内体相关的内吞作用。 将细胞与候选配体孵育，然后用含有β-半乳糖苷酶底物的裂解培养基裂解。 然后检测酶产物作为受体活化的量度。

公开(公告)号：US20100203555A1

公开(公告)日：2010-08-12

申请号：US12704399

申请日：2010-02-11

申请人： Thomas S. Wehrman ,  William Raab ,  Chin Yee Loh

发明人： Thomas S. Wehrman ,  William Raab ,  Chin Yee Loh

IPC分类号： G01N33/573

CPC分类号： G01N33/566 ,  G01N2333/726

摘要： A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of β-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of β-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the β-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.

摘要翻译： 使用表达至少辅助蛋白质的结合结构域和半乳糖苷酶的片段的融合蛋白的遗传构建体的细胞来测定受体的配体活化的方法，内含子相关蛋白的融合蛋白和内含子相关蛋白的互补片段 半乳糖苷酶和野生型受体。 受体的特征在于通过激动剂激活后与辅助蛋白结合结构域结合，然后与内体相关蛋白结合的内体相关的内吞作用。 将细胞与候选配体孵育，然后用含有β-半乳糖苷酶底物的裂解培养基裂解。 然后检测酶产物作为受体活化的量度。