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1.发明授权In vivo library-versus-library selection of optimized protein-protein interactions 失效体内文库对文库选择优化的蛋白质 - 蛋白质相互作用公开(公告)号：US07625700B2
公开(公告)日：2009-12-01
申请号：US11134253
申请日：2005-05-23
CPC分类号： C40B30/04 , C07K14/43595 , C07K2319/00 , C12N15/1055 , C12Q1/32 , G01N33/542 , G01N33/6845 , Y02A90/26
摘要： The present invention describes a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. It allows for the screening of a protein library against a second protein library and therefore finds numerous applications in the study of protein-protein interactions. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into E. coli. Interaction between the library polypeptides was required for reconstitution of the enzymatic activity of mDHFR, allowing bacterial growth. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We applied these selection processes to a library-versus-library sample of 2.0×106 combinations, and selected a novel leucine zipper pair which may be appropriate for use in further in vivo heterodimerization strategies.
摘要翻译： 本发明描述了快速和有效的体内文库对文库筛选策略，用于鉴定异构二聚化多肽的最佳相互作用对。 它允许对第二个蛋白质文库进行蛋白质文库的筛选，因此可以在研究蛋白质 - 蛋白质相互作用中发现许多应用。 两个亮氨酸拉链文库在与疏水核心相邻的位置半随机化，与酶二氢叶酸还原酶（mDHFR）的两个设计的片段中的任一个遗传融合，并共转化到大肠杆菌中。 需要文库多肽之间的相互作用来重构mDHFR的酶活性，从而允许细菌生长。 使用更弱的关联mDHFR片段，我们增加了选择的严格性。 我们将这些选择过程应用于2.0×10 6组合的文库与文库样品，并选择了可能适用于进一步体内异二聚化策略的新型亮氨酸拉链对。
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2.发明申请In vivo library-versus-library selection of optimized protein-protein interactions 审中-公开体内文库对文库选择优化的蛋白质 - 蛋白质相互作用公开(公告)号：US20100081580A1
公开(公告)日：2010-04-01
申请号：US12591731
申请日：2009-11-30
IPC分类号： C40B30/00
CPC分类号： C40B30/04 , C07K14/43595 , C07K2319/00 , C12N15/1055 , C12Q1/32 , G01N33/542 , G01N33/6845 , Y02A90/26
摘要： The present invention describes a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. It allows for the screening of a protein library against a second protein library, rather than against a single bait protein, and thus has numerous applications in the study of protein-protein interactions. Additionally, it allows for the application of different selection stringencies. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into E. coli. Interaction between the library polypeptides was required for reconstitution of the enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0×106 combinations, and selected a novel leucine zipper pair which may be appropriate for use in further in vivo heterodimerization strategies.
摘要翻译： 本发明描述了快速和有效的体内文库对文库筛选策略，用于鉴定异构二聚化多肽的最佳相互作用对。 它允许针对第二蛋白质文库筛选蛋白质文库，而不是针对单个诱饵蛋白质，因此在蛋白质 - 蛋白质相互作用的研究中具有许多应用。 此外，它允许应用不同的选择严格性。 两个亮氨酸拉链文库在与疏水核心相邻的位置半随机化，与酶二氢叶酸还原酶（mDHFR）的两个设计的片段中的任一个遗传融合，并共转化到大肠杆菌中。 需要文库多肽之间的相互作用来重构mDHFR的酶活性，从而允许细菌生长。 所得菌落的分析揭示了相对于原始文库的拉链序列的重要偏倚，这与选择稳定的异源二聚体对一致。 使用更弱的关联mDHFR片段，我们增加了选择的严格性。 我们通过在液体培养中多次传代合并的选定菌落，丰富了表现最好的亮氨酸拉链对，因为最佳对允许更好的细菌繁殖。 这种竞争性增长允许放大对中的小差异，并且以不同的速率富集不同的序列位置。 我们将这些选择过程应用于2.0×106组合的库与文库样本，并选择了可能适用于进一步体内异二聚化策略的新型亮氨酸拉链对。
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3.发明授权Vivo library-versus-library selection of optimized protein-protein interactions 失效体内文库与图书馆选择优化的蛋白质 - 蛋白质相互作用公开(公告)号：US06897017B1
公开(公告)日：2005-05-24
申请号：US09603885
申请日：2000-06-26
IPC分类号： C07K14/435 , C12N15/10 , C12Q1/32 , C12Q1/68 , C40B30/04 , G01N33/53 , G01N33/542 , G01N33/573 , G01N33/58 , G01N33/68 , C12N15/52 , C12P21/02
CPC分类号： C40B30/04 , C07K14/43595 , C07K2319/00 , C12N15/1055 , C12Q1/32 , G01N33/542 , G01N33/6845 , Y02A90/26
摘要： The present invention describes a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. It allows for the screening of a protein library against a second protein library, rather than against a single bait protein, and thus has numerous applications in the study of protein-protein interactions. Additionally, it allows for the application of different selection stringencies. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into E. coli. Interaction between the library polypeptides was required for reconstitution of the enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0×106 combinations, and selected a novel leucine zipper pair which may be appropriate for use in further in vivo heterodimerization strategies.
摘要翻译： 本发明描述了快速和有效的体内文库对文库筛选策略，用于鉴定异构二聚化多肽的最佳相互作用对。 它允许针对第二蛋白质文库筛选蛋白质文库，而不是针对单个诱饵蛋白质，因此在蛋白质 - 蛋白质相互作用的研究中具有许多应用。 此外，它允许应用不同的选择严格性。 两个亮氨酸拉链文库在与疏水核心相邻的位置半随机化，与酶二氢叶酸还原酶（mDHFR）的两个设计的片段中的任一个遗传融合，并共转化到大肠杆菌中。 需要文库多肽之间的相互作用来重构mDHFR的酶活性，从而允许细菌生长。 所得菌落的分析揭示了相对于原始文库的拉链序列的重要偏倚，这与选择稳定的异源二聚体对一致。 使用更弱的关联mDHFR片段，我们增加了选择的严格性。 我们通过在液体培养中多次传代合并的选定菌落，丰富了表现最好的亮氨酸拉链对，因为最佳对允许更好的细菌繁殖。 这种竞争性增长允许放大对中的小差异，并且以不同的速率富集不同的序列位置。 我们将这些选择过程应用于2.0×10 6组合的文库对文库样品，并选择了可能适用于进一步体内异二聚化策略的新型亮氨酸拉链对。
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4.发明申请PROTEIN FRAGMENT COMPLEMENTATION ASSAYS FOR THE DETECTION OF BIOLOGICAL OR DRUG INTERACTIONS 审中-公开用于检测生物或药物相互作用的蛋白质片段补充测定公开(公告)号：US20130022999A1
公开(公告)日：2013-01-24
申请号：US13488193
申请日：2012-06-04
CPC分类号： G01N33/6845 , A01K67/0271 , A01K2267/0331 , A01K2267/0393 , C07K14/43595 , C07K2319/00 , C12N15/1055 , G01N33/5008 , G01N33/542 , G01N33/573 , G01N33/58 , G01N33/581 , G01N33/68 , G01N33/6803 , Y02A90/26
摘要： The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule
摘要翻译： 本发明描述了一种用于检测生物分子相互作用的方法，所述方法包括：（a）选择选自蛋白质，荧光蛋白质，发光蛋白质和磷光蛋白质的合适的报道分子; （b）实现所述报道分子的片段化，使得所述片段化导致报告基因功能的可逆丧失; （c）将所述报告分子的片段与其它分子分开或附着; 随后（d）通过与所述片段融合的分子的相互作用使所述报道片段重新结合; 和（e）通过重建报告分子的活性来检测所述生物分子相互作用
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5.发明申请公开(公告)号：US20120208197A1
公开(公告)日：2012-08-16
申请号：US13137605
申请日：2011-08-29
申请人： Stephen William Watson Michnick , Barbara Belisle , Marnie L. MacDonald , John K. Westwick , Jane Elizabeth Lamerdin
发明人： Stephen William Watson Michnick , Barbara Belisle , Marnie L. MacDonald , John K. Westwick , Jane Elizabeth Lamerdin
IPC分类号： C12Q1/68 , G01N33/573 , G01N33/566
CPC分类号： G01N33/502 , C12Q1/6897 , G01N33/5008 , G01N33/5041
摘要： The cell-based assays described in the present invention can be used to directly assess the sensitivity and specificity of the gene annotation reagent against its target, mapping genes and to determine if a non-targeted gene participates in a pathway of interest or is functionally linked to another gene or protein. Preferred assay embodiments include fluorescence or luminescence assays in intact (live or fixed) cells. Such fluorescence or luminescence assays include high-throughput or high-content assays for protein activity, subcellular localization, post-translational modifications, or interactions of proteins. Suitable assays may include protein-protein interaction assays; protein translocation assays; and post-translational modification assays. The invention can be used to assess the efficacy of any gene silencing experiment, and to map novel genes into biochemical pathways, and to identify novel pharmaceutical targets. The results also demonstrate the feasibility of employing this strategy in genome-wide functional annotation efforts.
摘要翻译： 本发明中描述的基于细胞的测定可用于直接评估基因注释试剂对其靶标，测绘基因的灵敏度和特异性，并确定非靶向基因是否参与感兴趣的途径或功能上相关 到另一种基因或蛋白质。 优选的测定实施方案包括在完整（活的或固定的）细胞中的荧光或发光测定。 这种荧光或发光测定法包括用于蛋白质活性，亚细胞定位，翻译后修饰或蛋白质相互作用的高通量或高含量测定。 合适的测定可以包括蛋白质 - 蛋白质相互作用测定; 蛋白质易位测定; 和翻译后修饰测定。 本发明可用于评估任何基因沉默实验的功效，并将新基因映射到生物化学途径中，并鉴定新的药物靶点。 结果还表明在全基因组功能注释方面采用这一策略的可行性。
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6.发明申请Protein fragment complementation assays for the detection of biological or drug interactions 有权用于检测生物或药物相互作用的蛋白质片段互补测定法公开(公告)号：US20110287950A1
公开(公告)日：2011-11-24
申请号：US13137257
申请日：2011-08-01
IPC分类号： C40B30/00 , C07K14/34 , C07K14/435 , C12N9/96 , C12Q1/66 , C12Q1/48 , C12Q1/34 , C12Q1/26 , C07K14/21 , C07K14/00
CPC分类号： G01N33/6845 , A01K67/0271 , A01K2267/0331 , A01K2267/0393 , C07K14/43595 , C07K2319/00 , C12N15/1055 , G01N33/5008 , G01N33/542 , G01N33/573 , G01N33/58 , G01N33/581 , G01N33/68 , G01N33/6803 , Y02A90/26
摘要： We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E. coli doubling times illustrating the successful DHFR fragment reassembly rather than non-specific interactions between fragments. This assay could be used to study equilibrium and kinetic aspects of molecular interactions including protein-protein, protein-DNA, protein-RNA, protein-carbohydrate and protein-small molecule interactions, for screening cDNA libraries for binding of a target protein with unknown proteins or libraries of small organic molecules for biological activity. The selection and design criteria applied here is developed for numerous examples of clonal selection, colorometric, fluorometric and other assays based on enzymes whose products can be measured. The development of such assay systems is shown to be simple, and provides for a diverse set of protein fragment complementation applications.
摘要翻译： 我们描述了设计和实施蛋白质片段互补测定（PCAs）以检测体内和体外生物分子相互作用的策略。 该策略的设计，实现和广泛应用用大量酶进行了说明，具体细节提供了鼠二氢叶酸还原酶（DHFR）的例子。 由融合到GCN4亮氨酸拉链序列的鼠DHFR的N-和C-末端片段组成的融合肽在基本培养基中生长的大肠杆菌中共表达，其中内源性DHFR活性被甲氧苄啶抑制。 互补融合产物的共表达恢复了菌落形成。 存活只发生在两个DHFR片段存在并含有亮氨酸 - 拉链形成序列时，表明重组酶活性需要辅助亮氨酸拉链形成。 增加严重程度（Ile至Val，Ala和Gly）的DHFR片段 - 接口点突变体导致大肠杆菌倍增时间的顺序增加，说明成功的DHFR片段重组而不是片段之间的非特异性相互作用。 该测定可用于研究包括蛋白质蛋白质，蛋白质-DNA，蛋白质-RNA，蛋白质 - 碳水化合物和蛋白质 - 小分子相互作用在内的分子相互作用的平衡和动力学方面，用于筛选靶向蛋白质与未知蛋白质结合的cDNA文库 或用于生物活性的小有机分子的文库。 这里应用的选择和设计标准是针对克隆选择，色度，荧光测定和其他基于可以测量其产物的酶的测定的许多实例开发的。 这种测定系统的开发被证明是简单的，并且提供了多种蛋白质片段互补应用的集合。
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7.发明授权Protein fragment complementation assays for high-throughput and high-content screening 失效用于高通量和高含量筛选的蛋白质片段互补测定公开(公告)号：US07935493B2
公开(公告)日：2011-05-03
申请号：US11450379
申请日：2006-06-12
申请人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
发明人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
IPC分类号： G01N33/53 , G01N33/532 , G01N33/533 , G01N33/573 , G01N21/00 , G01N21/76 , C07K14/00
CPC分类号： G01N33/5011 , G01N33/5008 , G01N33/5041 , G01N33/542 , G01N33/6845 , G01N2500/02 , Y02A90/26
摘要： The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. Single-color and multi-color assays are disclosed. Further disclosed are universal expression vectors with cassettes that allow the rapid construction of assays for a large and diverse number of gene/reporter combinations. The development of such assays is shown to be straightforward, providing for a broad, flexible and biologically relevant platform for drug discovery.
摘要翻译： 本发明提供用于药物发现的蛋白质片段互补测定法，特别是鉴定激活或抑制细胞途径的化合物。 基于与适当的PCA报道子组合的相互作用的蛋白质对的选择，测定可以以高通量或高含量模式进行，并且可以用于化合物文库的自动化筛选。 可以通过cDNA文库筛选来选择相互作用的对; 通过基因相互作用作图; 或通过路径的先验知识。 可以使用本文提供的方法构建荧光和发光测定。 针对多种报道者描述了适合于高通量或高含量（高上下文）测定形式的PCA报告人的选择，具体提供了单体酶和荧光蛋白的实例。 描述了用于构建生物化学途径中的一个或多个步骤的这种测定方法; 测试来自组合，天然产物，肽，抗体，核酸或其他不同文库的化合物对感兴趣的蛋白质或途径的影响; 并使用筛选结果来鉴定激活或抑制感兴趣的蛋白质或途径的特定化合物。 公开了单色和多色测定。 进一步公开的是具有盒的通用表达载体，其允许快速构建用于大量和多样化数量的基因/报道子组合的测定。 显示出这种测定的发展是直接的，为药物发现提供了广泛，灵活和生物学上相关的平台。
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8.发明授权Protein fragment complementation assays in whole animals applications to drug efficacy, ADME, cancer biology, immunology, infectious disease and gene therapy 有权整个动物中的蛋白质片段互补测定应用于药物功效，ADME，癌症生物学，免疫学，传染病和基因治疗公开(公告)号：US07306914B2
公开(公告)日：2007-12-11
申请号：US10882292
申请日：2004-07-02
CPC分类号： A01K67/0271 , A01K2267/0331 , A01K2267/0393 , C07K14/43595 , C07K2319/00 , C12N15/1055 , C12Q1/025 , G01N33/5008 , G01N33/5088 , G01N33/542 , G01N33/573 , G01N33/58 , G01N33/581 , G01N33/68 , G01N33/6803 , G01N33/6845 , Y02A90/26
摘要： The instant invention describes a method for detecting protein-protein interactions in living organisms and/or cells, said method comprising: (a) synthesizing probe protein fragments from a protein which enables fluorescent or luminescent detection by dissecting the gene coding for the fluorescent or luminescent protein into a least two fragments; (2) constructing fusion proteins consisting of the probe protein fragments linked to protein domains that are to be tested for interactions; (3) coexpressing the fusion proteins; and (d) detecting reconstitution of the fluorescence or luminescence signal.
摘要翻译： 本发明描述了一种用于检测活体和/或细胞中的蛋白质 - 蛋白质相互作用的方法，所述方法包括：（a）从蛋白质合成探针蛋白片段，其通过解剖编码荧光或发光的基因进行荧光或发光检测 蛋白质至少两片; （2）构建融合蛋白，其由与待测试相互作用的蛋白质结构域连接的探针蛋白片段组成; （3）共表达融合蛋白; 和（d）检测荧光或发光信号的重构。
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9.发明授权Protein fragment complementation assays for the detection of biological or drug interactions 失效用于检测生物或药物相互作用的蛋白质片段互补测定法公开(公告)号：US06929916B2
公开(公告)日：2005-08-16
申请号：US10154758
申请日：2002-05-24
IPC分类号： A61K48/00 , C07K14/435 , C12N1/21 , C12N15/09 , C12N15/10 , C12Q1/02 , C12Q1/25 , C12Q1/68 , G01N33/15 , G01N33/50 , G01N33/542 , G01N33/573 , G01N33/58 , G01N33/68 , C12N15/63 , C12N15/11 , C07K14/00
CPC分类号： G01N33/6845 , A01K67/0271 , A01K2267/0331 , A01K2267/0393 , C07K14/43595 , C07K2319/00 , C12N15/1055 , G01N33/5008 , G01N33/542 , G01N33/573 , G01N33/58 , G01N33/581 , G01N33/68 , G01N33/6803 , Y02A90/26
摘要： The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule with the proviso that said protein is not ubiquitin.
摘要翻译： 本发明描述了一种用于检测生物分子相互作用的方法，所述方法包括：（a）选择选自蛋白质，荧光蛋白质，发光蛋白质和磷光蛋白质的合适的报道分子; （b）实现所述报道分子的片段化，使得所述片段化导致报告基因功能的可逆丧失; （c）将所述报告分子的片段与其它分子分开或附着; 随后（d）通过与所述片段融合的分子的相互作用使所述报道片段重新结合; 和（e）通过重建报告分子的活性来检测所述生物分子相互作用，条件是所述蛋白质不是泛素。
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公开(公告)号：US20110207162A1
公开(公告)日：2011-08-25
申请号：US12923256
申请日：2010-09-10
IPC分类号： C12Q1/02
CPC分类号： C40B30/04 , G01N33/5035 , G01N33/6845
摘要： The present invention provides functional annotation of novel genes by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. The instant invention also provides an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among network component proteins are detected and probed with stimulators and inhibitors of the network and subcellular location of the interacting proteins is determined. Additionally, applicants' use this strategy to map a signal transduction network that controls the G0 to G1 transition in eukaryotes. Analysis of 148 combinations of 65 protein pairs in mammalian cells allows applicants' to propose a model of network architecture. The results demonstrate the feasibility of employing this strategy in genome-wide functional annotation efforts.
摘要翻译： 本发明通过检测其编码的蛋白质与已知蛋白质的相互作用，然后进行测定来验证基因参与特定的细胞功能，来提供新型基因的功能注释。 本发明还提供了一种实验策略，其允许用单个报道系统检测蛋白质相互作用和功能测定。 检测网络组分蛋白之间的相互作用并用刺激物和网络的抑制剂进行探测，并测定相互作用的蛋白质的亚细胞定位。 此外，申请人使用这种策略来映射控制真核生物中G0到G1转换的信号转导网络。 分析哺乳动物细胞中65个蛋白质组的148个组合允许申请人提出一个网络架构模型。 结果表明在全基因组功能注释工作中采用这一策略的可行性。
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