公开(公告)号：US20050208577A1

公开(公告)日：2005-09-22

申请号：US11134253

申请日：2005-05-23

申请人： Stephen Michnick ,  Joelle Pelletier ,  Katja Arndt ,  Andreas Pluckthun

发明人： Stephen Michnick ,  Joelle Pelletier ,  Katja Arndt ,  Andreas Pluckthun

IPC分类号： C07K14/435 ,  C12N15/10 ,  C12Q1/32 ,  C12Q1/68 ,  C40B30/04 ,  G01N33/53 ,  G01N33/542 ,  G01N33/573 ,  G01N33/58 ,  G01N33/68

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。

公开(公告)号：US20070148682A1

公开(公告)日：2007-06-28

申请号：US11650466

申请日：2007-01-08

申请人： Stephen Michnick ,  Joelle Pelletier ,  Ingrid Remy

发明人： Stephen Michnick ,  Joelle Pelletier ,  Ingrid Remy

IPC分类号： C12Q1/68 ,  G06F19/00 ,  C12Q1/37

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。

公开(公告)号：US20090318377A1

公开(公告)日：2009-12-24

申请号：US12373476

申请日：2007-07-13

申请人： Louis-Philippe Vezina ,  Nathalie Landry ,  Joelle Pelletier ,  Sylvain Savard

发明人： Louis-Philippe Vezina ,  Nathalie Landry ,  Joelle Pelletier ,  Sylvain Savard

IPC分类号： A61K31/704 ,  A61P3/06

CPC分类号： A61K31/575 ,  A23L33/105 ,  A61K31/704

摘要： A cholesterol-lowering preparation comprising medicagenic acid saponin is disclosed. The amount of medicagenic acid saponin in the preparation is greater than 50% by weight to produce the cholesterol-lowering effect in an animal. A method of purifying a preparation of at least 30% medicagenic acid saponin is also disclosed. The preparation may be purified from alfalfa plants and used as a treatment for lowering cholesterol and triglycerides in an animal, in particular a human.

摘要翻译： 公开了包含药用皂苷的降胆固醇制剂。 制剂中的药用植物皂苷的量大于50重量％，以在动物中产生降低胆固醇的作用。 还公开了一种纯化至少30％的药用植物皂苷的制剂的方法。 该制剂可以从苜蓿植物中纯化并用作降低动物，特别是人类中的胆固醇和甘油三酸酯的治疗剂。