公开(公告)号：US07625700B2

公开(公告)日：2009-12-01

申请号：US11134253

申请日：2005-05-23

申请人： Stephen William Watson Michnick ,  Joelle N Pelletier ,  Katja M. Arndt ,  Andreas Pluckthun

发明人： Stephen William Watson Michnick ,  Joelle N Pelletier ,  Katja M. Arndt ,  Andreas Pluckthun

IPC分类号： C12Q1/68 ,  C12Q1/70 ,  C12P21/04 ,  C12N15/00 ,  C07H21/02 ,  C07H21/04

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。