A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.
A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.
复制标题
DOI：
10.1038/srep08070
复制
发表时间：
2015-01-28
影响因子：
4.6
通讯作者：
Guan L
中科院分区：
文献类型：
--
作者：
Tikhonova EB;Ethayathulla AS;Su Y;Hariharan P;Xie S;Guan L
采用基于聚合酶链式反应的组合组装策略，设计并构建了具有高度多样性的锚蛋白七重复蛋白(ANK-N5C)DNA文库。采用细菌蜜二糖发酵试验进行体内功能筛选。我们分离到一种转录阻断剂，通过特异性地阻止α操纵子的激活，完全抑制大肠杆菌Melibiose-Galactosidase(MELA)和Melibiose Performance ase(Melb)的表达。高分辨晶体结构测定表明，所设计的ANK-N5C蛋白具有典型的锚蛋白折叠，其特异性转录阻断剂ANK-N5C-281形成了结构域互换的二聚体。功能测试表明，转录因子家族成员、DNA结合转录激活因子MERR的活性被ANK-N5C-281蛋白抑制。所有ANK-N5C蛋白都有一个表面电势为负的凹面结合区，这表明所设计的ANK-N5C文库蛋白可能有助于发现识别具有正表面电势的结构基序的结合基序，就像DNA结合蛋白一样。总体而言，我们的结果表明，所建立的文库是发现新型生物活性试剂的有用工具。
A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.
登录
查看更多内容
MelB 的基于结构的 Na(+)/蜜二糖同向机制。
影响因子：
16.6
作者：
Ethayathulla, Abdul S.;Yousef, Mohammad S.;Amin, Anowarul;Leblanc, Gerard;Kaback, H. Ronald;Guan, Lan
通讯作者：
Guan, Lan
影响因子：
5.6
作者：
Binz, HK;Stumpp, MT;Plückthun, A
通讯作者：
Plückthun, A
影响因子：
3.7
作者：
Epa VC;Dolezal O;Doughty L;Xiao X;Jost C;Plückthun A;Adams TE
通讯作者：
Adams TE
影响因子：
2.1
作者：
Elrobh, Mohamed S.;Webster, Christine L.;Busby, Stephen J. W.
通讯作者：
Busby, Stephen J. W.
影响因子：
3.6
作者：
Grainger, DC;Belyaeva, TA;Busby, SJW
通讯作者：
Busby, SJW