Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase.
Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase.
复制标题
DOI：
10.1016/j.nbt.2011.11.015
复制
发表时间：
2012-06-15
影响因子：
5.4
通讯作者：
Kay, Brian K.
中科院分区：
文献类型：
--
作者：
Huang, Renhua;Fang, Pete;Kay, Brian K.
Fyn是一种非受体蛋白酪氨酸激酶，属于高度保守的激酶家族Src家族激酶（SFKs）。Fyn在炎症过程和神经元功能中起重要作用。为了产生可用于探测Fyn的合成亲和试剂，用Fyn的SH 3结构域亲和选择纤连蛋白III型（FN 3）单体的噬菌体展示文库，并分离三种结合剂。三种结合剂之一G9特异性结合Fyn的SH 3结构域，但不结合Src家族的其他成员（即，Blk、Fgr、Hck、Lck、林恩、Src、是），尽管它们共享51-81%的氨基酸同一性。另外两个主要结合Fyn SH 3结构域，与Yes SH 3结构域有一定的交叉反应性。如通过等温滴定量热法测量的，G9结合剂具有166 ± 6 nM的解离常数，并且仅结合在阵列中检查的150个人SH 3结构域中的Fyn SH 3结构域。有趣的是，尽管G9单体在其随机化的BC和FG环中缺乏脯氨酸，但它在SH 3结构域上与富含脯氨酸的配体相同的位点结合，如竞争测定所揭示的。本研究中鉴定的G9单抗体可作为高选择性的探针用于检测和纯化细胞Fyn激酶。
Fyn is a non-receptor protein tyrosine kinase that belongs to a highly conserved kinase family, Src family kinases (SFKs). Fyn plays an important role in inflammatory processes and neuronal functions. To generate a synthetic affinity reagent that can be used to probe Fyn, a phage-display library of fibronectin type III (FN3) monobodies was affinity selected with the SH3 domain of Fyn and three binders were isolated. One of the three binders, G9, is specific in binding to the SH3 domain of Fyn, but not to the other members of the Src family (i.e., Blk, Fgr, Hck, Lck, Lyn, Src, Yes), even though they share 51-81% amino acid identity. The other two bind principally to the Fyn SH3 domain, with some cross-reactivity to the Yes SH3 domain. The G9 binder has a dissociation constant of 166 ± 6 nM, as measured by isothermal titration calorimetry, and binds only to the Fyn SH3 domain out of 150 human SH3 domains examined in an array. Interestingly, although the G9 monobody lacks proline in its randomized BC and FG loops, it binds at the same site on the SH3 domain as proline-rich ligands, as revealed by competition assays. The G9 monobody, identified in this study, may be used as a highly selective probe for detecting and purifying cellular Fyn kinase.
登录
查看更多内容
影响因子：
64.8
作者：
LIM, WA;RICHARDS, FM;FOX, RO
通讯作者：
FOX, RO
影响因子：
7.7
作者：
Kärkkäinen, S;Hiipakka, M;Saksela, K
通讯作者：
Saksela, K
影响因子：
5.6
作者：
Huang J;Makabe K;Biancalana M;Koide A;Koide S
通讯作者：
Koide S
影响因子：
7.3
作者：
Liu BA;Shah E;Jablonowski K;Stergachis A;Engelmann B;Nash PD
通讯作者：
Nash PD
影响因子：
64.5
作者：
MAIN, AL;HARVEY, TS;CAMPBELL, ID
通讯作者：
CAMPBELL, ID