Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.
Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.
复制标题
Shark Attack：通过逐步体外亲和力成熟衍生自鲨鱼 vNAR 结构域的高亲和力结合蛋白
DOI：
10.1016/j.jbiotec.2014.04.023
复制
发表时间：
2014
影响因子：
4.1
通讯作者：
Kolmar H
中科院分区：
文献类型：
--
作者：
Zielonka S;Weber N;Becker S;Doerner A;Christmann A;Christmann C;Fritz J;Schäfer E;Steinmann B;Empting M;Ockelmann P;Lierz M;Kolmar H
描述了一种用于抗原特异性鲨鱼vNAR结构域的逐步体外亲和力成熟的新方法，其完全依赖于来自非免疫鲨鱼的半合成库。以酵母表面展示技术为平台，从CDR3随机化的竹鲨vNAR文库中筛选出目标特异性分子。各种抗原结合vNAR结构域通过针对几种治疗相关抗原进行筛选而容易地分离，所述抗原包括上皮细胞粘附分子（EpCAM）、肝配蛋白A型受体2（EphA2）和人丝氨酸蛋白酶HTRA1。通过使靶富集的群体的CDR 1多样化来证明EpCAM和HTRA 1的亲和力成熟，这允许快速选择纳摩尔结合剂。EpCAM特异性vNAR分子以可溶性蛋白的形式产生，并通过热位移测定和生物层干涉法进行更广泛的表征。基本上，我们证明了高亲和力的结合剂可以在体外产生，而不会在很大程度上损害vNAR支架所需的高热稳定性。
A novel method for stepwisein vitroaffinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterizedviathermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generatedin vitrowithout largely compromising the desirable high thermostability of the vNAR scaffold.
登录
查看更多内容
影响因子：
3
作者：
Duan, Y;Wu, C;Kollman, P
通讯作者：
Kollman, P
DOI：
10.1007/978-1-61779-968-6_23
发表时间：
2012-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Dudgeon, Kip;Rouet, Romain;Christ, Daniel
通讯作者：
Christ, Daniel
影响因子：
2.9
作者：
Ewert, S;Honegger, A;Plückthun, A
通讯作者：
Plückthun, A
影响因子：
3.7
作者：
Glotzbach B;Reinwarth M;Weber N;Fabritz S;Tomaszowski M;Fittler H;Christmann A;Avrutina O;Kolmar H
通讯作者：
Kolmar H
影响因子：
5.6
作者：
Walker LM;Bowley DR;Burton DR
通讯作者：
Burton DR