Protease-resistant peptide ligands from a knottin scaffold library.
Protease-resistant peptide ligands from a knottin scaffold library.
复制标题
DOI：
10.1021/cb200039s
复制
发表时间：
2011-08-19
影响因子：
4
通讯作者：
Daugherty, Patrick S.
中科院分区：
文献类型：
--
作者：
Getz, Jennifer A.;Rice, Jeffrey J.;Daugherty, Patrick S.
结蛋白家族中的肽已被证明具有固有的稳定性，使其成为开发治疗和诊断剂的有吸引力的支架。鉴于其对蛋白酶的显著稳定性，环肽kalata B1被用作支架，以创建在E.杆菌通过在kalata B1支架的环内随机化7个氨基酸构建超过109个变体的文库，并使用荧光激活细胞分选进行筛选以鉴定对人凝血酶的活性位点特异的肽配体。重折叠的凝血酶结合剂在溶液中表现出高纳摩尔亲和力，解离速率慢，并且能够抑制凝血酶的酶活性。重要的是，在与胰蛋白酶和胰凝乳蛋白酶孵育两小时后，80%的基于结蛋白的凝血酶抑制剂保持完整，表明修饰kalata B1序列不会损害其稳定性。此外，结蛋白变体介导的凝血酶的亲和力增强了20倍，当与由单个二硫键约束的相同的7个残基结合表位相比时。我们的研究结果表明，从kalata B1支架衍生的肽库可以产生高亲和力的蛋白配体，保留显着的蛋白酶抗性与亲本支架。更一般地说，这种策略可以证明适用于体内应用的稳定的肽配体的发展是有用的。
Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 109 variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin’s enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications.
登录
查看更多内容
影响因子：
4.6
作者：
Lehmann, Andreas
通讯作者：
Lehmann, Andreas
影响因子：
6.8
作者：
Gai, S. Annie;Wittrup, K. Dane
通讯作者：
Wittrup, K. Dane
DOI：
10.1093/protein/12.9.797
发表时间：
1999-09-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Christmann, A;Walter, K;Kolmar, H
通讯作者：
Kolmar, H
影响因子：
7.3
作者：
Gunasekera, Sunithi;Foley, Fiona M.;Daly, Norelle L.
通讯作者：
Daly, Norelle L.
DOI：
10.1073/pnas.0504613102
发表时间：
2005-09-27
影响因子：
11.1
作者：
Clark, RJ;Fischer, H;Craik, DJ
通讯作者：
Craik, DJ