期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 106, 期 27, 页码 10999-11004出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0902964106
关键词
directed evolution; protein ligation; Crk-II; SCA; coevolution
资金
- National Institutes of Health [GM086868, GM55843]
Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cell-based selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据