期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 99, 期 25, 页码 15926-15931出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.212627499
关键词
computational protein design; protein engineering; mutagenesis; directed evolution; beta-lactamase
We present a combined computational and experimental method for the rapid optimization of proteins. Using beta-lactamase as a test case, we redesigned the active site region using our Protein Design Automation technology as a computational screen to search the entire sequence space. Byeliminating sequences incompatible with the protein fold, Protein Design Automation rapidly reduced the number of sequences to a size amenable to experimental screening, resulting in a library of approximate to200,000 mutants. These were then constructed and experimentally screened to select for variants with improved resistance to the antibiotic cefotaxime. In a single round, we obtained variants exhibiting a 1,280-fold increase in resistance. To our knowledge, all of the mutations were novel, i.e., they have not been identified as beneficial by random mutagenesis or DNA shuffling or seen in any of the naturally occurring TEM beta-lactamases, the most prevalent type of Gram-negative beta-lactamases. This combined approach allows for the rapid improvement of any property that can be screened experimentally and provides a powerful broadly applicable tool for protein engineering.
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