• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Selection for improved protein stability by phage display.
【24h】

Selection for improved protein stability by phage display.

机译：通过噬菌体展示来改善蛋白质稳定性的选择。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display. Copyright 1999 Academic Press.
机译：通过定向诱变和随机诱变相结合，使用在确定的位置随机分配的寡核苷酸和两轮DNA改组，生成了单链Fv片段（scFv）突变体库。该文库基于已经折叠良好且稳定的scFv片段4D5Flu。为了进一步改善该框架并测试各种选择策略的效率，在不同的选择压力下进行了噬菌体展示选择，以提高热力学稳定性。将展示噬菌体在高温下的孵育与将噬菌体暴露于高浓度的氯化胍相比。由于展开过程的不可逆性，在经过两轮诱变和选择后，温度胁迫指导的选择产生了最稳定的scFv突变体。它仅具有两个突变（His（L27d）Asn和Phe（L55）Val），显示出热力学稳定性提高了约4 kcal / mol，在大肠杆菌中的表达产量提高了三倍，结合常数比在大肠杆菌中提高了20倍。 4D5Flu野生型。本研究获得的选择结果描述了噬菌体展示中不同稳定性应激选择方法的优缺点。版权所有1999，学术出版社。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号