期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 335, 期 4, 页码 1007-1018出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2003.11.014
关键词
aldolase antibody; catalytic antibody; enamine; phage display; in vitro evolution
资金
- NCI NIH HHS [CA27489] Funding Source: Medline
Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The k(cat) values of the antibody-catalyzed retro-aldol reactions were correlated with the K-d values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower K-d value displayed a higher k(cat) value in the retroaldol reaction, and a linear relationship was observed in the plots of log k(cat) versus log K-d. These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities. (C) 2003 Elsevier Ltd. All rights reserved.
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