Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

被引：115

作者：

Gronemeyer, Thomas ^{[1
]}

Chidley, Christopher ^{[1
]}

Juillerat, Alexandre ^{[1
]}

Heinis, Christian ^{[1
]}

Johnsson, Kai ^{[1
]}

机构：

[1] Ecole Polytech Fed Lausanne, Inst Chem Sci & Engn, CH-1015 Lausanne, Switzerland

来源：

PROTEIN ENGINEERING DESIGN & SELECTION | 2006年 / 19卷 / 07期

关键词：

directed evolution; O-6-alkylguanine-DNA alkyltransferases; phage display; protein labeling;

D O I：

10.1093/protein/gzl014

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The specific reaction of O-6-alkylguanine-DNA alkyltransferase (AGT) with O-6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives.

引用

页码：309 / 316

页数：8

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