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PEDS Advance Access originally published online on April 25, 2006
Protein Engineering Design and Selection 2006 19(7):309-316; doi:10.1093/protein/gzl014
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Thomas Gronemeyer, Christopher Chidley, Alexandre Juillerat, Christian Heinis and Kai Johnsson1

École Polytechnique Fédérale de Lausanne (EPFL), Institute of Chemical Sciences and Engineering CH-1015 Lausanne, Switzerland

To whom correspondence should be addressed. E-mail: kai.johnsson{at}epfl.ch

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-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.

Keywords: directed evolution/O6-alkylguanine-DNA alkyltransferases/phage display/protein labeling

Received December 13, 2005; revised February 13, 2006; accepted March 14, 2006.


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