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PEDS Advance Access originally published online on May 31, 2005
Protein Engineering Design and Selection 2005 18(6):273-278; doi:10.1093/protein/gzi033
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org

Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

Daisuke Kajihara1,2, Takahiro Hohsaka2,3,4 and Masahiko Sisido1,4

1Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, 2School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 and 3PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

4 To whom correspondence should be addressed. E-mail: hohsaka{at}jaist.ac.jp; sisido{at}cc.okayama-u.ac.jp

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.

Keywords: cell-free translation/four-base codon/green fluorescent protein/non-natural amino acid/random mutation

Received February 17, 2005; revised April 28, 2005; accepted May 3, 2005.

Edited by Don Hilvert


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