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PEDS Advance Access published online on February 2, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzl056
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

The creation of a novel fluorescent protein by guided consensus engineering

Mingha Dai*, Hugh E. Fisher*, Jamshid Temirov*, Csaba Kiss, Mary E. Phipps, Peter Pavlik, James H. Werner and Andrew R.M. Bradbury1

Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA

1 To whom correspondence should be addressed. E-mail: amb{at}lanl.gov

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with > 62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.

Keywords: fluorescent proteins/consensus engineering/Azami green/protein stability

Received November 29, 2006; accepted December 6, 2006.


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