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Published August 28, 2007 | Published + Supplemental Material
Journal Article Open

Evolution of a fluorinated green fluorescent protein

Abstract

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved {approx}650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.

Additional Information

© 2007 by the National Academy of Sciences. Edited by David H. Baker, University of Illinois at Urbana–Champaign, Urbana, IL, and approved July 17, 2007 (received for review March 5, 2007). Published online on August 23, 2007, 10.1073/pnas.0701904104 We thank Jin K. Montclare and Frances H. Arnold for helpful discussion. We also thank Mona Shahgholi for assistance with MALDI-MS. This work was supported by National Institutes of Health Grant GM62523 and Office of Naval Research Grant N00014-03-1-0793 (to D.A.T) and by a Samsung Scholarship (to T.H.Y.). Author contributions: T.H.Y. and D.A.T. designed research; T.H.Y. performed research; T.H.Y. and D.A.T. analyzed data; and T.H.Y., A.J.L., and D.A.T. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/cgi/content/full/0701904104/DC1.

Attached Files

Published - YOOpnas07.pdf

Supplemental Material - YOOpnas07suppfig10.pdf

Supplemental Material - YOOpnas07suppfig11.pdf

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Supplemental Material - YOOpnas07suppfig9.pdf

Supplemental Material - YOOpnas07supptext.pdf

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Additional details

Created:
August 22, 2023
Modified:
October 16, 2023