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Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):13887-90. Epub 2007 Aug 23.

Evolution of a fluorinated green fluorescent protein.

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Division of Chemistry and Chemical Engineering, Joseph J. Jacobs Institute for Molecular Engineering for Medicine, California Institute of Technology, 1200 East California Boulevard, MC210-41, Pasadena, CA 91125-4100, USA.


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 approximately 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.

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