Evolution of a fluorinated green fluorescent protein.
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.
Yoo
Tae Hyeon
TH
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.
Link
A James
AJ
Tirrell
David A
DA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000378:metabolism; D017931:DNA Primers; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins
Angew Chem Int Ed Engl. 2001 Apr 17;40(8):1494-1496
11317312
Angew Chem Int Ed Engl. 2004 Dec 3;43(47):6426-63
15578784
Nat Biotechnol. 2006 Jan;24(1):79-88
16369541
Angew Chem Int Ed Engl. 2006 Jul 3;45(27):4518-21
16763955
Biochim Biophys Acta. 2006 Jul;1764(7):1147-58
16782415
Nature. 2001 Jan 11;409(6817):253-7
11196654
Biotechnol Bioeng. 2003 Sep 20;83(6):729-34
12889037
J Am Chem Soc. 2006 Jan 11;128(1):337-43
16390163
Curr Opin Biotechnol. 2003 Dec;14(6):603-9
14662389
Nat Biotechnol. 2004 Apr;22(4):445-9
14990965
Annu Rev Biochem. 1998;67:509-44
9759496
Biochemistry. 1997 Jun 3;36(22):6786-91
9184161
Proc Natl Acad Sci U S A. 2004 Oct 26;101(43):15324-9
15486092
J Am Chem Soc. 2003 Sep 17;125(37):11164-5
16220915
Nat Biotechnol. 1996 Mar;14(3):315-9
9630892
Biochemistry. 2004 Nov 9;43(44):14238-48
15518574
Nat Biotechnol. 1996 Oct;14(10):1246-51
9631087
Science. 2005 Oct 28;310(5748):638-42
16254179
Chembiochem. 2006 Aug;7(8):1251-7
16758500
Nature. 1995 Feb 23;373(6516):663-4
7854443
J Am Chem Soc. 2001 Nov 7;123(44):11089-90
11686725
Curr Opin Struct Biol. 1997 Dec;7(6):821-7
9434902
Gene. 1996;173(1 Spec No):33-8
8707053
J Biol Chem. 2000 Sep 22;275(38):29200-6
10844000
Angew Chem Int Ed Engl. 2004 Dec 17;44(1):34-66
15599909
Nat Biotechnol. 2002 Jan;20(1):87-90
11753368
Curr Opin Struct Biol. 2005 Feb;15(1):50-6
15718133
Chem Rev. 2002 Mar;102(3):759-81
11890756