Directed evolution of an extremely stable fluorescent protein.
In this paper we describe the evolution of eCGP123, an extremely stable green fluorescent protein based on a previously described fluorescent protein created by consensus engineering (CGP: consensus green protein). eCGP123 could not be denatured by a standard thermal melt, preserved almost full fluorescence after overnight incubation at 80 degrees C and possessed a free energy of denaturation of 12.4 kcal/mol. It was created from CGP by a recursive process involving the sequential introduction of three destabilizing heterologous inserts, evolution to overcome the destabilization and finally 'removal' of the destabilizing insert by gene synthesis. We believe that this approach may be generally applicable to the stabilization of other proteins.
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA.
Csaba
C
Kiss
Jamshid
J
Temirov
Leslie
L
Chasteen
Geoffrey S
GS
Waldo
Andrew R M
AR
Bradbury
D000595:Amino Acid Sequence; D001483:Base Sequence; D019143:Evolution, Molecular; D049452:Green Fluorescent Proteins / Q000138:chemical synthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017422:Sequence Analysis, DNA; D013696:Temperature
D049452:Green Fluorescent Proteins