Directed evolution of an extremely stable fluorescent protein
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA
1 To whom correspondence should be addressed. E-mail: amb{at}lanl.gov
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°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.
Keywords: directed evolution/fluorescent protein/thermostability
Received February 9, 2009; revised February 9, 2009; accepted February 16, 2009.