Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein

Abstract

The green fluorescent protein (GFP) from Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of cells and organisms. Despite many early successes using this reporter, wild type GFP is suboptimal for most applications due to low fluorescence intensity when excited by blue light (488 nm), a significant lag in the development of fluorescence after protein synthesis, complex photoisomerization of the GFP chromophore and poor expression in many higher eukaryotes. To improve upon these qualities, we have combined a mutant of GFP with a significantly larger extinction coefficient for excitation at 488 nm with a re-engineered GFP gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yield an enhanced GFP which provides greater sensitivity in most systems.