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.

Optimized green fluorescent protein variants provide improved single cell resolution of transgene expression in ascidian embryos

“…Many of the commercially available GFP protein variants have been codon optimized for improved expression in mammalian cells because the wildtype sequence is not efficiently translated (Yang et al, 1996). We compared the codons present in several humanized fluorescent proteins (HeGFP, HDsRed) with the sequences for wildtype GFP and wild-type DsRed (all abbreviations are listed in Table 1) with the predicted codon usage for the ascidian Ciona intestinalis, generated using the program CodonW (Peden, 1999) based on all of the predicted genes present in Release 1 of the C. intestinalis genome (Dehal et al, 2002).…”

“…Previous studies have demonstrated that the expression of fluorescent proteins (Yang et al, 1996), as well as many other kinds of proteins (Gustafsson et al, 2004) is significantly enhanced by optimizing the codons for specific cell types, such as human cells. Over the past few years, our own experiences in expressing fluorescent proteins in transgenic ascidian embryos have supported this hypothesis.…”

“…More recently, fluorescent proteins from other cnidarians, particularly corals and sea anemones, have been discovered that emit light in the orange to red range (reviewed by Matz et al, 2002;Shaner et al, 2004). Many fluorescent protein color variants, particularly those available commercially, have been optimized for maximal protein translation in mammalian cells by inserting nearly 200 silent mutations to reduce the use of rare codons, thus eliminating constraints on protein production (Yang et al, 1996). Whereas these proteins are translated efficiently in mammalian cells, the altered codon usage may not be ideal for maximal protein expression in nonmammalian cells.…”

Rapid diagnosis and quantification of herpes simplex virus with a green fluorescent protein reporter system

“…This derivative, thus, has a single, strong, red-shifted excitation peak at 488 nm, which is well suited for detection by fluorescence microscopy and flow cytometry. Moreover, the EGFP fluoresces 35-fold more intensively than what wt GFP does as measured by standard FITC filtering procedures (Cormack et al, 1996;Yang et al, 1996).…”

“…The enhanced GFP (EGFP), for example, contains double-amino-acid substitutions, i.e. Phe-64 to Leu and Ser-65 to Thr (Cormack et al, 1996;Yang et al, 1996). This derivative, thus, has a single, strong, red-shifted excitation peak at 488 nm, which is well suited for detection by fluorescence microscopy and flow cytometry.…”