Maturation time of enhanced green fluorescent protein (EGFP)

Range ~60 Table - link Min
Organism Bacteria Escherichia coli
Reference Iizuka R, Yamagishi-Shirasaki M, Funatsu T. Kinetic study of de novo chromophore maturation of fluorescent proteins. Anal Biochem. 2011 Jul 15 414(2):173-8. p.176 table 1PubMed ID21459075
Primary Source J.A. Sniegowski, J.W. Lappe, H.N. Patel, H.A. Huffman, R.M. Wachter, Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein, J. Biol. Chem. 280 (2005) 26248–26255.PubMed ID15888441
Method (Footnote 'e' beneath table:) "Rate of GFP prepared from inclusion bodies at 30°C. Values were taken from [primary source]." (Primary source p.26248 right column 2nd paragraph:)"The kinetics of de novo chromophore formation has been determined by monitoring the rate of fluorescence acquisition. The GFP-S65T maturation process, inclusive of protein folding and the ensuing chemical steps, has been shown to proceed in vitro with a time constant of 122 min [ref 15]. In this experiment, maturation was induced by rapid dilution of urea-solubilized inclusion bodies at room temperature. De novo protein folding was monitored independently by a trypsin resistance assay and was estimated to proceed with a time constant of 14 min (t1/2~10 min)."
Comments "During earlier years, the maturation rate of GFP was determined as the rate of fluorescence development after admission of air to anaerobically expressed GFP [ref 5]. Currently, conventional methods to study the maturation kinetics are based on triggering protein folding of urea-solubilized inclusion bodies [ref 9] and reoxidation of the chromophore after chemical reduction of the mature chromophore [refs 9–13]. However, inconsistent data have been reported (Table 1)."
Entered by Uri M
ID 107001