Rapid purification of EGFP, EYFP, and ECFP with high yield and purity.
Most current high throughput purification procedures for the green fluorescent protein (GFP) suffer from poor yields and low purity. An improved purification procedure that delivers highly pure protein (>95% homogeneity) in high yields (>70% of the initial fluorescent protein content) has been developed. The purification procedure requires only two steps: the cell lysate is heated to 60 degrees C for 4 min in ammonium sulfate and triethylamine, followed by hydrophobic interaction chromatography using isopropanol during the elution phase. The resulting pure product exhibits the same fluorescence profile as the crude sample. This procedure has been demonstrated on three commercial variants of GFP from Aequorea victoria, enhanced green, enhanced yellow, and enhanced cyan fluorescent protein (Becton-Dickinson). The yield and purity of material are superior to other recently described methods.
McRae
Shelley R
SR
Faculty of Science and the Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Qld 4111, Australia. s.mcrae@griffith.edu.au
Brown
Christopher L
CL
Bushell
Gillian R
GR
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification*; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; C485192:green fluorescent protein, Aequorea victoria; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins