PURIFICATION OF RECOMBINANT GREEN FLUORESCENT PROTEIN FROM ESCHERICHIA COLI USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY.

□ In this study, an efficient purification system was developed using a packed-bed column for the purification of recombinant green fluorescent protein (GFP) from clarified feedstock. A series of hydrophobic ligands [phenyl (low sub), phenyl (high sub), butyl, and octyl] was screened at bench scale and the optimum concentration of salt in binding buffer was determined by precipitating the sample with (NH4)2SO4. The optimum salt concentration used in binding buffer is 30%(w/v) (NH4)2SO4in 20 mM sodium phosphate buffer at pH 8.0. Phenyl (high sub) ligand showed the best result of GFP purification in terms of yield and purity. Maximum recovery (82%) of recombinant GFP was achieved with phenyl (high sub) ligand. The optimum elution buffer for scaled-up process was determined by conducting a stepwise elution with 0–15%(w/v) (NH4)2SO4salt solution and the result showed that the GFP absorbed at the ligand was mostly eluted by using a buffer with the absence of (NH4)2SO4. The effect of GFP concentration in initial sample on the dynamic binding capacity was also studied by running a scaled-up hydrophobic interaction chromatography using HiPrep™ HIC Phenyl (high sub) 16/10 20mL packed-bed column connected to Äkta FPLC. [ABSTRACT FROM AUTHOR]