Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfp(uv)) from Escherichia coli

BACKGROUND: Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfp(uv)) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfp(uv) from the over-expressing cells. MATERIAL AND METHODS: Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h(-1) - 1.10 h(-1)) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfp(uv), absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfp(uv) released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfp(uv) in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). RESULTS: The sonication maximum released amount obtained from the cells was 327.67 μg gfp(uv)/mL (20.73 μg gfp(uv)/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfp(uv)/mL (29.74 μg gfp(uv)/mg BSA) was obtained. The specific mass range of gfp(uv) released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. CONCLUSIONS: The selective permeation of gfp(uv) by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfp(uv) extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.