Selective permeation and organic extraction of recombinant green fluorescent protein (gfpuv) from Escherichia coli.
BACKGROUND
Transformed cells of Escherichia coli DH5-alpha with pGFPuv, induced by IPTG (isopropyl-beta-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) 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 gfpuv from the over-expressing cells.


METHODS
Cultures (37 degrees C/100 rpm/ 24 h; mu = 0.99 h(-1)-1.10 h(-1)) of transformed (pGFP) Escherichia coli DH5-alpha, expressing the green fluorescent protein (gfpuv, 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 gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75 degrees C) cells were subjected to three freeze/thaw (-20 degrees C/ 0.83 degrees C/min) cycles interlaid by sonication (3 pulses/6 seconds/25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM beta-mercaptoethanol beta-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 microg gfpuv/mL (20.73 microg gfpuv/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 microg gfpuv/mL (29.74 microg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 microg/mg and 135.10 microg/mg.


CONCLUSIONS
The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.
Vessoni Penna
Thereza Christina
TC
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Brazil. tcvpenna@uspb.br
Ishii
Marina
M
D000645:Ammonium Sulfate; D005591:Chemical Fractionation / Q000379:methods*; D002625:Chemistry, Organic; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D005615:Freezing; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D055601:Organic Chemistry Phenomena; D010539:Permeability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D013010:Sonication; D020002:tert-Butyl Alcohol
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D020002:tert-Butyl Alcohol; D000645:Ammonium Sulfate