title: Selective permeation and organic extraction of recombinant green fluorescent protein (gfpuv) from Escherichia coli.
abstract: 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.
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Brazil. tcvpenna@uspb.br
authors.forename: Thereza Christina
authors.initials: TC
authors.lastname: Vessoni Penna
authors.forename: Marina
authors.initials: M
authors.lastname: Ishii
mesh_terms: 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
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D020002:tert-Butyl Alcohol; D000645:Ammonium Sulfate