Poster Presentation 3-50

Permeation Associated to Three Phase Partitioning Method

on the Release of Green Fluorescent Protein (gfp_uv) from Escherichia coli

Eb Chiarini, Thereza Christina Vessoni Penna, and Irene A. Machoshvili

Department of Biochemical and Pharmaceutical Technology

School of Pharmaceutical Science

University of São Paulo.

Rua Antonio de Macedo Soares, 452, zip code: 04607-000

São Paulo/SP, Brazil

Telephone:  0055-11-38183694; Fax:  0055-11-38156386; E-mail:  tcvpenna@usp.br

Transformed cells (0.92 – 1.44 mg/mL) of Escherichia coli DH5-a expressing green fluorescent protein (gfpuv) were submitted to four cycles (1^o, 2^o, 3^o, 4^o) of freezing (-20 ºC/ 0.83 ºC/ min)/ thawing interlaid by sonication (3 pulses/6 s/ 25 vibrations).  The intracellular permeate with gfp_uv in buffer solution (Tris-HCl 25 mM pH 8.0 + b-mercaptoethanol (1 mM) + PMSF (0.1 mM)) was submitted to the three-phase partitioning (TPP) method.  The permeate content of gfp_uv (l_excitation 394 nm and l_emission 509 nm) ranged as follows:  315.03-832.56 mg/mL (1st cycle); 174.44-504.17 mg/mL (2nd cycle); 39.10-350.98 mg/mL (3rd cycle); 7.78-174.80 mg/mL (4th cycle).  The extracted contents of gfp_uv by TPP ranged as follows:  20.47-374.13 mg/mL (1st cycle); 93.40-442.29 mg/mL (2nd cycle); 31.90-359.22 mg/mL (3rd cycle); 17.66-115.30 mg/mL (4th cycle).  Cells (1.46 mg/mL) of E. coli in buffer solution were directly submitted to three successive extractions by TPP method. The extracted contents of gfp_uv obtained from the 1^st, 2^nd, and 3^rd interface extractions were, respectively, the following:  22.55-54.23 mg/mL; 33.32-91.17 mg/mL and 7.16-91.67 mg/mL; and 24.96-97.80 mg/mL in the mixture of those extracts.  The gfp_uv contents in the mixtures of the extracts after being eluted through methyl HIC column, were the following:  (i) 2.48-12.88 mg/mL for TPP method; and (ii) 12.69-62.92 mg/mL for permeation associated to TPP methods.  The selective permeation of gfp_uv followed by TPP extraction was equivalent to the amount of gfp_uv extracted from the cells directly by TPP; although the permeate extracted releases showed better elution through the HIC column.

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