Monday, May 5, 2008
9-20

Evaluation of Green Fluorescent Protein Purification Using Different Purification Methods

Marina Ishii, Luiz Carlos M. das Neves, Mariane F. Minaguti, Carla F. M. Souza, and Thereza C. V. Penna. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, B.16, 05508-900, São Paulo, Brazil, São Paulo, Brazil

Due to the ability to emit fluorescence in a wide range of environmental conditions without use of substrates, the Green Fluorescent Protein (GFP) is a potential tool for monitoring industrial processes comparing its fluorescence intensity to the process by a rapid and low cost method.  Nevertheless, the purification step is crucial for uses in applied biotechnology. This work aims to evaluate the purification of GFPUV and GFP+ obtained from distinct sources (E. coli DH5-alpha and Bacillus subtilis W1012) by two different methods (hydrophobic interaction and ion exchanger chromatography columns). GFPUV and GFP+ were extracted from  cells by a three-phase-partitioning method. Aliquots of 0.5 mL were loaded onto 1 mL N-Butil Column or a HiTrap ion exchanger chromatography columns (Q Sepharose XL, DEAE and ANX resins) (GE Healthcare Biosciences®, Uppsala, Sweden) pre-equilibrated with 10mM tris-EDTA buffer (pH 8.0). GFP elution range was tested with fractions of a salt gradient from 0.05 to 0.30 M NaCl in 10 mM phosphate buffer (pH 7.0). High concentrations of protein were eluted in fractions between 0.2 M and 0.3 M NaCl for all resins evaluated. Results demonstrated columns have capability to recovery GFPUV and GFP+ between 25% and 140% according the kind of column and source of GFP. Nevertheless, N-Butil and Q XL resin resulted in a high loading capacity and better purification, showing that it is well suited for GFPUV (139.4%) or GFP+ (90%) purification, providing a final product with high purity plus high fluorescence intensity.

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