Monday, April 30, 2007

Optimization of green fluorescent protein purification by three anion exchange chromatography matrices

Marina Ishii1, Mariane Freitas Minaguti1, Thereza Christina Vessoni Penna1, and Olívia Cholewa2. (1) Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, Bl. 16, São Paulo, 05508-900, Brazil, (2) Molecular Probes, Inc.

Green fluorescent protein (GFP) is widely known as a molecule capable of monitoring industrial processes due to its ability to emit fluorescence under a broad temperature and pH range.  Improving GFP purification by a rapid and low cost method that delivers high yields (>70% of the initial fluorescent intensity) of pure protein (> 95%) is necessary to meet the demand for use in applied biotechnology.  This work aims to compare three different pre-packed anion exchange chromatography resins to optimize GFP purification and determine the most appropriate matrix.  GFP (pI = 4.7 - 5.1) was extracted from E. coli DH5-alpha cells by a three-phase-partitioning method and 0.5 mL aliquots were loaded onto 1 mL HiTrap ion exchanger chromatography columns (strong ion exchange resin Q Sepharose XL, weak ion exchange resins, DEAE and ANX, 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 GFP were eluted in fractions between 0.2 M and 0.3 M NaCl for all resins evaluated. GFP recovery was 26.5%, 62.4% and 139.4% for ANX, DEAE and Q XL matrices, respectively. The Q XL resin resulted in a high loading capacity and better purification, showing that it is well suited for GFP purification, providing a final product with high purity plus high fluorescence intensity.