(93ao) Colorful Protein Separation Using Anion Exchange Chromatography

The purification of biological compounds is critical to the success of the biotechnology and pharmaceutical industries. This study specifically focuses on the separation of proteins using DEAE Sepharose fast flow anion exchange resin. Enhanced Green Fluorescent Protein (EGFP, pI = 5.6), Flavodoxin from C. anabaena (pI = 4.2), and DsRed2 (pI = 6.25) were produced recombinantly in E. coli and purified using standard techniques. Two combinations of these proteins were mixed to serve as models for a moderate and challenging separation. Process parameters including buffer pH, salt concentration, and salt gradient were varied to optimize the separation. By operating at a pH of 7.5 and using a salt gradient from 45 to 55 mM, a resolution of 0.487 was achieved for the separation of DsRed2 and EGFP. Complete separation of EGFP and Flavodoxin was achieved using a salt gradient at pH 6.6, with EGFP eluting at 0.21 M NaCl and Flavodoxin eluting at 0.06 M NaCl. The effect of operating conditions on the quality of separation will be discussed as a model for difficult separations in the biotechnology industry. Application of this research as a teaching tool will also be discussed