CHROMATOGRAPHY FOR RAPID BUFFER EXCHANGE AND REFOLDING OF SECRETORY LEUKOCYTE PROTEASE INHIBITOR

A DEAE-cellulose stationary phase in a rolled configuration was used t o separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active fo rm. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl s uppressed binding of protein to this stationary phase. Separation of a n initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 degrees C, the fractionated r SLPI gave a 46% yield of properly refolded protein. The protein concen tration was 6.4 times higher than that reported in a previously publis hed method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The resu lts show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will signif icantly enhance process throughput by minimizing tankage, water requir ements, and process time.