Refolding of low molecular weight urokinase plasminogen activator by dilution and size exclusion chromatography - A comparative study

Overexpression of the serine protease domain of urokinase plasminogen activator (u-PA) in Escherichia coli (BL-21) results in the production of inclusion bodies. Batch dilution refolding of the u-PA fragment was investigated. The effect of denaturant concentration, redox potential, pH, and temperature on the recovery of u-PA activity was determined. It was found that u-PA is very susceptible to aggregation and therefore required a high concentration of urea (3 M) in the refolding buffer. It has recently been established that size exclusion chromatography can perform the buffer exchange to initiate protein refolding while minimizing aggregation. Using the best refolding buffer (3 M urea, 50 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.5 mM reduced glutathione, 0.5 mM oxidized glutathione) determined from batch dilution refolding, the effects of sample concentration, sample volume, and flow rate were investigated. It was found that size exclusion refolding is particularly sensitive to volume of denatured sample applied to the column. Finally, a comparison between batch dilution and size exclusion refolding established that size exclusion refolding resulted in a higher recovery of u-PA activity, below batch dilution factors of 40.