Molecular characterisation of size exclusion chromatography refolded urokinase-plasminogen activator

We have used five physical and structural techniques to characterise the chromatographic refolding of the serine protease fragment of recombinant human urokinase plasminogen activator (u-PA) from inclusion bodies. Sephacryl S-300 was used to refold GdnHCl-solubilised and DTT-denatured u-PA from a starting concentration of 8 mg/ml. Dynamic light scattering results showed that protein aggregates were the first component to be eluted followed by unfolded u-PA and finally compact, folded u-PA. This was confirmed by analytical size exclusion chromatography, which also indicated that for each elution fraction, a population of u-PA species existed which varied in their hydrodynamic radius. At later elution volumes, u-PA was eluted as a monomeric, active molecule. The use of intrinsic fluorescence measurements across the elution fractions showed a correlation between stabilisation of the fluorescence signal and peak u-PA activity. Far UV circular dichroism identified the increasing formation of β-sheet structures in the refolding molecule. The use of tryptic digestion-RPLC showed identical digestion patterns between SEC and batch refolded u-PA. Finally, analysing the tryptic digestion profiles by mass spectrometry identified four peptide sequences on the surface of the u-PA molecule (IRSKEGR, VSHFLPWIR, GCALKDKPGVYTR and IIGGEFTTIENQPWF) that became more accessible to digestion as folding progressed.