Evaluation of refolding conditions for a recombinant human interleukin-3 variant (daniplestim)

The refolding of daniplestim, a human interleukin-3 variant (SC-55494) from Escherichia coli inclusion bodies, was optimized using a reversed-phase HP LC method developed to permit quantification of the reduced and oxidized fo rms of daniplestim, The presence of cysteine or dithiothreitol accelerated refolding of daniplestim from E, coli inclusion body slurries dissolved in urea or guanidine solutions and was complete in 4-6 h, Regardless of the di ssolution and refolding protocol used to renature daniplestim, equivalently bioactive protein was produced. Under refolding conditions, no covalent mo dification of daniplestim by cysteine or cyanate was observed, The folding process was characterized further by following the unfolding of purified da niplestim by far-UV CD and fluorescence spectroscopies under both oxidizing and reducing conditions at pH values between 7 and 11. Formation of the si ngle disulphide bond had a large stabilizing effect on daniplestim structur e (approximate to 4-5 kCal at pH 9.5). This thermodynamic stabilization dro ve the refolding process towards the native form, even under conditions whe re the reduced protein was largely unfolded. From these data, scaleable ref olding conditions for daniplestim were established.