IMPROVED PROTEIN REFOLDING USING HOLLOW-FIBER MEMBRANE DIALYSIS

We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration me mbrane to refold bovine carbonic anhydrase, loaded into the lumen spac e, by removing the denaturant through controlled dialysis via the shel l side space. When challenged with GdnHCl-denatured carbonic anhydrase , 70% of the loaded protein reptated through the membrane into the cir culating dialysis buffer. Reptation occurred because the protein, in i ts fully unfolded configuration, was able to pass through the pores. T he loss of carbonic anhydrase through the membrane was controlled by t he dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min re duced the denaturant around the protein to a concentration that allowe d the return of secondary structure, increasing the hydrodynamic radiu s, thus preventing protein transmission. Under these conditions a maxi mum of 42% of carbonic anhydrase was recovered (from a starting concen tration of 5 mg/mL) with 94% activity. This is an improvement over ref olding carbonic anhydrase by simple batch dilution, which gave a maxim um reactivation of 85% with 35% soluble protein yield. The batch refol ding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25 degrees C the temperature sensiti vity was considerably reduced. In order to reduce the convection force s that give rise to aggregation and promote refolding the dialyzate wa s slowly heated from 4 to 25 degrees C. This slow, temperature-control led refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membran e, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield. (C) 1998 John Wiley & Sons, Inc.