Urea gradient size-exclusion chromatography enhanced the yield of lysozymerefolding

Protein refolding is still a bottleneck for large-scale production of valua ble proteins expressed as inclusion bodies in Escherichia coli. Usually bio logically active proteins cannot be obtained with high yield at a high conc entration after refolding. In order to meet the challenge of protein refold ing a urea gradient gel filtration-refolding system was developed in this a rticle. A Superdex 75 column was pre-equilibrated with a linear decreased u rea gradient, the denatured protein experienced the gradual decrease in ure a concentration as it went through the column. The refolding of denatured l ysozyme showed this method could significantly increase the activity recove ry of denatured lysozyme at high protein concentration. The activity recove ry of 90% was obtained from the initial protein concentration up to 17 mg/m l within 40 min. (C) 2001 Elsevier Science B.V. All rights reserved.