SummaryA new dual-gradient ion exchange chromatographic method was developed to improve the refolding yield of human lysozyme produced in Escherichia coil as an inclusion body. The dissolved and stretched polypeptide chain in a concentrated non-ionic denaturant was adsorbed onto an ion exchange column and induced to refold by gradually decreasing the denatu rant concentration and increasing pH in the flowing buffer. The dual gradients of denaturant concentration and pH provided a gradual change of the solution environment along the chromatographic column for the protein to refold, resulting in enhanced activityyield and purity.A post-separation was also studied using size-exclusion chromatography to remove protein aggregates and mis-folded proteins after the refolding step.

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