BACKGROUND: Solubilization of inclusion bodies using high concentration of chaotropes followed by suboptimal refolding results in lower recovery of bioactive proteins from E. coli. Growing evidence indicates that aggregation of expressed proteins into inclusion bodies is molecular specific in nature and proteins in inclusion bodies have native-like secondary structure. Protecting these pre-existing native-like secondary structures of proteins using mild solubilization conditions is one of the key steps in improved recovery of bioactive protein from inclusion bodies of E. coli. RESULTS: Inclusion bodies of seven proteins (hGH, oGH, Pyk, PKS, aldolase, enolase and superoxide dismutase) were solubilized at pH 12 in the presence of 2mol L-1 urea and were subsequently refolded and purified. Enrichment of inclusion bodies after cell lysis, mild solubilization at alkaline pH and pulsatile refolding resulted in lower aggregation of protein during refolding and improved the final recovery of protein from inclusion bodies of E. coli. The efficiency of protein recovery from inclusion bodies was 30-40% for most of the proteins. This was almost two times higher than that achieved when inclusion bodies were solubilized in 8 mol L-1 urea and subsequently refolded. Purified hGH and Pyk were functionally active in respective in vitro assays. CONCLUSION: Non-denaturing solubilization of inclusion body proteins at alkaline pH is a viable alternative to the use of high molar concentration chaotropes for efficient recovery of inclusion body proteins. Mild solubilization and pulsatile refolding contribute synergistically to reduce the extent of aggregation during refolding and result in high throughput recovery of bioactive proteins from inclusion bodies of E. coli. (C) 2008 Society of Chemical Industry.