Production of correctly folded and biologically active proteins in Escherichia coli can be a challenging process Frequently proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded Into the correct structure To address this a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests which confirmed the reliability of the predictions Finally the refolded protein was purified and its biological activity was tested in vitro The screen was applied for the refolding of Interleukin 17F (IL-17F) stromal-cell-derived factor-1 (SDF1 alpha/CXCL12) B cell-attracting chemokine 1 (BCA-1/CXCL13) granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a This procedure identified refolding conditions for all the tested proteins For the proteins where refolding conditions were already available the optimized conditions identified in the screening process increased the yields between 50% and 100% Thus the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein (C) 2010 Elsevier Inc All rights reserved