An optimization of the refolding of endostatin (ES), by a study of the conditions that can affect (i) dissociation of inclusion bodies (IBs) and (ii) renaturation under high hydrostatic pressure (HHP), is described. IBs produced by bacteria cultivated at 25 degrees C were shown to be more soluble than those produced at 37 degrees C and their dissociation by application of 2.4 kbar at 20 degrees C was shown to be further enhanced at -9 degrees C. A red shift in intrinsic fluorescence spectra and an increase in binding of the hydrophobic fluorescent probe bis-ANS show subtle changes in conformation of ES in the presence of 1.5 M GdnHCl at 2.4 kbar, while at 0.4 kbar the native conformational state is favored. The 25% refolding yield obtained via compression of IBs produced at 37 degrees C by application of 2.4 kbar, was increased to 78% when conditions based on the insights acquired were utilized: dissociation at 2.4 kbar and -9 degrees C of the IBs produced at 25 degrees C, followed by refolding at 0.4 kbar and 20 degrees C. Besides providing insights into the conformational transitions of ES structure under HHP, this work proposes innovative conditions that are likely to have wide applicability to the HHP-induced refolding of proteins in general. (C) 2013 Elsevier Ltd. All rights reserved.