An analysis of the factors that affect the dissociation of inclusion bodies and the refolding of endostatin under high pressure

AbstractAn 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°C were shown to be more soluble than those produced at 37°C and their dissociation by application of 2.4kbar at 20°C was shown to be further enhanced at −9°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 1.5M GdnHCl at 2.4kbar, while at 0.4kbar the native conformational state is favored. The 25% refolding yield obtained via compression of IBs produced at 37°C by application of 2.4kbar, was increased to 78% when conditions based on the insights acquired were utilized: dissociation at 2.4kbar and −9°C of the IBs produced at 25°C, followed by refolding at 0.4kbar and 20°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