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 °C were shown to be more soluble than those produced at 37 °C and their dissociation by application of 2.4 kbar 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 1.5 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 °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 °C of the IBs produced at 25 °C, followed by refolding at 0.4 kbar 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. © 2013 Elsevier Ltd. All rights reserved.
Chura-Chambi, R. M., Cordeiro, Y., Malavasi, N. V., Lemke, L. S., Rodrigues, D., & Morganti, L. (2013). An analysis of the factors that affect the dissociation of inclusion bodies and the refolding of endostatin under high pressure. Process Biochemistry, 48(2), 250–259. https://doi.org/10.1016/j.procbio.2012.12.017