Protein refolding is a bottleneck in the production of therapeutic proteins from inclusion bodies In recent years several studies have described on-column refolding of recombinant proteins DT389-hIL13 is a recombinant protein that targets the glioma In our study the recombinant protein DT389-hIL13 was expressed in Escherichia coli (E colt) The Isolated inclusion bodies were refolded using size exclusion chromatography (SEC) and further purified using anion exchange chromatography Three different methods of SEC on-column refolding were studied In vitro tests on U251 cells showed that the recombinant protein could effectively inhibit the proliferation of 13251 cells especially the protein refolded by urea and pH gradient method The half-maximal inhibitory concentration (IC50) of 0 887 nM was achieved with this new method unlike an IC50 of 11 4 nM achieved in the non-gradient method (C) 2010 Elsevier Inc All rights reserved