Abstract 385: Optimization of refolding recombinant human endostatin from E. coli
Authors: Lixi Zhao, Yongping Jiang
Journal: Cancer Research (2010)

Abstract Background: human endostatin is a fragment from human collagen 18. The antitumor activity of endostatin is well-established. However, preclinical and clinical studies of human endostatin have been limited since the production of a large quantity of soluble human endostatin is difficultly obtained. The eukaryotic expression systems of recombinant endostatin are limited with high costs and low production yields. On the other hand, prokaryotic (E.coli) expression systems are hurdled with very low yield of refolded endostatin. Obviously, it is pre-required to optimize the conditions for enhancing the refolding of recombinant human endostatin (rh-endostatin) from E. coli before the application of endostatin to clinical studies. Objectives: to optimize the conditions for enhancing the refolding of rh-endostatin from E. coli and test the biological activities of refolded endostatin . Methods: The rh-endostatin inclusion body was obtained by fermenting E.coli (strain M15) containing human endostatin cDNA which was ligated to plasmid pQE3. The partial purified inclusion bodies of rh-endostatin were dissolved with 6 M guanidine-HCL following by combination of dilution and dialysis of the dissolved endostatin. The refolded endostatin was then purified by cation-exchange chromatography and concentrated by ultrafiltration. The biological activities of purified endostatin were assessed by using endostatin-specific monoclonal antibody and chick embryo chorioallantoic membrane assay. Results: fifteen grams per liter of re-endostatin inclusion body was achieved by fermentation of E.coli with recombinant endostatin fragment in 20L bio-reactor. And a 63% (w/w) refolding yield was achieved after optimizing the conditions of refolding method after a numbers of comparison studies. Three conditions were critical to obtain high yield of refolded rh-endostatin: the starting refolding protein concentration, urea concentration, and pH in dialysis buffers during the refolding process. Our studies indicated that the best optimizing conditions of refolded rh-endostatin were achieved with 0.3mg/ml rh-endostatin in the starting refolding solution, 3.5M urea concentration, and pH 8.0 in a series dialysis buffers. The high yield of refolded protein was observed by SDS-PAGE and protein concentration assay. The purified endostatin was reacted with endostatin-specific monoclonal antibody and significantly reduced the formation of blood vessels in re-endostatin group compared with saline group in chick embryo chorioallantoic membrane assay, indicating that refolded rh-endostatin significantly inhibited the process of angiogenesis. Conclusion: The method of highest refolding yield of rh-endostatin from E. coli so far was developed in this study. This optimized method will greatly increase the application of our human endostatin to preclinical and clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 385.