In vitro stability of Endostar / 中国药科大学学报

ABSTRACT

Aim: To compare the stability of Endostar~(TM) and endostatin under different temperatures and pH using polyacrylamide gel electrophoresis( PAGE) and Western blot and to compare the activity of Endostar kept at 4 ℃ and 37 ℃ by inhibition of endothelial cell proliferation. Methods: Endostar and endostatin expressed by Phicha pastoris were kept at 4 ℃, and 37 ℃ for 96 hours, respectively. The electrophoresis of the samples was detected by reduced and non-reduced PAGE. The results were further confirmed by Western blot with rabbit anti-Endostar polyclonal antibody. The inhibitory effect of Endostar stored at different temperatures on HUVEC was also exam-ined by cell-based assay. Results: Single band at 20 kD was detected in all lanes of SDS-PAGE gel loaded with endostatin and Endostar samples under reducing condition. In acidic native PAGE with three different pH values, endostatin showed a smear characteristic, whereas Endostar showed a unique band in acidic non-continuous native PAGE. Although the smear phenomenon was also observed under two conditions of constant native elec-trophoresis, the major band of Endostar could be detected. Similar electrophoretic behavior was found for endosta-tin and Endostar stored at both 37 ℃ and 4 ℃ . Western blot showed similar results to those by PAGE. Furthermore, Endostar stored at these two temperatures also had identical inhibitory effect on proliferation of HUVEC. Conclusion: Endostar and endostatin exhibit similar thermostability at regular conditions, but Endostar was more stable than endostatin expressed in P. pastoris under acidic condition.