In vitro stability of Endostar

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 examined 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 electrophoresis，the major band of Endostar could be detected.Similar electrophoretic behavior was found for endostatin 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.