%0 Journal Article %J Archives of Medical Science %@ 1734-1922 %V 8 %N 4 %D 2012 %F Shang2012 %T Basic researchExpression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation %X Introduction: Carbonic anhydrase III (CAIII) is remarkably abundant in slow skeletal muscles. It has multiple biological activities which could dissipate or resist some fatigue-related substances. In this study, we purified trans-activating transcriptional activator (TAT) fused CAIII protein and investigated its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation. Material and methods: The CAIII and TAT-CAIII genes were constructed, cloned into plasmid pET28a and expressed in Escherichia coli BL21 (DE3). The fusion proteins were purified with a nickel-nitrilotriacetic acid affinity chromatography column and then verified by Western blot and phosphatase activity staining subsequently. The C2C12 cells were treated respectively with serum-free medium containing 1 M TAT-CAIII or 1 M CAIII for 1 h and the intracellular distributions of fusion proteins were observed by indirect immunofluorescence. The effect of TAT-CAIII on C2C12 cell apoptosis induced by hypoxia/reoxygenation was detected by flow cytometry. Results : The CAIII and TAT-CAIII fusion proteins were expressed and purified successfully. After being cultured for 1 h, green fluorescence was visible in TAT-CAIII group cells under the fluorescence microscope, while no fluorescence was found in the CAIII group. Compared with the oxygen-glucose deprivation group, the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation in the TAT-CAIII group decreased significantly (p < 0.001). Conclusions : The purified TAT-CAIII could be transferred into cells efficiently and clearly decreased the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation, which indicated that it had antioxidative activity. This study lays an experimental basis for future research on the relationship between CAIII and muscle fatigue. %A Shang, Xiliang %A Bao, Yuanyuan %A Chen, Shiyi %A Ren, Huimin %A Huang, He %A Li, Yunxia %P 711-718 %9 journal article %R 10.5114/aoms.2012.30295 %U http://dx.doi.org/10.5114/aoms.2012.30295