Basic research
Expression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation
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Submission date: 2011-07-31
Final revision date: 2011-12-28
Acceptance date: 2012-01-24
Online publication date: 2012-09-08
Publication date: 2012-08-31
Arch Med Sci 2012;8(4):711–718
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.