关键词: P21Cip1/WAF1, Flag标签, 蛋白酶体, 降解

Abstract: BACKGROUND ＆ AIM: N-terminal ubiquitination may play a role in p21Cip1/WAF1 degradation via proteasome-dependent proteolytic pathway. We studied the influence of adding a N-terminal flag tag on the stability of p21 protein. MATERIAL AND METHODS: NIH3T3 cell stably expressing flag-p21 was set up by retrovirus pWB3flag-p21 infection followed by Blasticidin selection. Half-life of p21 protein was tested by western blot with rabbit polyclonal anti-p21 antibody, and proteasome inhibitor MG-132 was employed to test the role of proteasome pathway on the degradation of p21 protein. RESULTS: Half-life of endogenous p21 in NIH3T3 cells was about 30 minutes, while the half-life of exogenous flag-p21 fusion protein expressed in the same tested cells was obviously increased. Although MG-132 significantly increased the amount of endogenous p21 protein, there was no detectable change of flag-p21 expression under the same condition. CONCLUSION: N-terminal ubiquitination gvas necessary for effective p21 proteasome-dependent degradation. That adding flag tag at the amino acid termini may stabilize p21 protein should be considered.

Key words: P21Cip1/WAF1, flag tag, proteasome, degradation