Expression of TAT-Smad7-HA fusion protein and validation of its transduction activity / 第三军医大学学报

ABSTRACT

Objective To construct,express,purify,identify and label the TAT-Smad7-HA fusion protein (protein transduction domain of trans-activator,human Smad7 and hyaluronic acid) and to validate its transduction activity in the cultured human primary keloid fibroblast cells. Methods TAT-PTD,Smad7 and HA fragments were sequentially inserted into pET32a(+) to construct the pTAT-Smad7-HA prokaryotic expression vector. After the expression of target fusion protein was induced to express by IPTG,affinity purification,Western blot analysis,enterokinase cleavage,target protein capture and FITC labeling were subsequently performed by turns to obtain the FITC-TAT-Smad7-HA fusion protein and to further observe its transduction activity in the human primary KFB cells in vitro. Results The prokaryotic expression vector for the TAT-Smad7-HA fusion protein,named as pTAT-Smad7-HA was successfully constructed,and the target fusion protein was efficiently induced to express,covering more than 25% of the total bacterial proteins,successfully purified with a purity of more than 95% purity,desalted by desalting column,identified by Western blotting,thioredoxin removed and FITC labeled. Finally,a fusion protein of FITC-TAT-Smad7-HA with the approximately molecular weight of 50?103 was successfully purified and its high transduction activity in KFB cells was validated. Conclusion The highly-purified FITC-TAT-Smad7-HA fusion protein and the validation of its high transduction activity in KFB cells have provided an experimental foundation for further studies on the role the human Smad7 protein playing in the TGF-?/Smads signal transduction pathway and further elucidation of the pathogenesis of keloid formation.