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

enterokin

ase 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 50×10

3

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-β/Smads signal transduction pathway and further elucidation of the pathogenesis of keloid formation.