Current research on the biological functions of Arabidopsis AtESD4 primarily relies on esd4 mutants and in vivo experiments, while its recombinant protein expression and purification system remains unexplored. This study established a prokaryotic expression and purification system for AtESD4, identifying the optimal expression vector and induction time. The open reading frame(ORF)of AtESD4 was amplified from an Arabidopsis cDNA library by PCR and cloned into prokaryotic expression vectors(pRSF-duet, pMAL-C2X, and pGEX-4T-1)using a one-step cloning method. Recombinant proteins were expressed in Escherichia coli, purified by affinity chromatography, and analyzed via SDS-PAGE to evaluate the effects of different vectors and induction times (8 h, 16 h, and 24 h)on expression efficiency and purity. Results demonstrated that AtESD4 recombinant proteins predominantly formed inclusion bodies, with significantly higher abundance in bacterial precipitates than in supernatants. Among the three vectors, pMAL-C2X yielded AtESD4 fusion proteins with optimal quality and purity under 24 h induction, whereas pRSF-duet and pGEX-4T-1 showed inferior performance.