Abstract: Objective:This study aimed to express soluble human LysoPLD in Escherichia coli.Methods:Human LysoPLD gene sequence (GenBank: L46720.1) was retrieved from NCBI and synthesized chemically. The codon optimized sequence was cloned into pET-28a vector and transformed to the E.coli BL2(DE3) strain which was co-expressed with maltose binding protein fusion tag (MBP) or co-expressed with a pTF16 chaperone plasmid, then induced by isopropyl thiogalactoside (IPTG). The downstream purification process was established, including ion column elution, ammonium sulfate salting out, hydrophobic column purification, and amylose resin purification. The LysoPLD purity was determined by polyacrylamide gel electrophoresis (SDS-PAGE) and the enzymatic properties were determined by catalytic reaction of p-hydroxypalmitate.Results:The plasmids pET28a-MBP-LysoPLD and pET28a-pTf16-LysoPLD were constructed, then transformed into the E.coli BL21(DE3) strain respectively. BL21(DE3)-pET28a-MBP-LysoPLD was induced by 0.6 mmol/L IPTG, and incubated overnight to obtain MBP-LysoPLD protein; BL21(DE3)-pET28a-pTF16-LysoPLD was cultured in LB medium containing 0.5 µg/mL L-arabinose then induced by 0.1 mmol/L IPTG, the soluble LysoPLD was obtained. After downstream purification, the purity of the human LysoPLDs expressed through both methods were more than 80%. It was found that using p-hydroxypalmitate as substrate, the optimum reaction conditions including optimal temperature, pH and Ca²⁺ concentration for both LysoPLDs were basically the same.Conclusion:In this study, two different methods for improving soluble expression of human LysoPLD in E.coli were established and proved to be successful. The purified human LysoPLDs were enzymatically active, and the enzymatic activity were basically the same.