Heterologous expression of the <i>Stellaria media</i> plant defensin SmD1 in <i>Escherichia coli</i>

SmD1 isolated from seeds of common chickweed <i>Stellaria media</i> has strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC₅₀ ≤ 1 μM). However, the low production of plant defensins in natural strains limits their large-scale actual production. In this study, defensin gene <i>SmD1</i> was successfully heterologously expressed in <i>Escherichia coli</i> BL21 (DE3) for efficient production of plant defensins. The defensin gene <i>SmD1</i> fused with thioredoxin was cloned into pET22b (+) vector. Then, it was transformed into <i>E. coli</i> BL21 (DE3) and expressed solubly after induction of isopropyl-β-D-thiogalactopyranoside (IPTG). At 50 °C, active SmD1 was released by 50% (v/v) formic acid hydrolysis of the cleavage of Asp-Pro bond between fused proteins. The recombinant protein SmD1 was purified by Ni-IDA column and showed significant antifungal activities against fungi. The induction conditions was optimized, and the results showed that the antimicrobial activity reached its maximum when the IPTG had a concentration of 0.6 mmol/L, a temperature of 25 °C, an induction time of 12 h and an <i>OD₆₀₀</i> of 0.8.