Heterologous expression of the Stellaria media plant defensin SmD1 in Escherichia coli

TL;DR

Researchers successfully produced a plant defensin in bacteria, improving its potential for large-scale use against plant pathogens.

Contribution

The study demonstrates efficient heterologous expression and purification of SmD1 in E. coli for scalable antifungal defensin production.

Findings

SmD1 was successfully expressed in E. coli BL21 (DE3) with thioredoxin fusion.

Optimized induction conditions maximized antimicrobial activity of recombinant SmD1.

Purified SmD1 showed significant antifungal activity against target pathogens.

Abstract

SmD1 isolated from seeds of common chickweed Stellaria media 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 SmD1 was successfully heterologously expressed in Escherichia coli BL21 (DE3) for efficient production of plant defensins. The defensin gene SmD1 fused with thioredoxin was cloned into pET22b (+) vector. Then, it was transformed into E. coli 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 OD600 of 0.8.

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