Cloning and Prokaryotic Expression of Cassava MePOD10

The key gene associated with Class III peroxidase (POD) in stress responses of animals and plants, MePOD10 from cassava was cloned for bioinformatics, prokaryotic expression, and protease kinetics analyses.

The coding sequence (CDS) of MePOD10 of South China 124 cassava cultivar (SC124) was amplified and subjected to bioinformatics analysis. A MePOD10-pET28a fusion expression vector was constructed and transformed into BL21 competent cells for protein induction.The protein was confirmed on its expression by SDS-PAGE and western blot and purified for enzymatic activity determination and kinetic analysis.

The length of MePOD10 CDS was 981 bp encoded 326 amino acids with a molecular weight of 35 069.60 Da and a theoretical isoelectric point of 6.59. The unstable and hydrophobic MePOD10 contained a conserved domain of plant POD sharing the highest amino acid sequence similarity of 93.25% with that of rubber trees. Induced by 1 mmol·L⁻¹ isopropyl-β-D-thiogalactoside (IPTG) at 37 ℃ for 6 h, the protein expressions in the supernatant and precipitate were examined. The purified supernatant showed greater enzymatic activity than control. When guaiacol was used as a substrate, the catalytic activity of MePOD10 increased rapidly with increasing substrate concentration to reach a peak.

MePOD10 was determined to contain conserved POD domain. MePOD10-pET28a could be correctly expressed under the induction of 1 mmol·L⁻¹ IPTG at 37 ℃ for 6 h, and the purified fusion protein exhibited a significant catalytic activity.