To investigate the function of the prokaryotic expression product of the peroxiredoxin 3 gene (OsPrx3) from Oryza sativa L. in inhibiting DNA oxidative damage in vitro.

Molecular recombination was used to construct a prokaryotic expression vector for OsPrx3 from ‘Dahonggu’ red rice and its site-directed mutant OsPrx3mC51A. The target protein was expressed in Escherichia coli and its in vitro hydroxyl radical scavenging activity was determined. Using the supercoiled form (SF) of the pMD18 plasmid as the substrate, DNA nicking experiments were conducted to identify the function of OsPrx3 in inhibiting DNA oxidative damage.

OsPrx3 was successfully expressed in E. coli. The scavenging rate of hydroxyl radical by OsPrx3 at 1.0 mg/mL was nearly 40% after 2 h of reaction. The results of hydroxyl radical scavenging assay revealed that OsPrx3mC51A was almost completely inactivated, confirming that cysteine residue at position 51 was the key catalytic site of this enzyme activity. The DNA nicking experiments using the pMD18 plasmid at 0.06 μg/μL showed that OsPrx3 at low concentrations (0.06–0.18 μg/μL) inhibited oxidative damage to the target DNA in a concentration-dependent manner. The highest inhibition rate of 88.9% was found at an OsPrx3 concentration of 0.18 μg/μL, which was 1.7-fold higher than that at 0.06 μg/μL. At optimal OsPrx3 concentration of 0.15 μg/μL, the inhibition rate of DNA oxidative damage was 77.5%. When a SF-DNA/nicked DNA (NF-DNA) ratio of 1.0 was set as the redox equilibrium point, the inhibitory effect of OsPrx3 at the optimal concentration could be maintained for at least 180 min, and the SF-DNA/NF-DNA ratio in the system still reached 1.5 after 180 min. Taken together, the prokaryotically expressed OsPrx3 could potently inhibit DNA oxidative damage in vitro. This study provides an experimental basis for the discovery and utilization of beneficial protein resources from red rice.

To determine whether the prokaryotic expression product of the red rice OsPrx3 gene, which encodes a peroxiredoxin, can enhance oxidative stress resistance in animals.

OsPrx3 was cloned and expressed in Escherichia coli. After ingestion of the target protein, the animal model Caenorhabditis elega was evaluated for its motility, body length, oxidative stress resistance, reactive oxygen species (ROS) and fat contents, and oxidation-related gene expression levels.

This gene was cloned and expressed in E. coli successfully, and the purified expressed protein had high antioxidant activity, which at 1 μg/mL scavenged more than 40% of H₂O₂ in five minutes. Ingestion of the target protein significantly increased the resistance to oxidative stress within 15 minutes after oxidative treatment and reduced ROS and cellular lipid content in C. elegans compared to the control group, although there were no significant changes in motility or body length. Additionally, the expression levels of several critical antioxidant genes were evidently increased in the treated C. elegans.

The OsPrx3 protein had high in vitro antioxidant potency, and when ingested by C. elegans, significantly enhanced the resistance to oxidative stress.

To investigate the antioxidant function of the prokaryotic expression product of the heme-binding protein (HBP) gene, rHBP2, cloned from red rice.

We conducted molecular cloning of target genes and construction of expression vectors, transformation and expression in Escherichia coli, followed by isolation of the target protein rHBP2 and identification of its heme binding capacity and antioxidant activity in vitro. Additionally, we assessed the effect of feeding rHBP2 on oxidative and heat stress responses and oxidative stress-responsive gene expression in Caenorhabditis elegans N2.

Sequence and molecular structure analysis showed that the full-length coding sequence (CDS) of rHBP2 was 651 bp, encoding a protein containing 216 amino acid residues. The conservativeness of its amino acid sequence exceeded 90% among the compared rice plants. The molecular structure of rHBP2 exhibited both internal and external compatibility, and the pocket formed by several folded sheets might provide an important site for heme binding. The typical alpha helix was located on the periphery, potentially facilitating other potential interactions. rHBP2 was successfully expressed in E. coli, with a molecular weight of approximately 25 kDa. RHBP2 demonstrated a strong binding affinity for 1.0 mmol/L hemin and high hydroxyl radical scavenging activity, which scavenged nearly 30% of hydroxyl radical at a concentration of 1 mg/mL rHBP2. After 1.5 h exposure to H₂O₂ and 8 h exposure to 35 ℃, the survival rates of nematodes fed rHBP2 were 5.7 and 2.4 times higher than those of the control group, respectively, indicating that rHBP2 significantly enhanced nematode tolerance to oxidative and thermal stress. Moreover, nematodes fed rHBP2 exhibited significantly lower endogenous reactive oxygen species (ROS) levels and higher expression levels of oxidative stress-responsive genes such as SOD-3 and CAT-1. These results suggested that rHBP2 had potent antioxidant function. This study provides an experimental basis for discovering and utilizing antioxidant proteins from red rice.