*2.4. Expression and Purification of Functional Recombinant FhSOD1 and FhSOD3 in Escherichia coli*

The cytoplasmic (*FhSOD1*) and extracellular (*FhSOD3*, with the signal peptide sequence removed) sequences were codon optimised for expression in *Escherichia coli* and individually cloned into pET-28a(+) vectors with a C-terminal His-tag (GenScript, Piscataway, NJ, USA). The vectors were electro-transformed into kanamycin-resistant ClearColi BL21 (DE3) (ThermoFisher Scientific, Waltham, MA, USA) cells, with transformants selected on LB-Miller + kanamycin (50 μg/mL) agar plates after growth overnight at 37 ◦C.

Recombinant cells harbouring *FhSOD1* and *FhSOD3* were grown in LB-Miller broth at 37 ◦C and 180 rpm with 50 μg/mL kanamycin until the OD600 was between 0.7 and 0.8. Isopropyl-β-d-thiogalactopyranoside (IPTG; ThermoFisher Scientific) was added to the culture medium at 0.5 mM to induce protein expression, and cultures were incubated at 21 ◦C for a further 21 h. A 1 mL aliquot of each culture was removed at T0 and T21 to monitor protein production. Following centrifugation at 10,000× *g* for 10 min at 4 ◦C, the recovered bacteria were resuspended in sterile ST buffer (10 mM Tris, 150 mM NaCl, pH 8.0) and stored at −20 ◦C overnight. Cell pellets were thawed on ice then treated with 10 mg/mL lysozyme for 30 min. After incubation, 750 μL 10% Sodium lauroyl sarcosinate (sarcosyl) was added to the pellets prior to sonication at 70% amplitude with six cycles of 10 s burst/10 s rest on ice.

To recover soluble protein, the supernatant was collected after centrifugation at 15,000× *g* for 30 min at 4 ◦C, diluted to a final volume of 50 mL in lysis buffer (sodium phosphate buffer, pH 8, 10 mM imidazole) and purified using the Profinia Affinity Chromatography Protein Purification System (Bio-Rad, Hercules, CA, USA) with the corresponding mini profinity IMAC and mini Bio-Gel P—6 desalting cartridges (Bio-Rad). Proteins were eluted into 4 mL of 1× PBS, aliquoted, and stored at –70 ◦C until use. Protein concentration and purity were verified immediately after purification using the Bradford Protein Assay (Bio-Rad) and by 4–20% SDS-PAGE gels (Bio-Rad) stained with Biosafe Coomassie (Bio-Rad), respectively. To further confirm the expression and purification of the recombinant proteins, Western blots were performed using a monoclonal mouse anti-polyhistidine antibody (1:10,000) (Sigma-Aldrich, St. Louis, MO, USA) as a primary antibody, followed by incubation with a secondary antibody alkaline phosphatase conjugated goat to mouseanti-IgG diluted 1:5000 (Sigma-Aldrich). The gels and Western blots were visualised using a G:BOX Chemi XRQ imager (Syngene, Bengaluru, India).

The tertiary states of the recombinant FhSOD1 and FhSOD3 proteins were resolved by size-exclusion chromatography (gel filtration) performed on a high performance Superdex 75 10/300 GL (Tricorn) column, with a flow rate of 400 μL/min and eluted into 1× PBS. Three proteins of different molecular sizes were resolved in the column as standards, namely conalbumin (76 kDa), carbonic anhydrase (29 kDa), and aprotinin (6.5 kDa) (GE Healthcare). Upon determination of the retention parameters, rFhSOD1 and rFhSOD3 were added to the column, with 200 μL aliquots of each purification fraction collected and stored at 4 ◦C for enzyme activity (see below). The 3D structure of the corresponding native FhSOD1 (D915\_003308) and FhSOD3 (D915\_009739) amino acid sequences was predicted by the AlphaFold Protein Structure Database [25,26].
