# The Infestation of Olive Fruits by _Bactrocera oleae_ ([PERSON])
## 3 Materials and Methods
### OePPO Genes Cloning, Heterologous Protein Expression, Purification, and Functional Characterization  
The coding sequences of the selected candidate genes, _OePPO1_ (OE6A068152) and _OePPO2_, (OE6A114203) were synthesized with _E. coli_ codon optimization (GenScript, Piscataway, NJ, USA) and cloned into a pGEX6P.1 vector as EcoRI-XhoI fragments. Two constructs were obtained to be produced as glutathione-S-transferase (GST) fusion proteins: GST-OePPO1 and GST-OePPO2.  
Protein expression and purification were carried out according to [PERSON] et al. [49] with minor modifications. BL21(DE3) lacIq E. coli cells containing OePPO1 or OePPO2 constructs were grown at 37 \({}^{\circ}\)C in Luria Bertani media (LB) with 0.5 M NaCl to OD\({}_{600}\) of 0.6, then supplemented with 0.5 mM isopropyl-b-D-thiogalactoside (IPTG) and 0.5 mM CuS and grown for 20 h at 19 \({}^{\circ}\)C. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 1 mM EDTA, glycerol 10% (_v/v_), 1 mM benzidine, 1 mM phenylmethanesulfonyl fluoride), and lysed by sonication. The resulting crude protein lysate was clarified by centrifugation prior to chromatographic purification with Sepharose-GSH beads (GE Healthcare, Chicago, IL, USA) following the manufacturer's instructions. The soluble fraction obtained was added to the beads for GST-OePPOs purification and incubated for 2 h at 4 \({}^{\circ}\)C in a 360 \({}^{\circ}\) rotator. Then, recombinant protein-bound-beads were incubated in preScission Protease proteolytic digestion buffer (GE Healthcare) (50 mM Tris HCl pH 7.0, 150 mM NaCl, 1 mM EDTA) and 160 U/mL of protease. The enzymatic digestion was carried out overnight at 4 \({}^{\circ}\)C on a 360\({}^{\circ}\) rotator to eliminate the GST fusion protein from the OePPO1 and OePPO2 proteins. The purified proteins were collected as eluates after centrifugation. OePPO1 and OePPO2 purified protein buffer was exchanged to 100 mM Tris-maleate pH 6.8 and 10% glycerol buffer on a PD-10 column (Sephadex G-25, GE Healthcare, Chicago, IL, USA) and further concentrated using a Vivaspin centrifugal concentrator (MWCO 30 kDa, Merck, Darmstadt, Germany). Purity of the recombinant proteins was evaluated by SDS-PAGE and the protein concentration was determined by the Bradford assay [50].  
The PPO enzyme assays for functional characterization of PPO recombinant proteins were performed in 150 \(\upmu\)L of reaction buffer (50 mM Tris-maleate pH 6.8, 1 mM SDS) containing 10-20 \(\upmu\)g of recombinant protein, using as substrates (2.5 mM) two olive phenolic components: hydroxytyrosol (Sigma-Aldrich, St. Louis, MO, USA) and oleupropein (Extrasynthese, Genay, France). The mixture was incubated at 25 \({}^{\circ}\)C for 5 min and then the reaction was terminated by the addition of 150 \(\upmu\)L methanol. The reaction mixture was centrifuged and filtered (0.45 \(\upmu\)m) and the supernatant was analyzed by HPLC using the same equipment and chromatographic conditions previously described for the analysis of phenolic compounds. In all cases, controls were carried out using the same reaction mixture, but carrying untransformed _E. coli_ BL21 protein extracts.