**5. Materials and Methods**

#### *5.1. Strains, Plasmids, Enzymes*

The expression vector pD912-AK with the synthesized insert of interest (codonoptimized) was purchased from ATUM 2.0 (Newark, NJ, USA), and *P. pastoris* strain X-33 (Invitrogen, Waltham, MA, USA) was used as the expression host. The *Escherichia coli* strain DH5α and restriction enzyme SacI were purchased from Thermo Fisher (Waltham, MA, USA). *E. coli* cells with plasmids were cultured at 37 ◦C in Luria–Bertani medium (yeast extract, 5 g/L; tryptone, 10 g/L; NaCl, 10 g/L; agar, 15 g/L) containing 25 μg/mL Zeocin (Invitrogen, Waltham, MA, USA). Papain was purchased from Sigma-Aldrich (St. Louis, MO, USA) and human cathepsin L from R&D Systems (Minneapolis, MN, USA).

### *5.2. Sequence Source and In Silico Characterization*

The Cystatin-Hv cDNA sequence (transcript Hviz00340) was obtained from the sialotranscriptome of *Haementeria vizzotoi* leech [23]. The signal peptide sequence, determined by SignalP 4.0 [43], was excluded from further analysis and from the insert synthesis. Theoretical pI and Mw were determined using the Expasy platform. Identity and similarity percentages of the full-length amino acid sequence were obtained by BLAST search (NCBI database), and multiple sequence alignments were performed on a sequence of Cystatin-Hv versus known cystatins, using Clustal Omega [44].

#### *5.3. Expression and Purification of Recombinant Protein*

The vector pD912-AK: Cystatin-Hv was linearized with SacI and electroporated into competent *P. pastoris* X-33 cells. Transformants were screened on YPD medium plates containing 25 μg/mL Zeocin, and the presence of Cystatin-Hv insert was confirmed by PCR. Expression was carried out in replicates inoculating 50 mL of BMGY medium [1.0% yeast extract, 2.0% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × <sup>10</sup><sup>−</sup>5% D-biotin (*w*/*v*), and 1% glycerol (*v*/*v*)] and cultivated under the influence of 350 rpm orbital shaking at 28 ◦C for 24 h. Cells were harvested by centrifugation at 450× *g* for 5 min at 4 ◦C and resuspended in 50 mL of BMMY medium [1.0% yeast extract, 2.0% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × <sup>10</sup>−5% D-biotin (*w*/*v*), and 0.5% methanol (*v*/*v*)] to absorbance at 600 nm of 5.0. Incubation was carried out at 30 ◦C and 350 rpm

orbital shaking for 44 h, with further additions of methanol to a final concentration of 0.5% every 12 h, approximately. Samples (1 mL) were taken during the assay, submitted to protein precipitation with methanol/chloroform [45], and analyzed by SDS-PAGE.

Cells were removed from the supernatant by centrifugation (3500× *g* for 15 min at 4 ◦C) and filtration (0.45 μm). The supernatant was dialyzed (5 kDa molecular exclusion) and concentrated with 20 mM Tris-HCl pH 8.0 using Cogent μScale TFF System (Merck, Darmstadt, Germany) and submitted to ion-exchange chromatography in a Mono Q 5/50 GL (GE Healthcare) 1 mL column connected to an AKTA Avant system (GE Healthcare), equilibrated with 20 mM Tris-HCl pH 8.0. The sample was added, and the column was washed with 15 CV (column volume) of 20 mM Tris-HCl pH 8.0 (0.5 mL/min), followed by the elution step supported by a crescent linear gradient of 20 mM Tris-HCl pH 8.0, 1.0 M NaCl along 30 CV (0.5 mL/min). Fractions (~500 μL) were pooled, analyzed by SDS-PAGE, and the one presenting inhibitory activity towards papain was applied on Superdex 75 10/300 column (GE Healthcare), being eluted with 20 mM Tris-HCl pH 8.0 along 2 CV (1 mL/min). Fractions with expected molecular weight, single band, were pooled, quantified using the bicinchoninic acid (BCA) Protein Assay Kit (Pierce, WA, USA) and further analyzed for inhibitory activity against papain and cathepsin L.
