*5.3. Plasmid Construction-Expression and Purification of the Recombinant Dromaserpin*

All manipulation of microorganisms was developed in the BSL-2 area, as authorized by CIBio-IBu (Internal Biosafety Committee of Butantan Institute, Brazil) and CTNBio (National Technical Biosafety Commission, Brazil) (Registration number CQB-039/98 of 13 February 2014). The chosen full coding cDNA sequence was used for plasmid construction after the deletion of the sequence coding for signal peptide. Codon optimization, gene synthesis and molecular cloning of rDromaserpin coding sequence into plasmid pET28a (Novagen/EMD Millipore, Darmstadt, Hesse, Germany) were conducted by GenOne (GenOne Inc. Rio de Janeiro, RJ, Brazil). Recombinant pET28a\_Dromaserpin plasmid, coding for the C-terminal His-tagged protein, was transformed into chemically competent *E. coli* BL21 (DE3) strain. The *E. coli* cultures were inoculated in 1 L of 2 YT culture medium (supplemented with 20 μg/mL kanamycin) at 30 ◦C with 250 rpm agitation. Protein expression was induced with 1 mM isopropyl β-D-Thiogalactoside (IPTG), at OD600 0.6, and cells remained incubated for 3 h at 30 ◦C following the induction. Expression was confirmed by resolving samples on a 12.5% SDS-PAGE. Bacterial cells were harvested, washed with 150 mM NaCl and re-suspended in lysis buffer (50 mM Tris-HCl pH = 8; 500 mM NaCl, 1% Triton; 10 mM β-mercaptoethanol). Whole cell extracts were obtained through a Panda Plus 2000 (GEA NIRO, Erie, PA, USA) high pressure homogenizer disrupter, three times at 1000 bars, and the suspension was clarified through centrifugation at 16,000× *g* rpm for 1 h at 4 ◦C. Histidine tagged protein was subsequently purified from the soluble fraction using a HisTrap HP (5 mL; GE Healthcare, GE Healthcare, Uppsala, Uppland, Sweden) column of Immobilized Metal Affinity Chromatography (IMAC) (AKTA AVANT; GE Healthcare GE Healthcare, Uppsala, Uppland, Sweden). Fractions containing the eluted protein were pooled and subjected to Q-sepharose ion-exchange chromatography (AKTA AVANT; GE Healthcare, GE Healthcare, Uppsala, Uppland, Sweden) using a HiTrap Q FF (1 mL; GE Healthcare, GE Healthcare, Uppsala, Uppland, Sweden) column. The purified protein was applied to a Superdex 75 (1 mL; GE Healthcare, GE Healthcare, Uppsala, Uppland, Sweden) column used to exchange the buffer to 1 × Phosphate-buffered saline (PBS), pH 7.4 supplied with 10% glycerol. To evaluate its purity, the purified protein was resolved on a 12.5% SDS-PAGE and stained with Coomassie Brilliant Blue. Finally, rDromaserpin concentration was determined by its absorbance at 280 nm using a Biodrop spectrophotometer (Biochrom™ BioDrop μLite Micro-Volume, Braeside, Australia).

#### *5.4. Structural Characterization of the rDromaserpin*

#### 5.4.1. Determination of Molecular Size by Gel Filtration

Gel Filtration experiments were performed with purified rDromaserpin using Superdex™ 75 HR 10/300 column (GE Healthcare, Uppsala, Uppland, Sweden) on an ÄKTA purifier liquid-chromatography system. Chromatography was carried out at 4 ◦C in 1× PBS pH 7.4 and 10% glycerol, at a flow of 0.5 mL/min. Protein elution was monitored by measuring absorbance at 280 nm. Apparent molecular masses of protein eluted from the column were deduced from a calibration curve obtained by loading 100 μL of the following standards: Conalbumin (75,000 Da), Ovalbumin (44,000 Da, 30.5 Å), Carbonic anhydrase (29,000 Da), Ribonuclease A (13,700 Da, 16.4 Å), Aprotinin (6500 Da), as well as blue dextran (for the void volume V0). According to the calibration curve obtained with standards, we calculated the molecular weight of rDromaserpin as described in the Equation (1).

$$\log(\text{MW}) = -0.1615 \text{ Ve} + 3.4268 \tag{1}$$

#### 5.4.2. Circular Dichroism (CD) Spectroscopy

CD spectra were recorded on a JASCO J-810 spectropolarimeter, equipped with a thermoelectric sample temperature controller (Peltier system) equilibrated to 20 ◦C. The rDromaserpin samples at 0.73 μM were diluted 10 × in Milli-Q water. The diluted buffer without protein was used to calibrate the equipment. The scans were collected at Far-UV region (from λ = 190 nm to λ = 260 nm) after seven accumulations, using a pathlength quartz cuvette of 1.0 mm. Spectra were corrected by subtracting a buffer blank, normalized to residue molar absorption, measured in mdeg (M−<sup>1</sup> cm<sup>−</sup>1), and adjusted to the input buffer. The mean molar residual ellipticity θλ (deg cm2 dmol−1) was calculated as described in the Equation (2), based on a molecular mass of 43,159.27 Da, where *θRaw* <sup>λ</sup> is ellipticity in millidegrees, C is molar rDromaserpin concentration in M, N is number of amino acid residues, and l is the path length of the cuvette in mm.

$$\theta\_{\lambda} = \frac{\theta\_{\lambda}^{\text{Raw}} \times 10^6}{\mathbb{C} \times N \times L} \tag{2}$$

The estimation of secondary structure was performed by BeStSel website algorithm (http://bestsel.elte.hu/index.php (accessed on 9 April 2021)) using the CD spectra values ranging from 190 nm to 250 nm.

#### 5.4.3. Comparative Modeling and Structural Analysis of Dromaserpin

The three-dimensional structure (3D) model of Dromaserpin was predicted using a comparative modeling approach. Using its predicted amino acid sequence, we performed a homology search among the 3D structures available in PDB database (https://www. rcsb.org/ (accessed on 22 March 2020)) using BLAST from NCBI (https://blast.ncbi.nlm. nih.gov/Blast.cgi (accessed on 22 March 2020)). For homology modeling, we selected the crystal structure of Conserpin in an uncleaved (PDB code: 5cdx) and a latent (PDB code: 5cdz) state, which presents 40.62% identity with Dromaserpin, covering 94% of the sequence. Aligned regions were selected using 5cdx as template on Swiss-Model online tool (https://swissmodel.expasy.org/ (accessed on 22 March 2020)). A 3D model was generated by PyMOL software (http://www.pymol.org/ (accessed on 22 March 2020)). Subsequently, the overall quality of the Serpin 3D-structure models was evaluated by analyzing each correspondent Ramachandran plot, calculated by MolProbity program (http://molprobity.biochem.duke.edu/ (accessed on 30 September 2021)). From these generated models, we determined the content of the secondary structure using Stride (http://webclu.bio.wzw.tum.de/cgi-bin/stride/stridecgi.py (accessed on 22 March 2020)).
