*4.6. Protein Expression and Purification*

The nucleotide sequence of Cryptoxin-1 was optimized for expression in *E. coli* and the construction pET24b-Cryptoxin-1 was performed using Invitrogen™ Gene Synthesis (GeneArt™ Thermo Fisher Scientific, Waltham, MA, USA).

For their expression, chemically competent *E. coli* BL21 Star™ (DE3) (Invitrogen®) were transformed with pET24b-Cryptoxin-1 construction or pET-42a (Novagen) containing the sequence of glutathione S-transferase (GST). For each experiment, a cell colony grown overnight from LB-agar plates was transferred into a liquid LB medium and grown overnight at 30 ◦C in the presence of 50 μg/mL kanamycin. This culture was diluted 1:50 into 200 mL of fresh LB broth/kanamycin. When the cell suspension reached an optical density of 0.6 at 30 ◦C (OD 600 nm) it was induced with a final concentration of 1 mM of isopropyl-b-D-thiogalactoside (IPTG). Cells were then grown for four hours after which the cells were collected by centrifugation at 10,000× *g* for 10 min at 10 ◦C (ultracentrifuge Beckman). The whole-cell pellets were then resuspended in a binding buffer (20 mM sodium phosphate pH 7.4 and 500 mM NaCl) and lysed on ice by an ultrasonication device (amplitude of 20% with 3 s pulse and 4 s interval between each pulse) for 60 s, and the process was repeated 5 times. Cell debris were removed from the protein solution by centrifugation at 10,000× *g* for 10 min in a Beckman ultracentrifuge. The entire amount of the supernatant containing the soluble protein was purified with high-performance immobilized metal affinity chromatography (IMAC) using HisTrapHP 5 mL column pre-packed (Cytiva™, Marlborough, MA, USA) coupled to the ÄKTA™ start protein purification system and then desalted into phosphate-buffered saline (PBS) buffer pH 7.4 with HiTrap® Desalting Columns (Cytiva™, Marlborough, MA, USA). The endotoxin was removed with Pierce™ High-Capacity Endotoxin Removal Spin Columns (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol. Quantification of recombinant proteins was performed using the BCA Pierce™ Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol.

#### *4.7. Mass Spectrometry Analysis*

Mass spectrometry of Cryptoxin-1 was performed on the MALDI-TOF Autoflex Speed (Bruker Corporation, Billerica, MA, USA) equipment following pre-established protocols for protein analysis. In summary, 0.5 μL of a saturated solution of sinapinic acid in ethanol was mixed with 100 ng of Cryptoxin-1. After drying, 1 μL of a TA30 (0.1% trifluoroacetic acid/acetonitrile in a proportion of 70/30) was added to the mixture and applied to the Ground Steel plate for analysis. Data acquisition was performed in a linear mode with positive polarity, with the following parameters: Ion Source 1—19.50 kV, Ion Source 2— 17.60 kV, Lens—9.0 kV, Pulsed Ion Extraction 170 ns, Mass Range 5—70 kDa, Laser Frequency 500 Hz, Gain Detector 10.0×. The results were analyzed using the online software mMass version 5.5.0, 2013 (Martin Strohalm© Open Source Mass Spectrometry Tool).
