*2.1. Peptide Expression and Purification*

To study the properties of peptides and then to develop peptide-based drugs, it is necessary to obtain their recombinant analogues at sufficient qualities and quantities. The plasmid vector pET32b(+) containing the gene of thioredoxin ensures high yields of cysteine-containing polypeptides with native conformations, and was, therefore, chosen to create an expression construct. The synthetic gene encoding magnificamide was cloned into pET32b(+) using restriction sites KpnI and XhoI (Figure 1a). The resulting plasmid was transferred into *Escherichia coli* BL21(DE3) cells by electroporation and expressed as a fusion protein Trx-magnificamide (Figure 1b).

**Figure 1.** (**a**) Map of the pET32b(+)-magnificamide expression plasmid. A synthetic gene encoding the magnificamide and enterokinase sites was cloned using the restriction sites for KpnI and XhoI. (**b**) The scheme of fusion protein Trx-magnificamide and sequence of magnificamide (UniProtKB—C0HK71).

The fusion protein was isolated from the cell lysate by metal affinity chromatography, desalted, hydrolyzed by enterokinase, and then the recombinant magnificamide (r-magnificamide) was purified by RP-HPLC (Figure 2). After HPLC two fractions which inhibited PPA were obtained, one of them contained the mature r-magnificamide (Figure 3a); the other one contained peptide with incorrect folding (Figure 3b). The average yield of target peptide was equal to 4 mg per 1 L of cell culture (OD A600 = 0.6–0.8).

**Figure 2.** The RP-HPLC elution profile of r-magnificamide, obtained as the result of hydrolysis of the fusion protein Trx-magnificamide by enterokinase, on a Jupiter C4 column (Phenomenex, Torrance, CA, USA) equilibrated by 0.1% TFA, pH 2.2, in a gradient of acetonitrile concentration (0%–70%) for 70 min at 2 mL/min. Fraction 1 containing the mature peptide r-magnificamide (4770 Da) (Figure 3a) is filled by dark grey color; fraction 2 containing peptide with incorrect folding (4777 Da) (Figure 3b) is filled by light grey color.

**Figure 3.** Mass spectra, *m*/*z*, of the peptides isolated by RP-HPLC (Figure 2): (**a**) mature r-magnificamide from fraction 1 and (**b**) incorrectly folded r-magnificamide from fraction 2. *m*/*z*—mass-to-charge ratio; a. u.—arbitrary units.

#### *2.2. Secondary Structure of Peptides*

To calculate the secondary structural elements of recombinant and native magnificamide, the circular dichroism spectroscopy method was used. The spectra in the far UV region (190–240 nm) were characterized by a minimum at 212 nm and a maximum at 203 nm. In the 225–235 nm range, distinct shoulders were observed on the spectra's curves due to the contribution of the disulfide groups' absorption (Figure 4). The similarity between the circular dichroism (CD) spectra of magnificamide and its recombinant analogue suggested that the recombinant peptide should be functional and can be used successfully to study its biological activity. Moreover, the calculation of secondary structure elements using Provenzer–Glockner method [20] revealed the complete identities of the peptides at the secondary structure level (Table 1). It should be noted that content of α-helices of the magnificamide was significantly lower than that of helianthamide, which might be reflected in the difference in their spatial structures and biological activity.

**Figure 4.** Circular dichroism (CD) spectra of native magnificamide (black line) and recombinant magnificamide (grey line) in 0.01M phosphate buffer, pH 7.0, far or peptide bond UV region.


**Table 1.** Secondary structural elements of the natural and recombinant magnificamide and helianthamide (percentages).

α β <sup>β</sup>
