*4.1. Obtaining the Recombinant Magnificamide*

The synthetic gene encoding the target peptide was cloned into a pET32b(+) (Novagen, Germany) vector using the restriction sites for KpnI and XhoI by JSC "Eurogen" company, Moscow, Russia. The resulted construct was checked by sequencing to verify the open reading frame.

The expression construction pET32b(+)/*magnificamide* was used for the transformation of BL21(DE3) *E. coli* cells by electroporation using a Multiporator (Eppendorf, Hamburg, Germany) device. Cells were screened on LB agarose plates containing 100 μg/mL carbenicillin (Invitrogen, Carlsbad, CA, USA). Next the transformed cells were cultured in LB medium (1 L) containing 100 μg/mL carbenicillin at 37 ◦C to the optical density of (at A600) 0.6–0.8. Isopropyl-β,D-thiogalactopyranoside (IPTG) (Invitrogen, Carlsbad, CA, USA) was added to a final concentration of 0.2 mM for induction of expression. The cells were grown for 16 h at 18 ◦C for the production of the fusion protein in a soluble form and then cells were precipitated from the culture medium by centrifugation at 8000 rpm for 6 min. Cells were lysed by ultra-sonication on a Sonopuls 2070 instrument (Bandeling, Berlin, Germany). The fusion protein Trx-magnificamide was isolated from the cell lysate by metal affinity chromatography on the Ni-NTA-agarose (Qiagen, Hilden, Germany) in native conditions. Then the fusion protein was desalted using Amicon Ultra-15 Centrifugal Filter Units 3000 NMWL (Millipore, Burlington, MA, USA), followed by hydrolysis by Enterokinase (NEB, Ipswich, MA, USA) according to the manufacturer's instructions. Recombinant magnificamide was purified from the reaction mixture by RP-HPLC, using a Jupiter C4 column (Phenomenex, Torrance, CA, USA), equilibrated with 0.1% TFA at pH 2.2, in a gradient of acetonitrile (with concentrations from 0%–70%) for 70 min at 2 mL/min. The retention time of the target peptide was 30 min.
