Expression and Purification of Goat Cathelicidins

The recombinant plasmids for expression of the goat cathelicidins were constructed with the use of pET-based vector as described previously (Panteleev and Ovchinnikova, 2017 (link)). The target peptides were expressed in E. coli BL21 (DE3) as chimeric proteins that included 8 × His tag, the E. coli thioredoxin A with the M37L substitution (TrxL), methionine residue, and a mature cathelicidin. The ChMAP-28 amino acid sequence was translated from mRNA for the corresponding precursor protein (GenBank: AJ243126.1) as a 27-residue peptide without the C-terminal glycine, a common amidation signal in cathelicidins. The transformed E. coli BL21 (DE3) cells were grown up to OD₆₀₀ 1.0 at 37°C in lysogeny broth (LB) containing 20 mM glucose, 1 mM MgSO₄, and 0.1 mM CaCl₂, 100 μg/ml of ampicillin and then were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.3 mM. The cells were cultivated for 5 h at 30°C with intense agitation. Then the cells were pelleted by centrifugation and sonicated in immobilized metal affinity chromatography (IMAC) loading buffer containing 6 M guanidine hydrochloride. The clarified lysate was applied on a column packed with Ni Sepharose (GE Healthcare). The recombinant protein was eluted with the buffer containing 0.5 M imidazole. Then the eluate containing the fusion protein was acidified (up to pH 1.0) and cleaved by 100-fold molar excess of cyanogen bromide over methionine for 20 h at 25°C in the dark. The reaction products were lyophilized, dissolved in water, titrated to pH 5.0, and loaded on a semi-preparative Reprosil-pur C18-AQ column (10 mm × 250 mm, 5-μm particle size, Dr. Maisch GmbH). Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed with a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. The peaks were monitored at 214 and 280 nm. The collected fractions were analyzed by MALDI-TOF mass-spectrometry using Reflex III instrument (Bruker Daltonics). The fractions containing the target peptides were lyophilized and dissolved in water. The synthetic melittin (>98% pure) was kindly provided by Dr. Sergey V. Sychev (M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia). The recombinant tachyplesin-1 was obtained as described previously (Panteleev and Ovchinnikova, 2017 (link)). The peptides concentrations were estimated using UV absorbance.