Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest .

BACKGROUND

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests.

METHODS

AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive () and gram-negative ( and ) bacteria.

RESULTS

AgCecropB was expressed in BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive bacteria growth.

CONCLUSIONS

The first cecropin B-like peptide was described in and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit growth .