Abstract
Cecropin-B (Cec-B) is an Antimicrobial Peptide (AMP) found in insects. Recombinant production of Cec-B peptide in Escherichia coli (Rosetta™ DE3), and studying its anticancer effect on Hepatocellular Carcinoma Cell line (HCC). The Cec-B gene of Drosophila melanogaster was synthesized by PCR assembly using the Simplified Gene Synthesis (SGS) method. To express the recombinant peptide in E. coli (Rosetta™ DE3); the synthesized gene was cloned into pET-15b expression vector. The recombinant peptide was expressed as insoluble aggregates called Inclusion Bodies (IBs) using 2mM lactose inducer. IBs were solubilized in a denatured form using 8 M urea followed by in-vitro protein refolding using rapid dilution method. The refolded Cec-B was purified using cation-exchange SP-FF column. Cytotoxicity of recombinant Cec-B (rCec-B) was reported on normal human lung cell line (WI-38), and Hepatocellular carcinoma cell line (HepG2). The Cec-B gene was expressed and purified at concentration 1.212±0.1 mg/ml which represents 48.49±4% of the total proteins injected to the column (2.5±0.2 mg/ml). The safe dose of purified rCec-B on normal WI-38 cells was calculated to be 1.57 mg/ml. The half-maximal Inhibitory Concentration (IC50) of rCec-B on HepG2 cell line was calculated to be 25 μg/ml. Scanning Electron Microscope (SEM) showed that untreated and treated HepG2 cells had cell diameters from 11-12.92 μm and 14.18-21.58 μm, respectively. The results of this study revealed a successful expression of the rCec-B peptide using a pET-based expression system with a simple purification step. The purified peptide could be considered as a hopeful anticancer drug against HCC.
