Recombinant expression of cec-b peptide in escherichia coli with a sig-nificant anticancer effect on hepatocellular carcinoma

Theodor Bilharz Research Institute

Bibliographic Information

Authors: Okasha H.; Nasr S.M.; Samir S.

Journal: Current Pharmaceutical Biotechnology

Publisher: Bentham Science Publishers

Publication Date: July 2021

Volume / Issue: Volume 22 / Issue 9

Pages: 1235–1245

ISSN: 13892010

DOI: 10.2174/1389201022666210104121709

Scopus: View on Scopus

PubMed: 33397234

Document Type: Article

Authors and Affiliations

Okasha H., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt; Nasr S.M., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt; Samir S., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt

Abstract

Background: Cecropin-B (Cec-B) is an Antimicrobial Peptide (AMP) found in insects. Objectives: Recombinant production of Cec-B peptide in Escherichia coli (Rosetta™ DE3), and studying its anticancer effect on Hepatocellular Carcinoma Cell line (HCC). Methods: 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. Cy-totoxicity of recombinant Cec-B (rCec-B) was reported on normal human lung cell line (WI-38), and Hepatocellular carcinoma cell line (HepG2). Results: The Cec-B gene was expressed and purified at concentration 1.212±0.1 mg/ml which rep-resents 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. Conclusion: 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 consid-ered as a hopeful anticancer drug against HCC. © 2021 Bentham Science Publishers.

Keywords

Anticancer peptide; Cecropin-B; Escherichia coli; Hepatocellular carcinoma; Peptide; Recombinant expression; Antimicrobial Cationic Peptides; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Cloning, Molecular; Humans; Inclusion Bodies; Insect Proteins; Liver Neoplasms; Recombinant Proteins; 4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid; agarose; alcohol; amphotericin B deoxycholate; ampicillin; antineoplastic agent; arginine; buffer; carbon dioxide; cecropin B; chloramphenicol; dithiothreitol; DNA ligase; doxorubicin; edetic acid; formazan; glutaraldehyde; gold; lactose; oligonucleotide; osmium tetraoxide; palladium; phosphate buffered saline; restriction endonuclease; sodium chloride; trometamol; type II site specific deoxyribonuclease; urea; antimicrobial cationic peptide; cecropin B protein, Insecta; insect protein; recombinant protein; agar gel electrophoresis; antineoplastic activity; Article; bacterial cell; cell inclusion; cell viability; centrifugation; cytotoxicity; DNA extraction; Drosophila melanogaster; expression vector; fast protein liquid chromatography; fetal bovine serum; gene expression; gene synthesis; Hep-G2 cell line; hepatocellular carcinoma cell line; IC50; incubation time; ionic strength; liver cell carcinoma; microtiter plate assay; molecular weight; MTT assay; nonhuman; polyacrylamide gel electrophoresis; polymerase chain reaction; protein refolding; purification; scanning electron microscopy; spectrophotometry; WI-38 cell line; biosynthesis; chemistry; drug effect; genetics; human; isolation and purification; liver tumor; metabolism; molecular cloning; pathology; tumor cell line

Citation Information

Scopus Citations: 14

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