Recombinant Expression of Cec-B Peptide in Escherichia coli with a Significant Anticancer Effect on Hepatocellular Carcinoma.
Antimicrobial Cationic Peptides
/ genetics
Antineoplastic Agents
/ metabolism
Carcinoma, Hepatocellular
/ metabolism
Cell Line, Tumor
Cell Survival
/ drug effects
Cloning, Molecular
Escherichia coli
/ genetics
Humans
Inclusion Bodies
/ chemistry
Insect Proteins
/ genetics
Liver Neoplasms
/ metabolism
Recombinant Proteins
/ biosynthesis
Cecropin-B
Escherichia coli
anticancer peptide
hepatocellular carcinoma
peptide
recombinant expression
Journal
Current pharmaceutical biotechnology
ISSN: 1873-4316
Titre abrégé: Curr Pharm Biotechnol
Pays: Netherlands
ID NLM: 100960530
Informations de publication
Date de publication:
2021
2021
Historique:
received:
01
09
2020
revised:
22
10
2020
accepted:
11
11
2020
pubmed:
6
1
2021
medline:
11
8
2021
entrez:
5
1
2021
Statut:
ppublish
Résumé
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.
Sections du résumé
BACKGROUND
BACKGROUND
Cecropin-B (Cec-B) is an Antimicrobial Peptide (AMP) found in insects.
OBJECTIVES
OBJECTIVE
Recombinant production of Cec-B peptide in Escherichia coli (Rosetta™ DE3), and studying its anticancer effect on Hepatocellular Carcinoma Cell line (HCC).
METHODS
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. Cytotoxicity of recombinant Cec-B (rCec-B) was reported on normal human lung cell line (WI-38), and Hepatocellular carcinoma cell line (HepG2).
RESULTS
RESULTS
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.
CONCLUSION
CONCLUSIONS
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.
Identifiants
pubmed: 33397234
pii: CPB-EPUB-113002
doi: 10.2174/1389201022666210104121709
doi:
Substances chimiques
Antimicrobial Cationic Peptides
0
Antineoplastic Agents
0
Insect Proteins
0
Recombinant Proteins
0
cecropin B protein, Insecta
80451-05-4
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1235-1245Subventions
Organisme : Theodor Bilharz Research Institute
ID : 28/K (2018)
Informations de copyright
Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.