Lab-scale Preparation of Recombinant Human Insulin-like Growth Factor-1 in Escherichia coli and its Potential Safety on Normal Human Lung Cell Line

Theodor Bilharz Research Institute

Bibliographic Information

Authors: Mohamed O.A.; Samir S.; Omar H.; Hassan E.A.; Abdelazeem E.

Journal: Recent Patents on Biotechnology

Publisher: Bentham Science Publishers

Publication Date: September 2022

Volume / Issue: Volume 16 / Issue 3

Pages: 266–280

ISSN: 18722083

DOI: 10.2174/1872208316666220412105822

Scopus: View on Scopus

PubMed: 35418294

Document Type: Article

Authors and Affiliations

Mohamed O.A., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute (TBRI), Giza, Egypt; Samir S., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute (TBRI), Giza, Egypt; Omar H., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute (TBRI), Giza, Egypt; Hassan E.A., Biochemistry Department, Faculty of Science, Ain-Shams University, Cairo, Egypt; Abdelazeem E., Biochemistry Department, Faculty of Science, Ain-Shams University, Cairo, Egypt

Abstract

Background: Insulin-like growth factor-1 (IGF-1) is structurally similar to insulin and acts as an endocrine hormone secreted by the liver. Objective: Production of recombinant human IGF-1 (rhIGF-1) in Escherichia coli (E.coli) and evaluation of its proliferation stimulatory activity. Methods: hIGF-1 gene cloned into pBSK (+) simple vector was transformed into TOP 10 chemically competent cells of E. coli. Polymerase chain reaction (PCR) was achieved using specific hIGF-1 gene primers to confirm the successful transformation. To express the rhIGF-1 in E. coli (Rosetta (DE3) pLysS); the hIGF-1 gene was cloned into the pET-15b expression vector and then the recombinant pET-15b/IGF-1 vector was transformed into a chemically prepared competent expression bacterial cells; Rosetta (DE3) pLysS. The rhIGF-1 was expressed as insoluble aggregates called inclusion bodies (IBs) using a 2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer. IBs were solubilized in a denatured form using 6 M guanidinium hydrochloride (GdmCl), followed by in vitro protein refold-ing using the rapid dilution method. The refolded hIGF-1 was purified using the HiTrap-ANX anion exchange column. Western blot and ELISA using rabbit polyvalent anti-hIGF-1 were performed to confirm the protein antigenic identity. Cell proliferation activity of rhIGF-1 was testified on normal human lung cell line (WI-38). Results: rhIGF-1 was purified from the HiTrap-ANX column at a concentration of 300 µg/ml. Western blot showed a single 7.6 kDa band obtained in the induced Rosetta (DE3) pLYsS. ELISA confirmed the molecular identity of the rhIGF-1 epitope, the concentration of purified rhIGF-1 obtained from the ELISA standard curve using rhIGF-1 reference protein as a standard was 300 µg/ml, and activity on WI-38 cells was 2604.17I U/mg. Conclusion: Biologically active native rhIGF-1 protein was successfully expressed. Pa-tents related to the preparation of IGF-1 were mentioned along the text. © 2022 Bentham Science Publishers.

Keywords

ELISA; Escherichia coli; hIGF-1; human lung cell line; proliferation assay; recombinant expression; Western blot; Animals; Cell Line; Humans; Insulin-Like Growth Factor I; Irritable Bowel Syndrome; Lung; Patents as Topic; Rabbits; Recombinant Proteins; Antigens; Biological organs; Cell culture; Cell proliferation; Cloning; Genes; Insulin; Polymerase chain reaction; Purification; growth factor; recombinant human insulin; recombinant somatomedin C; somatomedin C; recombinant protein; Cell lines; Human lung cells; Insulin-like growth factor-1; Rosetta; Western blots; anion exchange; anion exchange chromatography; antibacterial activity; Article; bacterial cell; calibration; cell count; cell inclusion; cell proliferation assay; cross linking; denaturation; dilution; enzyme activity; enzyme linked immunosorbent assay; expression vector; gene amplification; gene expression; human; human cell; lung cell line; lymphocyte; molecular weight; MTT assay; nonhuman; plasmid; polyacrylamide gel electrophoresis; protein denaturation; protein expression; protein purification; Western blotting; WI-38 cell line; animal; genetics; irritable colon; Leporidae; metabolism; patent; Proteins

Citation Information

Scopus Citations: 6

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