Interdisciplinary gene manipulation, molecular cloning, and recombinant expression of modified human growth hormone isoform-1 in E. coli system

Theodor Bilharz Research Institute

Bibliographic Information

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

Journal: International Journal of Biological Macromolecules

Publisher: Elsevier B.V.

Publication Date: February 2024

Volume / Issue: Volume 257

Article No.: 128637

ISSN: 1418130

DOI: 10.1016/j.ijbiomac.2023.128637

Scopus: View on Scopus

PubMed: 38061513

Document Type: Article

Authors and Affiliations

Nasr S.M., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, 12411, Egypt, School of Biotechnology, Badr University in Cairo, Badr City, Cairo, 11829, Egypt; Samir S., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, 12411, Egypt; Okasha H., Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, 12411, Egypt

Abstract

Background: Growth hormone (GH) is a hormone that promotes growth, cell reproduction, and cell restoration in humans and animals. Objectives: Production of recombinant human growth hormone (rhGH) in Escherichia coli (E. coli) and assessment of its characteristics and proliferation stimulatory activity. Methods: The hGH gene was cloned into a pET 3a expression vector and transformed into a competent E. coli cell. The refolded hGH was purified, Western blot and batch fermentation were performed. Cell cytotoxicity was tested on Vero cells, and MALDI-TOF and Nano-LC-ESI MS/MS were used for protein and target peptide analysis. Results: Induced rhGH was purified with a concentration of 511.9 mg/ml. Western blot confirmed the molecular identity of rhGH, showing a single 22 kDa band. The bacterial growth at OD600 after 24 h in batch fermentation was 9.78 ± 0.26, and wet cell weight (WCWg/L) was 15.2 ± 0.32. Purified rhGH activity on Vero cells was 0.535 IU/mg. LC-MS/MS analysis revealed a score of 70.51 % and coverage of 60.37 %. Conclusion: Biologically active native rhGH protein was successfully expressed in the Prokaryotic system. Our goal is to increase its production on a pilot level in the native form at a high activity effect identical to isoform 1. © 2023 Elsevier B.V.

Keywords

Batch fermentation; Escherichia coli; Growth hormone; Immunodetection; MALDI-TOF; Nano-LC-ESI MS/MS; Proliferation activity; Recombinant drug; Vero cells; Animals; Chlorocebus aethiops; Chromatography, Liquid; Cloning, Molecular; Human Growth Hormone; Humans; Protein Isoforms; Recombinant Proteins; Tandem Mass Spectrometry; Cell proliferation; Clone cells; Cloning; Fermentation; Genes; Molecular biology; Proteins; Purification; isoprotein; recombinant protein; Growth hormones; Isoforms; Nano-LC-ESI-MS/MS; animal cell; Article; bacterial cell; bacterial growth; concentration (parameter); controlled study; cytotoxicity; drug purification; drug safety; drug synthesis; electrospray mass spectrometry; expression vector; genetic manipulation; genetic transformation; heterologous expression; liquid chromatography; liquid chromatography-mass spectrometry; matrix assisted laser desorption ionization time of flight mass spectrometry; molecular cloning; molecular weight; nano liquid chromatography electrospray mass spectrometry; nonhuman; peptide analysis; pilot study; protein refolding; Vero cell line; weight; Western blotting; wet cell weight; animal; chemistry; genetics; human; metabolism

Citation Information

Scopus Citations: 10

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