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Interdisciplinary gene manipulation, molecular cloning, and recombinant expression of modified human growth hormone isoform-1 in E. coli system.
- Source :
-
International journal of biological macromolecules [Int J Biol Macromol] 2024 Feb; Vol. 257 (Pt 1), pp. 128637. Date of Electronic Publication: 2023 Dec 06. - Publication Year :
- 2024
-
Abstract
- Background: Growth hormone (GH) is a hormone that promotes growth, cell reproduction, and cell restoration in humans and animals.<br />Objectives: Production of recombinant human growth hormone (rhGH) in Escherichia coli (E. coli) and assessment of its characteristics and proliferation stimulatory activity.<br />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.<br />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 OD <subscript>600</subscript> 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 %.<br />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.<br />Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Sami Mohamed Nasr reports financial support, administrative support, equipment, drugs, or supplies, and travel were provided by Science and Technology Development Fund. Dr. Sami Mohamed Nasr reports a relationship with Theodor Bilharz Research Institute that includes: employment. Co-author Dr. Hend Okasha and member Dr. Safia Samir are employee at Theodor Bilharz research institute.<br /> (Copyright © 2023 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Chlorocebus aethiops
Humans
Escherichia coli genetics
Escherichia coli metabolism
Chromatography, Liquid
Vero Cells
Tandem Mass Spectrometry
Growth Hormone genetics
Growth Hormone metabolism
Growth Hormone pharmacology
Cloning, Molecular
Recombinant Proteins metabolism
Protein Isoforms metabolism
Human Growth Hormone chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1879-0003
- Volume :
- 257
- Issue :
- Pt 1
- Database :
- MEDLINE
- Journal :
- International journal of biological macromolecules
- Publication Type :
- Academic Journal
- Accession number :
- 38061513
- Full Text :
- https://doi.org/10.1016/j.ijbiomac.2023.128637