Back to Search Start Over

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

Authors :
Nasr SM
Samir S
Okasha H
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.)

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