A novel mutant recombinant human growth hormone (rhGH) production in Escherichia coli via genetic modifications

Document Type : Research Paper

Authors

Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran

Abstract

Human growth hormone (hGH) (also called somatotropin) is a single-chain polypeptide that is made and secreted into the blood circulation by the anterior part of the pituitary gland. The growth hormone is composed of 191 amino acids, two disulfide bonds, and four α-helices. Based on crystallographic studies, one hGH binds to two growth hormone receptors and forms a ternary complex. The hGH has two receptor-binding sites: site1 (high affinity) and site2 (low affinity). The primary objective of the present study was to enhance the binding affinity of receptor-binding site 1 through targeted substitution of eight specific amino acid residues (Arginine, lysine, aspartic acid, lysine, glutamine, histidine, isoleucine, and histidine with asparagine, alanine, serine, arginine, serine, asparagine, threonine, and aspartic acid). For this aim, the GH1 gene (which encodes hGH) was manipulated by introducing mutations (missense) using polymerase chain reaction (PCR). Then, mutant GH1 was cloned into the pGH vector (a plasmid vector) and, after propagation (in Escherichia coli DH5α), was subcloned into the pCold vector and expressed in Escherichia coli. The Western Blot technique was used to determine the production of mutant hGH. Protein purification and quantitative assessment were performed using Nickel-Sepharose affinity chromatography and Bradford assay, respectively. The biological activity of the mutant hGH was examined using the Ba/F3-rat-GHR cell line, which stably expresses the human growth hormone receptor (hGHR). Molecular docking analysis using HADDOCK indicated that the mutant hGH exhibited a higher binding affinity for hGHR compared to the wild-type hormone. Two recombinant growth hormones (R-GH1 and R-GH2) were obtained. Results suggested that recombinant hGHs induced the proliferation of Ba/F3-rat-GHR cell lines more potently than commercial (Zorbtive) hGH (P< 0.05). This study successfully engineered a mutant form of hGH with enhanced receptor-binding affinity, improved in vitro biological activity, and greater proliferative potency compared to commercial hGH, suggesting its potential for therapeutic applications.

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References

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Microbiology, Metabolites and Biotechnology
Volume 8, Issue 1
June 2025
Pages 47-52

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