Aras Part Medical International Press International Journal of Medical Parasitology and Epidemiology Sciences 2766-6492 7 2 2026 06 29 Enhanced Expression and Bioactivity of Recombinant Human Growth Hormone in Escherichia coli BL21 (DE3) via Codon Optimization 84 93 10.34172/ijmpes.6267 EN Barakat Abdulrazzaq Mutar https://orcid.org/0009-0008-4014-6828 Azhar Ali Sekhi Saba Salih Saihood Hams Hussain Hashim Al-Fattli Journal Article 10.34172/ijmpes.6267 2026 03 12 2026 05 18 Introduction: The production of recombinant human growth hormone (hGH) in Escherichia coli faces limitations, such as codon bias, low translation efficiency, and improper protein folding. The aim of the current study was to investigate the effects of a codon-optimized hGH gene on protein expression, yield, and biological activity in vitro. Methods: The hGH gene was codon-optimized to enhance the Codon Adaptation Index (from 0.62 to 0.91) and minimize rare codons (from 38 to 3) and GC content (from 67.4% to 53.2%), and was cloned into the pET21a(+) vector for expression in E.coli BL21(DE3). Bioactivity assays (Nb2 cell proliferation and IGF-1 induction in HepG2 cells), expression optimization, and inclusion body refolding were performed. Results: Codon optimization increased total hGH expression by approximately twofold (245 vs. 120 mg/L). Following refolding optimization, a yield of 165 mg/L of biologically active hGH was achieved with a 94% refolding efficiency. The refolded recombinant hGH retained 96% proliferative activity in Nb2 cells and 92% IGF-1 stimulation capacity in HepG2 cells compared to the WHO international standard. Conclusion: Overall, codon-optimizing hGH vastly improved the expression and yield of recombinant hGH proteins in E. coli. Combined with effective refolding strategies, this strategy presents a scalable and robust platform for producing human hormones at therapeutic scales, alleviating key biomanufacturing bottlenecks.