Abstract
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.