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

BACKGROUND:Growth hormone (GH) is a hormone that promotes growth, cell reproduction, and cell restoration in humans and animals. OBJECTIVES:Production of recombinant human growth hormone (rhGH) in Escherichia coli (E. coli) and assessment of its characteristics and proliferation stimulatory activity. 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. 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 OD600 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 %. 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.