Overexpression, overproduction, purification, and characterization of rhGH in Escherichia coli

Overexpression of insoluble human growth hormone (hGH) in cytoplasm was achieved by E. coli Rosetta-gami B(DE3) [pET21a (+)-hGH]). For overexpression of hGH, effects of eight factors including temperature, type and concentration of carbon source, IPTG and MgSO₄ , buffering capacity, induction time, yeast extract/peptone ratio on rhGH production were studied by Plackett-Burman screening. Maximum production of rhGH was 0.681 g/L, and results of statistical analysis showed that induction temperature and glucose have the greatest effect and the presence of MgSO₄ increases rhGH expression and reduces biomass concentration. So, the effect of ethanol and MgSO₄ concentrations on the rhGH production was examined according to the central composite experimental design. The ANOVA of the results showed rhGH production increases to 1.128 g/L in 4 g/L MgSO₄ and 1% ethanol. Then, the impact of glucose concentration and induction time on the rhGH production was evaluated in two levels in the fermenter by Taguchi statistical method. Under optimum conditions, OD_600nm 4 and 10 g/L glucose crude rhGH concentration 4.17 g/L was obtained, which is one of the highest value ever reported. Finally, rhGH was purified using the biophysical and biochemical techniques comprising circular dichroism, fluorescent spectroscopy, and dynamic light scattering, and it was confirmed that the produced protein is comparable to the commercial standard sample.