Human nerve growth factor a member of the neurotrophin family can be used to treat neurodegenerative diseases. As it has disulfide bonds in its structure, periplasmic expression of it using appropriate signal sequence is beneficial. Therefore, in this work β-nerve growth factor (β-NGF) was expressed in Escherichia coli using pET39b expression vector containing DsbA signal sequence. In an initial step, the effect of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose concentration as inducer on protein production was investigated using response surface methodology. Then the effect of different postinduction time and temperature on protein production was studied. Our results indicated that the highest β-NGF production was achieved with 1 mM IPTG and low concentrations of lactose (0-2% w/v), low cultivation temperature of 25°C and postinduction time of 2 hr. Also following β-NGF purification, bioassay test using PC12 cell line was done. The biological activity of the purified β-NGF showed a similar cell proliferation activity with the standard recombinant human β-NGF. In conclusion, the results indicated an optimized upstream process to obtain high yields of biologically active β-NGF.
A b s t r a c tHuman nerve growth factor β (β-NGF) is considered a major therapeutic agent for treatment of neurodegenerative diseases. We have previously reported the optimized conditions for β-NGF overproduction in Escherichia coli in a shake-flask culture. In this study the optimal %DO (dissolved oxygen) and post induction temperature values for improved production of β-NGF were found in the bioreactor scale using response surface methodology (RSM) as the most common statistical method. Also, for further enhancement of the yield, different post-induction periods of time were selected for testing. In all experiments, the productivity level and bacterial cell growth were evaluated by western blotting technique and monitoring of absorbance at 600 nm, respectively. Our results indicated that %DO, the post-induction time and temperature have significant effects on the production of β-NGF. After 2 hours of induction, the low post induction temperature of 32°C and 20% DO were used to increase the production of β-NGF in a 5-l bioreactor. Another important result obtained in this study was that the improved β-NGF production was not achieved at highest dry cell weigh or highest cell growth. These results are definitely of importance for industrial β-NGF production.K e y w o r d s: bioreactor, RSM, β-NGF over production, E. coli Hajihassan Z. et al.
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