Effect of oxidative stress on the production of recombinant human interferon-gamma in Escherichia coli.
To reduce the degradation pathway of rhIFN-gamma (recombinant human interferon-gamma), the susceptibility against oxidative stress during fermentation in Escherichia coli cells and in purification process were investigated. Fermentations of recombinant E. coli were performed at 5, 30 and 60% DO (dissolved oxygen) concentrations. The expressed rhIFN-gamma is purified in three-step chromatography processes with 99.9% purity under reducing and non-reducing conditions. Recombinant IFN-gamma production, carbonyl and dimeric contents and antiviral-specific activity of purified protein were monitored. The increase in carbonyl content was taken to be an indicator of protein oxidation. DO concentration did not show any noticeable effect on rhIFN-gamma production. During fermentation, the carbonyl-group content showed no significant increase at 5 and 30% DO but a 10-fold increase at 60% DO concentration was observed. The antiviral-specific activity of purified rhIFN-gamma was decreased 35 and 69% at 30 and 60% DO concentrations respectively. After the purification process, under non-reducing conditions, the activity of purified protein decreased to 30% of its original value. The degree of dimeric forms of protein was found to depend on the O2 concentration during fermentation and purification.
Mohammadian-Mosaabadi
Jafar
J
Biochemistry Group, Department of Biology, Faculty of Science, Tarbiat Modarres University, Tehran, PO Box 14155-4838, Iran.
Naderi-Manesh
Hossein
H
Maghsoudi
Nader
N
Khalilzadeh
Rasoul
R
Shojaosadati
Seyd Abbas
SA
Ebrahimi
Marzieh
M
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018384:Oxidative Stress / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D007371:Interferon-gamma; D010100:Oxygen