Production of interferon-alpha in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins.
Escherichia coli TG1 transformed with a temperature-regulated interferon-alpha expression vector was grown to high cell density in defined medium containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high cell density cultures resulted in the production of approximately 4 g interferon-alpha/l culture broth. Interferon-alpha was produced exclusively in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified interferon-alpha was approximately 300 mg/l with respect to the original high cell density culture broth (overall yield of approximately 7.5% active interferon-alpha). The purified recombinant interferon-alpha was found by different criteria to be predominantly monomeric and possessed a specific bioactivity of approximately 2.5 x 10(8) IU/mg based on viral cytopathic assay.
Babu
K R
KR
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
Swaminathan
S
S
Marten
S
S
Khanna
N
N
Rinas
U
U
D010582:Bacteriophage lambda / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D002479:Inclusion Bodies; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013678:Technology, Pharmaceutical / Q000191:economics
D016898:Interferon-alpha; D011994:Recombinant Proteins