Affinity purification of recombinant interferon-alpha on a mimetic ligand adsorbent.
A method for improved refolding and purification of recombinant human interferon-alpha (rh-IFN-alpha) from inclusion bodies is described. The optimal conditions of refolding were obtained by the addition of 0.5 M l-arginine to the refolding buffer. The rh-IFN-alpha was purified to near homogeneity utilizing a single-step chromatography on a mimetic dye-ligand matrix. Improved refolding, coupled to a single-column affinity purification strategy, resulted in a 10-fold increase in the yield of rh-IFN-alpha. This single-step purification protocol yielded approximately 50 mg of purified rh-IFN-alpha from 1 liter of shake flask culture. The rh-IFN-alpha prepared by this protocol was found to be essentially monomeric based on HPLC gel filtration and nonreducing SDS-PAGE. It had a specific activity of approximately 2.8 x 10(8) IU/mg, measured as inhibition of cytopathic effect of encephalomyocarditis virus on A549 human lung carcinoma cells.
Recombinant Gene Products Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
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Swaminathan
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Khanna
D000327:Adsorption; D000998:Antiviral Agents / Q000494:pharmacology; D001120:Arginine / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000187:drug effects; D004926:Escherichia coli; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007370:Interferon Type I / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D008024:Ligands; D008175:Lung Neoplasms / Q000473:pathology; D018716:Molecular Mimicry; D017510:Protein Folding; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D004396:Coloring Agents; D007370:Interferon Type I; D008024:Ligands; D011994:Recombinant Proteins; D001120:Arginine