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.
Swaminathan
S
S
Recombinant Gene Products Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Khanna
N
N
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