Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

Abstract

Increasing therapeutic applications for recombinant human interferon-γ (rhIFN-γ), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 ™ matrix in the purification of rhIFN-γ from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-γ monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50-100 μg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg g −1 cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 to 4 × 10 7 IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-γ in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

Expression, purification and characterization of human interferon-γ in Pichia pastoris

“…Activity assay of hIFN-γ. The antiviral activity of rhIFN-γ was evaluated as previously described (17). The human carcinoma cell line, HEP2C, was challenged with the encephalomyocarditis virus (EMCV) in the presence or absence of hIFN-γ.…”

Specific Expression of Interferon-�� Induced by Synergistic Activation Mediator-Derived Systems Activates Innate Immunity and Inhibits Tumorigenesis

“…Although the most conventional model in industrial production of overexpressing recombinant human interferon-γ is in E. coli, the cytoplasmic inclusion bodies and endotoxin secreted by the bacteria have been found to accumulate in the compound, making interferon-γ production frequently subject to substandard protein purity and higher background protein. In particular, the formation of protein maintaining conserved structure and complex functions is affected [11,12]. In addition, although recombinant human interferon-γ was shown to be involved in antiproliferative [13] and apoptotic [14] effects against cancer cells, it also needs an exogenous supplement.…”

A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity

“…La cuantificación por ambos métodos arrojó valores similares. El rendimiento obtenido, alrededor de 40 U/ml no es óptimo comparado con cultivos en alta densidad expresando por ejemplo, IFN-γ en células de mamíferos, que están en el orden de 10 5 U/ml [171], pero para la escala de este trabajo fue suficiente. De todos modos en nuestro laboratorio se está seleccionando la línea celular que exprese establemente el IFN-λr.…”