title: Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography.
abstract: Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), 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-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.
authors.affiliation: Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Reddy
authors.forename: Srinivasa G
authors.initials: SG
authors.lastname: Reddy
authors.forename: Venkata R
authors.initials: VR
authors.lastname: Narala
authors.forename: Sangita S
authors.initials: SS
authors.lastname: Majee
authors.forename: Sudhakar
authors.initials: S
authors.lastname: Konda
authors.forename: Sripad
authors.initials: S
authors.lastname: Gunwar
authors.forename: Raju C
authors.initials: RC
authors.lastname: Reddy
mesh_terms: D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003470:Culture Media; D017931:DNA Primers; D006801:Humans; D007371:Interferon-gamma / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins; D015183:Restriction Mapping; D012995:Solubility
chemical_list: D003470:Culture Media; D017931:DNA Primers; D011994:Recombinant Proteins; D007371:Interferon-gamma