Differential Purification by Immunoaffinity Chromatography of Two Carboxy-Terminal Portion-Deleted Derivatives of Recombinant Human Interferon-γ from<i>Escherichia coli</i>

Honda, Susumu; Asano, Tsuneo; Kajio, Tomoko; Nakagawa, Shizue; Ikeyama, Shuichi; Ichimori, Yuzo; Sugino, Hiromu; Nara, Kiyoshi; Kakinuma, Atsushi; Kung, Hsiang‐Fu

doi:10.1089/jir.1987.7.145

Cited by 25 publications

(8 citation statements)

References 26 publications

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“…Complex aggregate formation and misfolding is to be avoided in the isolation and purification procedures. Solubilized rhIFN-γ has been purified using a variety of chromatographic and affinity techniques, including size exclusion gelfiltration [25], CM-Sepharose [27], MonoS [28], S-Sepharose [29], and cation exchange [30] chromatography, sometimes incorporating monoclonal antibodies [31], and affinity tags [32].…”

Section: Introductionmentioning

confidence: 99%

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

Reddy

Narala

et al. 2007

Protein Expression and Purification

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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.

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Section: Introductionmentioning

confidence: 99%

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

Reddy

Narala

et al. 2007

Protein Expression and Purification

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“…The results indicate that both PoIFN-y resolve, upon deglycosylation, into two polypeptide monomers with Mr of 16,500 and 14,500 (Fig.4, lane 3 and 5). The latter band corresponds most probably to a well documented Cterminal truncation of about 15 amino acids (Honda et al, 1987).…”

Section: Structure Of Rgpoifn-y Monomersmentioning

confidence: 85%

“…from proteolysis more efficiently, since carbohydrate moieties have an important function in protease resistance (Sareneva et al, 1995, Gahmberg andTolvanen, 1996). In spite of this, the larger band (Mr 16, 500) obtained upon treatment with N-Gycosydase F could be less than the expected Mr (16,800), which suggests a slight truncation, probably in the C-terminus, like the one known in E.coli-derived human IFN-y (Honda et al, 1987). This small truncation, as well as the larger one which results into the 14,500 band, are probably due to a proteolytic effect inside the cell, since the addition of protease inhibitors in the medium for 7 days produced no detectable differences in the electrophoretic pattern (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

Cencič¹,

Lefèvre²,

Koren³

et al. 1999

Animal Biotechnology

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Tetracycline-controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon-gamma in the RK-13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon-gamma (rGPoIFN-gamma) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN-gamma cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN-gamma (up to 16 microg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of 35S-Methionine, labelled rGPoIFN-gamma with natural leukocytic IFN-gamma after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN-gamma species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N-glycosydase F. The biological activity of rGPoIFN-gamma was in the same range as that of natural leukocytic PoIFN-gamma (2 x 10(6) U/mg). Eventually, this recombinant mammalian IFN-gamma should constitute a very useful substitute for leukocyte PoIFN-gamma in in vitro or in vivo experiments.

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“…Although the protocols of rhIFN-γ refolding have been established, little research on refolding assisted with chaperone (especially mini-GroEL) has been reported. Additionally, all of these protocols required large refolding reactors and quantities of buffer (11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

On-Column Refolding of Recombinant Human Interferon-γ with an Immobilized Chaperone Fragment

et al. 2003

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The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag. The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies. In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini-GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN-gamma was up to 3.93 x 10(6) IU with the immobilized mini-GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN-gamma folding in vitro was developed.

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Differential Purification by Immunoaffinity Chromatography of Two Carboxy-Terminal Portion-Deleted Derivatives of Recombinant Human Interferon-γ fromEscherichia coli

Cited by 25 publications

References 26 publications

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

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

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

On-Column Refolding of Recombinant Human Interferon-γ with an Immobilized Chaperone Fragment

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