Expression of human immune interferon cDNA in E. coli and monkey cells

Gray, Patrick W.; Leung, David W. M.; Pennica, Diane; Yelverton, Elizabeth; Najarian, Richard C.; Simonsen, Christian C.; Derynck, Rik; Sherwood, Pamela J.; Wallace, Donald MackenzieHG; Berger, Shelby L.; Levinson, Arthur D.; Goeddel, David V.

doi:10.1038/295503a0

Cited by 665 publications

(207 citation statements)

References 67 publications

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“…These characteristics were consistent with homology to y-interferon. Restriction mapping indicated that the clones of interest were related to each other but not to the known y-interferon mRNA sequence immediately adjacent to the primer (18). A partial map of the "new" portion of the lymphocyte cDNA is shown at the top of Fig.…”

Section: Resultsmentioning

confidence: 99%

The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA.

Eschenfeldt¹,

Berger²

1986

Proc. Natl. Acad. Sci. U.S.A.

184

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Clones for human prothymosin a have been identified in cDNA libraries from staphylococcal enterotoxin A-stimulated normal human lymphocytes and from simian virus 40-transformed fibroblasts. The 1198-base-pair fibroblast clone has been sequenced. The encoded protein is highly acidic (54 residues out of 111) and shares >90% sequence homology with rat prothymosin a. The peptide "hormone" thymosin al appears at positions 2-29 of the prothymosin a amino acid sequence. There is no N-terminal signal peptide.

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Section: Resultsmentioning

confidence: 99%

The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA.

Eschenfeldt¹,

Berger²

1986

Proc. Natl. Acad. Sci. U.S.A.

184

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“…The first reported recombinant interferon-γ [44] consisted of 146 amino acids with the Nterminal portion of the molecule commencing with the sequence Cys-Tyr-Cys. Later, however, it was shown that the native human interferon-γ lacks the Cys-Tyr-Cys sequence at the Nterminal.…”

Section: Cloning and Expressionmentioning

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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“…In the current study it is shown that a basic peptide which corresponds to the sequence between residues 127 and 136 [37] at the C-terminus of IFN-g can inhibit the binding of this cytokine to cultured EAhy.926 cells. The C-terminal region is not involved in binding to the extracellular portion of the IFN-g receptor [38].…”

Section: Discussionmentioning

confidence: 67%

Examination of the mechanism by which heparin antagonizes activation of a model endothelium by interferon-gamma (IFN-γ)

Douglas

Rix

Dark

et al. 1997

Clinical and Experimental Immunology

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SUMMARYIFN-g increases the potential immunogenicity of vascular endothelial cells by up-regulation of intercellular adhesion molecule-1 (ICAM-1) and class I MHC antigen expression and by induction of class II MHC antigens and certain chemokines. In this study the mechanism by which the glycosaminoglycan (GAG) heparin antagonizes the activation of a model endothelium by IFN-g was investigated. Radioligand binding assays demonstrated that total binding of 125 I-IFN-g to the EAhy.926 endothelial hybridoma cell line was reduced in the presence of heparin or heparan sulphate (HS); the structurally dissimilar GAG chondroitin sulphate had no effect. Treatment of the cells with chlorate, a metabolic inhibitor of GAG sulphation, was found to reduce both the subsequent binding of IFN-g and its ability to induce expression of class II MHC antigens. Treatment with heparinase II dramatically reduced the binding of IFN-g, while chondroitin ABC lyase had no effect. A cationic peptide from the C-terminal region of IFN-g was also found to reduce binding of intact IFN-g to the cells. These results appear to demonstrate that IFN-g is sequestered at the surface of endothelial cells by electrostatic interaction between specific basic amino acid residues and sulphated domains on HS, the most abundant endothelial GAG. This interaction is competitively inhibited by heparin, which is structurally related to HS. These observations are consistent with the model that IFN-g is bound by membrane-associated HS before engagement with the high-affinity receptor and signal transduction. Inhibition of the interaction between proinflammatory cytokines and membrane-associated GAG molecules may provide a mechanism for inducing clinically useful immunosuppression.

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Expression of human immune interferon cDNA in E. coli and monkey cells

Cited by 665 publications

References 67 publications

The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA.

The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA.

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

Examination of the mechanism by which heparin antagonizes activation of a model endothelium by interferon-gamma (IFN-γ)

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