Purification and properties of a novel recombinant human hybrid interferon, delta-4 alpha 2/alpha 1.
The human interferon (huIFN) delta-4 alpha 2(5-62)/alpha 1(64-166) is a genetically engineered hybrid that consists of residues 5-62 of huIFN alpha 2 and residues 64-166 of huIFN alpha 1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN alpha subtypes. This novel recombinant hybrid was purified from Escherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17,800 and 17,100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7 x 10(7) units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) on various cell lines of different histological origin appeared to be more comparable to that of huIFN alpha 1 than huIFN alpha 2. The data suggest that huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) hybrid may be a useful tool for understanding huIFN structure-function relations.
D000998:Antiviral Agents; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis; D006131:Growth Inhibitors; D006801:Humans; D007370:Interferon Type I / Q000302:isolation & purification* / Q000494:pharmacology; D008970:Molecular Weight; D011994:Recombinant Proteins; D012995:Solubility; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis
D000998:Antiviral Agents; D004220:Disulfides; D006131:Growth Inhibitors; D007370:Interferon Type I; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds