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Producing Soluble Recombinant RNases and Assays to Measure Their Interaction with Interferon-γ In Vitro□

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Nuclease Methods and Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 160))

Abstract

Many cytokines alter the half-lives and stabilities of mRNAs for other cytokines (1) and enzymes that may be related to disease progression. For example, IL-1β stabilizes the mRNA for human interstitial collagenase-1 in normal fibroblasts, but not in breast cancer cells (2). Altering mRNA stability through direct control of specific RNase activity is a rapid way for cells to respond to environmental changes. Small differences in half-life can have dramatic effects on expressed products; a sixfold increase in μ heavy-chain mRNA half-life in differentiated B-cells largely accounted for a 100-fold increase in its messenger concentration compared to the undifferentiated cells (3). The high specific activity of RNases makes them ideal cellular targets for interaction with cytokines (4).

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Notes

  1. 1.

    *

    A “back of the envelope” calculation, based on a specific activity of 5×107 interferon units/mg protein, a molecular weight of 28,000 for the dimeric protein, 106 cells/mL in the IFN assay and a 10% binding efficiency to the cell receptors, would suggest that 100–400 molecules/cell are necessary to exert an antiviral effect

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  1. Human Biological Chemistry and Genetics, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX

    Catherine H. Schein

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  1. University of Texas Medical Branch, Galveston, TX

    Catherine H. Schein

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Schein, C.H. (2001). Producing Soluble Recombinant RNases and Assays to Measure Their Interaction with Interferon-γ In Vitro□. In: Schein, C.H. (eds) Nuclease Methods and Protocols. Methods in Molecular Biology™, vol 160. Humana Press. https://doi.org/10.1385/1-59259-233-3:113

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  • DOI: https://doi.org/10.1385/1-59259-233-3:113

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-679-6

  • Online ISBN: 978-1-59259-233-3

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