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Related Experiment Videos

Expression of enzymatically active reverse transcriptase in Escherichia coli.

N Tanese, M Roth, S P Goff

    Proceedings of the National Academy of Sciences of the United States of America
    |August 1, 1985
    PubMed
    Summary

    Researchers engineered gene fusions to produce large quantities of Moloney murine leukemia virus reverse transcriptase. This method yields high enzyme activity, even with altered protein termini, aiding in enzyme production for research.

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    Area of Science:

    • Molecular Biology
    • Virology
    • Biochemistry

    Background:

    • Murine retroviral reverse transcriptase is an ~80 kDa monomeric protein.
    • This enzyme is encoded by the central region of the viral pol gene.

    Purpose of the Study:

    • To develop a method for producing large quantities of murine retroviral reverse transcriptase.
    • To investigate the expression of functional reverse transcriptase from engineered gene fusions.

    Main Methods:

    • Constructed gene fusions between the Escherichia coli trpE gene and Moloney murine leukemia virus pol gene sequences.
    • Included the complete coding region for mature reverse transcriptase and variable additional sequences in the fusions.
    • Analyzed the expression and activity of the resulting fusion proteins.

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    Main Results:

    • Several gene fusion constructs expressed high levels of reverse transcriptase activity.
    • Significant enzyme activity was observed even when the NH2 and COOH termini of the expressed protein only approximated authentic termini.

    Conclusions:

    • Gene fusion technology provides an effective strategy for overproducing murine retroviral reverse transcriptase.
    • The expressed enzyme retains significant activity despite potential modifications at its termini, facilitating its use in research applications.