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Related Experiment Videos

An immunoaffinity purification procedure for SV40 large T antigen.

V Simanis, D P Lane

    Virology
    |July 15, 1985
    PubMed
    Summary

    Researchers developed a rapid method to purify SV40 large T antigen using a hybrid virus and immunoaffinity chromatography. This technique yields milligram amounts of pure antigen for sensitive detection assays.

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    Area of Science:

    • Molecular Biology
    • Virology
    • Biochemistry

    Background:

    • Simian virus 40 (SV40) large T antigen is a crucial viral protein involved in DNA replication and cellular transformation.
    • Efficient purification of biologically active SV40 large T antigen is essential for detailed functional studies and diagnostic applications.

    Purpose of the Study:

    • To develop a rapid and high-yield purification protocol for SV40 large T antigen.
    • To characterize the biological activities of the purified protein.
    • To establish a sensitive assay for detecting SV40 large T antigen.

    Main Methods:

    • Utilized an adenovirus-SV40 hybrid virus for overproduction of SV40 large T antigen.
    • Employed immunoaffinity chromatography with an anti-large T monoclonal antibody coupled to protein A Sepharose for purification.
    • Assessed protein purity, yield, and biological activities including p53-binding, ATPase, and DNA-binding.
    • Developed a competition radioimmunoassay using iodinated pure large T antigen.

    Main Results:

    • Achieved a rapid purification procedure completed within one day.
    • Isolated milligram quantities of SV40 large T antigen with excellent yield.
    • The purified protein retained essential biological functions: p53-binding, ATPase activity, and sequence-specific DNA-binding.
    • The pure protein was highly antigenic and stable for iodination, enabling sensitive radioimmunoassay development.
    • The radioimmunoassay reliably detected nanogram amounts of SV40 large T antigen.

    Conclusions:

    • A fast and efficient method for purifying functional SV40 large T antigen has been established.
    • The purified antigen is suitable for developing sensitive diagnostic and research tools.
    • This protocol facilitates further investigation into the molecular mechanisms of SV40 large T antigen.

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