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Affinity purification of sequence-specific DNA binding proteins.

J T Kadonaga, R Tjian

    Proceedings of the National Academy of Sciences of the United States of America
    |August 1, 1986
    PubMed
    Summary
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    This study presents a rapid and efficient method for purifying sequence-specific DNA binding proteins using custom DNA affinity resins. The technique significantly enhances protein purity, enabling the isolation of rare transcription factors like Sp1.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Protein Chemistry

    Background:

    • Sequence-specific DNA binding proteins play crucial roles in gene regulation.
    • Efficient purification of these proteins is essential for studying their function.
    • Existing methods can be time-consuming and may yield low purity.

    Purpose of the Study:

    • To develop a fast and effective method for the affinity purification of sequence-specific DNA binding proteins.
    • To enable the isolation of rare and low-abundance DNA binding proteins.

    Main Methods:

    • Chemically synthesized oligodeoxynucleotides with specific recognition sites were annealed and ligated.
    • The resulting DNA oligomers were covalently coupled to Sepharose CL-2B using cyanogen bromide to create an affinity resin.

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  • Partially purified protein fractions were incubated with competitor DNA and passed through the DNA-Sepharose resin.

    • Main Results:

    • The method achieved high-fold purification (500- to 1000-fold) for transcription factor Sp1, reaching an estimated 90% homogeneity with a 30% yield.
    • Sequential affinity chromatography steps demonstrated the effectiveness of the purification strategy.
    • Tandem affinity columns allowed for simultaneous purification of multiple DNA binding proteins from a single extract.

    Conclusions:

    • This DNA affinity purification method is a robust technique for isolating sequence-specific DNA binding proteins.
    • The approach is particularly valuable for purifying rare proteins, such as Sp1 and CAAT-binding transcription factor.
    • The method offers a significant advancement in protein purification for molecular biology research.