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9.9: Isolating Transcription Factors

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    4890
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    Transcription factors are extraordinarily diverse, and any one factor represents only a tiny fraction of the protein molecules present in the cell. This page describes how one can isolate and purify such rare molecules.

    Example: Isolating the lac Repressor

    An E. coli cell contains only 10-20 copies of the lac repressor. This represents a ratio of only 1 molecule in 50,000 protein molecules in the cell. However, the specificity of the lac repressor for the DNA sequence of the operator provides a mechanism for fishing it out of the mixture.

    Affinity Chromatography

    Fig. 9.9.1 Affinity Chromatography

    The following procedure is carried out for an affinity chromatograph:

    • Learn the sequence to which the repressor binds (by footprinting).
    • Synthesize a segment of DNA containing the sequence.
    • Attach this artificial molecule to beads of an inert, solid medium (the matrix).
    • Pour an extract of E. coli cells over the beads.
    • Only molecules specific for the DNA sequence — in this case, molecules of the lac repressor — will bind to the beads.
    • After irrelevant protein molecules have passed through the column, wash the beads with a buffer that will release the lac repressor molecules so they can be studied.

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