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Cyanogen bromide-activated coupling: DNA catalytic chromatography purification of EcoRI endonuclease

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Abstract

A method to purify enzymes utilizing their specific biological affinity and catalytic specificity is described. For this chromatographic technique, an enzyme binds immobilized substrate coupled to a column in the absence of a cofactor required for catalysis but permissive for substrate binding. After washing, the missing cofactor is added to the column mobile phase, and the enzyme converts substrate into product and elutes from the column. A single-step purification of EcoRI endonuclease using a sequence-specific DNA column (containing the GAATTC motif coupled to cyanogen bromide-activated Sepharose 4B) binds EcoRI in the absence of Mg2+ and elutes when Mg2+ is applied in a highly purified state. Although the method described is specific for EcoRI, it can be readily modified for the purification of DNA polymerases and other enzymes. Furthermore, many of the same materials are also used for transcription factor purification. This protocol can be completed within 4–6 d.

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Figure 1
Figure 2: EcoRI eluted by catalytic chromatography and loaded on a 1.5% agarose electrophoresis gel stained with ethidium bromide.
Figure 3: Cyanogen bromide-activated coupling: DNA catalytic chromatography purification of EcoRI endonuclease
Figure 4: For 20,000 U ml−1 EcoRI, 50% digestion occurred at 3,750-fold dilution.
Figure 5: SDS-PAGE (12%) was performed on proteins purified by catalytic chromatography and silver stained.

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Authors and Affiliations

  1. Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, 78249, Texas, USA

    Linda I Nagore, Daifeng Jiang, Shoulei Jiang, YanWen Zhou, Magda Loranc & Harry W Jarrett

  2. Department of Molecular Science, University of Tennessee Health Science Center, Memphis, 38163, Tennessee, USA

    Sucharita Mitra

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  1. Linda I Nagore
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  2. Sucharita Mitra
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  3. Daifeng Jiang
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  4. Shoulei Jiang
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  6. Magda Loranc
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  7. Harry W Jarrett
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Correspondence to Harry W Jarrett.

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Nagore, L., Mitra, S., Jiang, D. et al. Cyanogen bromide-activated coupling: DNA catalytic chromatography purification of EcoRI endonuclease. Nat Protoc 1, 2909–2915 (2006). https://doi.org/10.1038/nprot.2006.439

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