Protein purification with light via a genetically encoded azobenzene side chain

Peter Mayrhofer, Markus R. Anneser, Kristina Schira, Carina A. Sommer, Ina Theobald, Martin Schlapschy, Stefan Achatz and Arne Skerra ()
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Peter Mayrhofer: Technical University of Munich
Markus R. Anneser: Technical University of Munich
Kristina Schira: Technical University of Munich
Carina A. Sommer: Technical University of Munich
Ina Theobald: Technical University of Munich
Martin Schlapschy: Technical University of Munich
Stefan Achatz: Technical University of Munich
Arne Skerra: Technical University of Munich

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: Abstract Affinity chromatography is the method of choice for the rapid purification of proteins from cell extracts or culture supernatants. Here, we present the light-responsive Azo-tag, a short peptide comprising p-(phenylazo)-L-phenylalanine (Pap), whose side chain can be switched from its trans-ground state to the metastable cis-configuration by irradiation with mild UV light. Since only trans-Pap shows strong affinity to α-cyclodextrin (α-CD), a protein exhibiting the Azo-tag selectively binds to an α-CD chromatography matrix under daylight or in the dark but elutes quickly under physiological buffer flow when illuminating the column at 355 nm. We demonstrate the light-controlled single-step purification – termed Excitography – of diverse proteins, including enzymes and antibody fragments, without necessitating competing agents or harsh buffer conditions as normally applied. While affinity chromatography has so far been governed by chemical interactions, introducing control by electromagnetic radiation as a physical principle adds another dimension to this widely applied separation technique.

Date: 2024
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DOI: 10.1038/s41467-024-55212-y

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