Mayrhofer Peter, Anneser Markus R, Schira Kristina, Sommer Carina A, Theobald Ina, Schlapschy Martin, Achatz Stefan, Skerra Arne
Chair of Biological Chemistry, School of Life Sciences, Technical University of Munich, 85354, Freising, Germany.
Nat Commun. 2024 Dec 18;15(1):10693. doi: 10.1038/s41467-024-55212-y.
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.
亲和层析是从细胞提取物或培养上清液中快速纯化蛋白质的首选方法。在此，我们展示了光响应性偶氮标签，这是一种包含对（苯偶氮）-L-苯丙氨酸（Pap）的短肽，其侧链可通过温和紫外光照射从反式基态转变为亚稳态顺式构型。由于只有反式Pap对α-环糊精（α-CD）具有强亲和力，带有偶氮标签的蛋白质在日光下或黑暗中选择性地结合到α-CD层析基质上，但在355nm波长光照柱子时，在生理缓冲液流动下会快速洗脱。我们展示了对多种蛋白质（包括酶和抗体片段）进行光控一步纯化（称为激发成像法），无需使用通常所用的竞争剂或苛刻的缓冲条件。虽然迄今为止亲和层析一直受化学相互作用支配，但引入电磁辐射控制作为一种物理原理为这种广泛应用的分离技术增添了新的维度。
