Fig. 1: Light-controlled protein purification via Excitography.
From: Protein purification with light via a genetically encoded azobenzene side chain
A A protein mixture or cell extract containing the protein of interest (POI) equipped with the Azo-tag is applied to an α-CD affinity column. With the Pap side chain in the lower-energy trans-configuration, the POI binds to the α-CD groups of the column matrix whereas all contaminating proteins are quickly washed out. Subsequent illumination with near UV light triggers configurational switch of the azobenzene moiety to the more bulky cis-state which effects desorption from the affinity matrix. Thus, the POI is eluted in the chromatography buffer of choice, without adding reagents, salts or change of pH. B Interplay between the dynamic chemical equilibrium of non-covalent complex formation between α-CD and the Azo-compound (either free Pap or the Azo-tagged POI) according to the Law of Mass Action and the light-induced cis/trans-isomerization of the latter. The photostationary composition comprises a variable cis:trans-ratio depending on the wavelength and mode of irradiation, which may either stay fixed (when washing in the dark) or can be dynamic (that is, under continuous illumination). Due to the almost undetectable affinity between α-CD and the Azo-group in the cis-state, this isomer elutes with the buffer flow essentially without retardation (here depicted with a thick vertical arrow). On the other hand, in the trans-configuration – with its moderate KD value of around 100 µM – the Azo-compound is strongly retarded on the affinity matrix due to tight complex formation with the α-CD groups. However, there will always be some noticeable migration (thin vertical dotted arrow) due to the continuous re-equilibration between free and bound Azo-compound at each theoretical plate of the column (here depicted with horizontal dotted lines) during the buffer flow, which is governed by the dissociation and association kinetics. (For further details and the practical influence of illumination during the washing step, after sample load, see Supplementary Fig. S14).
