Controlled levels of protein modification through a chromatography-mediated bioconjugation

Abstract

This article introduces a method to control levels of protein modification through a chromatography-mediated bioconjugation.

Capture and Recycling of Sortase A through Site‐Specific Labeling with Lithocholic Acid

“…However, most βCD guests have binding affinities in the order of 10 3 –10 5 m −1 , which are too weak for efficient capture and separation unless multivalent coordination can be exploited . In our hands, common βCD binders, such as adamantane and azo compounds, could be used for chromatographic separation, but proved insufficient for complete resin capture. Lithocholic acid (LA) stands out as one of the strongest monovalent binders to βCD with a reported affinity of about 10 6 m −1 .…”

“…To capture and recover LA‐modified SrtA 7M selectively, we functionalized sepharose resin with βCD (see the Supporting Information for details). As a demonstration of the concept, we purposefully used a 56:44 mixture of unmodified to LA‐modified Pro‐SrtA 7M, even though higher levels of modification were achievable (see above).…”

Capture and Recycling of Sortase A through Site‐Specific Labeling with Lithocholic Acid

“…[20] However,m ost bCD guests have binding affinities [21] in the order of 10 3 -10 5 m À1 ,w hich are too weak for efficient capture and separation unless multivalent coordination can be exploited. [22] In our hands,common bCD binders, such as adamantane and azo compounds, [23] could be used for chromatographic separation, but proved insufficient for complete resin capture.L ithocholic acid (LA) stands out as one of the strongest monovalent binders to bCD with ar eported affinity of about 10 6 m À1 . [24] Although this binding affinity could be influenced by attached proteins,w ei dentified LA as an ideal affinity handle for the quantitative capture of modified proteins.H owever,t he extremely low solubility of LA in water (0.05 mm at 25 8 8C [25] )h as complicated direct bioconjugation, and so far only very few examples of synthetically attaching LA to proteins have been demonstrated.…”

“…To capture and recover LA-modified SrtA 7M selectively, we functionalized sepharose resin with bCD [23] (see the Supporting Information for details). As ad emonstration of the concept, we purposefully used a5 6:44 mixture of unmodified to LA-modified Pro-SrtA 7M, even though higher levels of modification were achievable (see above).…”

Supramolecular strategies for protein immobilization and modification

“…Since that initial report, other examples of cyclodextrin-mediated protein purification have been reported, [13] including a more general approach reported by Francis and coworkers in 2015. [14] Proteins were modified heterogeneously with an azobenzene derivative that has a high binding affinity for b-cyclodextrin. Chromatographic purification of the proteins with a cyclodextrin column achieved the separation of unmodified, singly, and doubly modified proteins.…”