Protein Conjugation via SpyStapler‐Mediated SpyTag/BDTag Coupling

Abstract

Genetically encoded peptide‐protein coupling reactions, such as the SpyTag/SpyCatcher chemistry, are recent additions to the expanding toolbox of protein bioconjugation. The alternative three‐component ligation system, e.g., SpyStapler‐mediated SpyTag/BDTag coupling, retains most advantages of the Tag/Catcher chemistry, yet requires only two short peptide tags in the genetic fusion for side‐chain ligation. Not only does this facilitate the construction of large protein conjugates directly from as‐expressed protein components with minimal disruption to their function, but it also provides an entirely new mode of bioconjugation via mechanical bonding, which could impart additional functional benefits such as improved activity and enhanced stability to the conjugate. Such features are attractive for improving the pharmacokinetic performance of protein therapeutics. Herein we describe protocols for SpyStapler‐mediated SpyTag/BDTag coupling for protein bioconjugation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Conjugation via isopeptide bond Support Protocol: Purification by size‐exclusion chromatography Basic Protocol 2: Conjugation via mechanical bond

Rational Design and Cellular Synthesis of Proteins with Unconventional Chemical Topology

“…[ 45‐46 ] Based on the three‐component system of SpyTag/BDTag/SpyStapler, we developed a biologically enabled active template for the precise synthesis of protein heterocatenanes by rewiring the connectivity between the three segments (Figure 4). [ 47‐48 ] By connecting the C‐ and N‐termini of SpyStapler to acquire the cyclic protein and fusing the BDTag and SpyTag to acquire the telechelic protein, protein heterocatenane would then be formed from the entwining of the two components and the cyclization of the telechelic protein by isopeptide bond.…”

Spatiotemporally Controlled Photolabeling of Genetically Unmodified Proteins in Live Cells

“…Particularly in combination with the fast-developing superresolution fluorescence microscopy 1,2 and single-molecule imaging, 3,4 live-cell fluorescent labeling of the protein of interest (POI) has become a powerful tool to obtain fundamental insights into molecular and cellular processes. 5−8 Many methods have been developed in this context, including fusing fluorescent proteins on the POI, 9,10 genetically engineering the POI with affinity peptide tags 11,12 and bioorthogonal chemistry handles, 13,14 and ligand-directed proximity modifications. 15−17 These elegant technologies enable the visualization of dynamic life processes with molecular resolution.…”

“…Live cell protein labeling is essential for elucidating protein functions and dynamics in the cells’ natural habitat. Particularly in combination with the fast-developing superresolution fluorescence microscopy , and single-molecule imaging, , live-cell fluorescent labeling of the protein of interest (POI) has become a powerful tool to obtain fundamental insights into molecular and cellular processes. − Many methods have been developed in this context, including fusing fluorescent proteins on the POI, , genetically engineering the POI with affinity peptide tags , and bioorthogonal chemistry handles, , and ligand-directed proximity modifications. − These elegant technologies enable the visualization of dynamic life processes with molecular resolution. However, it remains a significant challenge to label the POI in live cells with spatiotemporal control when attempting to resolve the dynamic function or track the intracellular fate of a protein in a specific time period at a specific location.…”