Site-specific N-terminal labeling of proteins using sortase-mediated reactions

Abstract

For many proteins, the N- or the C-terminus make essential contributions to substrate binding, for protein-protein interactions, or for anchoring the proteins to a membrane. In other circumstances, at least one of the termini is buried within the protein, rendering it inaccessible to labeling. The possibility of selective modification of one of the protein’s termini may present unique opportunities for biochemical and biological applications. We describe sortase-mediated reactions to selectively label the N-terminus of a protein with a variety of functional groups. If sortase, the protein of interest, and a suitably functionalized label are available, the reactions usually require less than 3 hours.

The Inflammasome Adaptor ASC Induces Procaspase-8 Death Effector Domain Filaments

“…Because purified procaspase-8 DEDs lacking the catalytic domain spontaneously form oligomers, we expressed a Y8A mutant that reduced self-association and allowed purification of monomers when fused to an N-terminal His 6 -MBP tag and a C-terminal SUMO tag. The fusion protein also contained a C-terminal sortase recognition motif for labeling with a fluorescent probe (43). The fluorescently labeled His 6 -MBP-procaspase-8 DEDs-Y8A-SUMO fusion protein was incubated either alone or with two different concentrations of ASC PYD, ASC CARD, or full-length ASC, also prepared as monomers by For each sample, the percentage of speck-containing cells in the same expression window of ASC-GFP expression is shown and is plotted against the expression levels of the mCherry-tagged proteins, which were also determined by flow cytometry and are expressed as mean fluorescence intensity (MFI).…”

HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

“…Sortase reaction nucleophiles GGG-TAMRA, GGG-biotin, and GGG-Alexa Fluor 647 were synthesized as described (30,31). In-solution sortase labeling reactions with SrtA staph7M were performed overnight at 4°C in 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl; upon reaction completion, His 6 -tagged sortase was removed from the reaction mixture using nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen).…”

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

“…Prior to performing conjugation reactions using the continuous flow system, suitability of the designated protein as a substrate for sortase A should be evaluated in small batch reactions. Detailed protocols for production of sortases, sortase substrate design, peptide synthesis, and sortase reactions in solution are provided in previous protocols 20,21 . Sortase A immobiliziation has been previously described using a different strategy than detailed in this protocol 22 .…”