Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins

Abstract

A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs.

Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A

“…Here we aimed to extend this ligating capability to label free N-terminal glycine-containing proteins in whole-cell lysates with a tagged (fluorophore/biotin) LPXTG depsipeptide SrtA substrate. To optimize labeling with SrtA in whole-cell lysates, we generated a high-activity S. aureus SrtA pentamutant (15, 25) and a synthetic TAMRA-ALPET-Haa depsipeptide (Haa = 2-hydroxyacetamide), and tested a range of concentrations through an in-gel fluorescence workflow (Fig. 1, supplemental Fig.…”

“…Samples (20–300 μg protein content) were adjusted to 1 mg/ml protein concentration in SrtA buffer. The SrtA reaction was carried out by adding 75 μ m ALPET-Haa substrate and 0.1 μ m SrtA pentamutant (a kind gift from Dr. M. Jamshidiha, Imperial College London, expressed and purified from Addgene (MA) plasmid 86962 (15)) to each sample and incubating with mild shaking overnight (16 h) at 4 °C. The reaction was stopped by addition of 5 m m EDTA.…”

Integrating single-molecule spectroscopy and simulations for the study of intrinsically disordered proteins

“…Sortase A catalyzes the ligation of an “LPETG” motif with a “GGG” motif [ 191 , 192 ]. In this way, a linker containing a fragment of a protein that harbors a single cysteine can be utilized to enable maleimide chemistry on the sole cysteine residue [ 193 ]. The rest of the protein that contains multiple cysteines can be ligated to the singular-cysteine containing protein fragment.…”

Broadening the scope of sortagging

“…For N-terminal protein labeling, Sarpong et al developed a procedure that combines TEV protease cleavage, SML and affinity purication. 124 The protein of interest with TEV recognition motif is cleaved yielding an N-terminal glycine. A label with sortase recognition sequence and affinity tag for isolation of the modied protein is subsequently linked.…”