Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe

Abstract

Summary Here, we describe a protocol to set up a screening assay for ADP-ribosyl binding proteins including proteins that possess O-glycosidase or N-glycosidase activities. The FRET-based assay measures the interaction of any ADP-ribosyl binding protein fused to CFP with a cysteine-ADP-ribosylated GAP-tag fused to YFP. Recombinant PtxS1 and PARP2 are used to mono-ADP-ribosylate and poly-ADP-ribosylate the GAP-tag. The protocol does not require specialized compounds or substrates, making it accessible and easy to adapt in any laboratory or for other proteins of interest. For complete details on the use and execution of this profile, please refer to Sowa et al. (2021) .

Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening

“…All plasmids and proteins used were expressed and purified as previously described (Alhammad et al, 2021;Dasovich et al, 2022;Sowa et al, 2021Sowa et al, , 2022. All compounds were repurchased from MolPort except for compounds 6 and 10, which were repurchased from Chem-Div.…”

Design, synthesis and evaluation of inhibitors of the SARS-CoV-2 nsp3 macrodomain

“…A FRET method was utilized for the profiling of 15c a panel of human and viral macrodomains to determine their specificity [22] , [27] , [28] . The assay is based on the site-specific introduction of cysteine-linked mono-ADP ribose to the C-terminal Gαi peptide (GAP) by Pertussis toxin subunit1 (PtxS1) fused to YFP.…”

Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening

“…FRET method was utilized for the profiling of MCD-628 a panel of human and viral macrodomains to determine their specificity (30,36). The assay is based on the site-specific introduction of cysteine-linked mono-ADP-ribose to the C-terminal Gαi peptide (GAP) by Pertussis toxin subunit1 (PtxS1) fused to YFP.…”

“…All plasmids and proteins used were expressed and purified as previously described (30, [35][36][37]. All compounds were repurchased from MolPort except for compounds 6 and 10, which were repurchased from ChemDiv.…”