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

Sven T Sowa; Albert Galera-Prat; Sarah Wazir; Heli I Alanen; Mirko M Maksimainen; Lari Lehtiö

doi:10.1016/j.xpro.2022.101147

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

STAR Protoc. 2022 Jan 29;3(1):101147. doi: 10.1016/j.xpro.2022.101147. eCollection 2022 Mar 18.

Authors

Sven T Sowa¹, Albert Galera-Prat¹, Sarah Wazir¹, Heli I Alanen¹, Mirko M Maksimainen¹, Lari Lehtiö¹

Affiliation

¹ Faculty for Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, 90220 Oulu, Finland.

Abstract

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).

Keywords: High Throughput Screening; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification; Signal Transduction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Diphosphate
Biological Assay*
Glycoside Hydrolases
Proteins*

Substances

Proteins
Adenosine Diphosphate
Glycoside Hydrolases