Expression, purification, and characterization of a membrane-associated cyclic oligo-adenylate degrader from Sulfolobus islandicus

Abstract

Summary Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus , membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020) .

A short prokaryotic argonaute cooperates with membrane effector to confer antiviral defense

“…Then, the plasmids were transformed into S. islandicus E233S1 to generate the corresponding expression strains (Table S2). The detailed procedures of S. islandicus E233S1 cultivation and transformation were described previously (Zhao et al, 2021). All the primers were synthesized by Tingke (Beijing, China).…”

“…SCVU plates were prepared before the assay (Zhao et al, 2021). To determine viral titer in the storage buffer, the storage buffer was serially diluted and mixed with 4 mL fresh S. islandicus C1C2 culture (OD 600 around 0.2).…”

“…S. islandicus E233S1 (Deng et al, 2009) was used as the genetic host to express the SiAgo system. The strain was grown at 78 °C in SCVU medium as indicated previously (basic salts medium supplemented with 0.2% (w/v) sucrose, 0.2% (w/v) casamino acids, 0.2% (w/v) uracil and a vitamin mixture) (Zhao et al, 2021). The strains carrying a pSeSD-derived expression plasmid were grown in SCV medium lacking uracil.…”