2xyt medium

Proteins were expressed in E. coli BL21 CodonPlus-RIL (Stratagene) in cultures up to 1 liter using 2xYT medium (Roth). Protein expression was induced with 0.2 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) at an optical density of 0.6–0.8 and performed at 18 °C overnight. Afterwards, expression cultures were divided into several aliquots, harvested by centrifugation, flash frozen, and stored at −80 °C until further use.

Cas13a was expressed from p2CT-His-MBP-Lbu_C2c2_WT, which was obtained from the Jennifer Doudna lab via Addgene (Addgene plasmid No. 83482; RRID:Addgene_83482; http://n2t.net/addgene:83482) [22 (link)]. The His-MBP tagged Cas13a plasmid was transformed into Rosetta E. coli. LB-carbenicillin plates were plated with a glycerol stock of the cells (50% glycerol, 50% saturated culture) and incubated at 37°C overnight. Single colonies were picked and resuspended in a preculture of 50 mL LB-medium supplemented with 100 μg/ml of the antibiotic carbenicillin. 10 mL of the preculture was further grown in 1 L 2x YT medium (Carl Roth) with 100 μg/ml carbenicillin. Cells were grown aerobically in a shaker at 37°C up to an optical density of 0.6-0.8, before inducing Cas13a production with 1 mM IPTG in an overnight culture at 16°C. Cells were pelleted the next day by centrifugation at 5000 rcf for 30 minutes at 4°C. The supernatant except 50 mL was discarded. The cell pellet was resuspended in the remaining 50 mL and centrifuged again at 4500 rcf for 10 min at 4°C. After discarding the supernatant, pellets were flash frozen with liquid nitrogen and stored until further use at -80°C.

IMPACT Kit, restriction enzymes, Crimson Taq DNA polymerase and T4 DNA ligase were purchased from New England Biolabs (Frankfurt am Main, Germany). The 2xYT-medium was obtained from Carl Roth (Karlsruhe, Germany). HBS-P+10x Buffer was supplied by GE Healthcare (Freiburg, Germany) and recombinant human RAGE Fc chimera from a mouse myeloma cell line by R&D Systems (Wiesbaden, Germany). Calibration standards for size exclusion chromatography, MGO (40% aqueous solution), sodium pyruvate, and liquid chromatography-mass spectrometry (LC-MS)-grade acetonitrile were purchased from Sigma-Aldrich (Taufkirchen, Germany). GluC and chymotrypsin were supplied by Roche (Mannheim, Germany). 2,5-Dihydroxyacetophenone (DHAP) and protein and peptide calibration standards for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS were obtained from Bruker Daltonik (Bremen, Germany). All other chemicals were, unless otherwise noted, purchased from Sigma-Aldrich or Acros (Geel, Belgium) and were at least of analytical grade. H₂O was purified water from a Synergi-185 laboratory water system (Millipore, Schwalbach, Germany).